From the clinical symptoms and signs of viral hepatitis can be divided into acute hepatitis, chronic hepatitis, severe hepatitis. Which by the hepatitis A virus and hepatitis E viral hepatitis, chronic hepatitis due to non-place, they will not develop into cirrhosis and liver cancer.
Heard that such and such a person's liver function is not normal, is usually the first reaction of the people that "he is not got hepatitis, will not be transmitted." As everyone knows, a considerable number of patients with abnormal liver function was not caused by hepatitis. In addition, even got hepatitis, do not panic, not all are infectious hepatitis, nor is it would develop into liver cirrhosis or liver cancer.
Misunderstanding 1 - liver disease is hepatitis
A great range of liver diseases, including infectious diseases accounted for the main part. Not only common viral hepatitis, as well as leptospirosis, parasitic infections (schistosomiasis, malaria and amoebic disease), liver abscess, liver granuloma change, hepatobiliary tuberculosis, leprosy, rickettsial infections, mycoplasma infections, relapsing fever, liver syphilis. Fungal infections, kala-azar, Clonorchis sinensis disease, liver and biliary ascariasis ascariasis, liver hydatid disease, biliary Giardia lamblia infection. In addition, many systemic infectious diseases also can cause small areas of focal necrosis of liver inflammation.
In addition to infection factors, other non-infectious factors also can cause changes in the liver.
● autoimmune diseases, such as autoimmune hepatitis;
● drugs and toxins causing liver injury, such as drug-induced, alcohol, industrial and environmental toxic liver disease, etc.;
● genetic and metabolic diseases, such as malnutrition, fatty liver, diabetes, etc.;
● hepatic vascular diseases, such as hepatic artery stenosis, hepatic artery aneurysm. Hepatic arteriovenous - venous fistula, etc.;
● liver tumors, such as primary liver cancer, metastatic liver cancer. Other liver tumor, a benign tumor such as liver and gallbladder.
Clinically, the disease can cause liver damage as many dozens, some of the very low incidence, not a few.
2010年3月18日星期四
A Short History of Viral Hepatitis
Far away in BC 460 ~ 375 years, Western medicine Hippocrates, the father of the "medical disease" mentioned that "epidemic jaundice."
1858 Bambecga that the opening in the common bile duct obstruction is a cause of inflammation, known as "obstructive jaundice." 1868 Virchow discovered that the common bile duct embolization caused by mucus, called "catarrhal jaundice." The beginning of 1940 proved to be the liver inflammation. Followed by Dr. Su Lian at first said that liver damage is mainly systemic infections, called "special package's disease." 1939-1945 years during World War II took place not strictly a result of syringe disinfection of serum hepatitis epidemic due to suspected viral. 1973 Feinstone, etc. from a patient with immuno-electron microscopy of hepatitis A virus found in feces, through fecal transmission, then known as infectious hepatitis (hepatitis A), by serum-borne is called serum hepatitis (hepatitis B).
Dane with the immune electron microscopy in 1970 to complete hepatitis B virus (HBV) particles. Of hepatitis B vaccine to prevent hepatitis B by KrugMam first proposed, has been widely used.
1979, hepatitis A antibodies can be detected.
70s of last century found a non-A, non-B hepatitis. Until September 1989, it will be transmitted by transfusion of non-A non-B hepatitis called hepatitis C infection through the digestive tract of non-A non-B hepatitis called hepatitis.
Rizzetto, etc. in Italy in 1977 found that patients with HBsAg-positive liver cell nucleus there is a new antigen, said the Q factor, was named in 1983, hepatitis D virus (HDV). Was recognized that it alone, the total overlap with the hepatitis B infection, jackal to the tiger, siding with the bully.
Today, these five types of viral hepatitis can be detected in a number of hospitals identified. Subsequently found in sporadic acute hepatitis and chronic post-transfusion hepatitis in many cases, using existing diagnostic techniques can be sensitive to exclude A, B, C, D, hepatitis E, therefore, there may have been-and G-type hepatitis . 1996 Linnen and other officially reported hepatitis G virus (HGV).
In addition to the above, there are a number of viral infection can cause inflammation of the liver disease, mostly systemic symptoms, but mainly, the liver inflammation, mostly secondary, not typical. Such as intestinal virus, rubella virus, measles virus, adenovirus, HSV, VZV, EB virus can cause viral hepatitis.
Engineering and viral hepatitis prevention and treatment of human immunodeficiency
Non-antigen-specific immune drugs increases the immune prevention of infectious diseases in this new method, known as the twenty-first century, the human immune engineering research.
The twenty-first century, the body's immune latest research project
As early as 1987, experimental study, we found that in the antigen stimulated levamisole liniment can quickly enhance the body's specific exemption. In rats, horses to prevent filarial infection study, we observed that as long as the skin can be painted levamisole 1-2 times mice and horses to improve the immune response against filarial-specific.
The specific method is: Immunization-treated mice by 10mg/kg levamisole applicator, and in applicator 7 days after infection of larvae per 50 mice were inoculated subcutaneously with a dose of their skin on both sides of the groin. Immunohistochemistry according to three horse / kg subcutaneously worm volume, was inoculated 5 days before 5 days after inoculation respectively 20mg/kg levamisole applicator. Control group, rats and horses do not applicator, according to the amount of the same methods, and insect larvae were inoculated infection.
The results showed that: 56 immunohistochemistry mice, only 4 cases the disease, 18 disease control group, all rats (P <0.001);>
Immunohistochemistry Ma compared with the control group occurred 10 days before Marty protective antibody peak, and the higher antibody concentration by statistical P <0.01.>
This experiment confirmed that, through the non-specific immune liniment levamisole drug, to enhance the body's resistance, when there is antigen (infection of the original invasion or pathogen inoculation), the same as vaccination, but vaccine coverage without the system, to clear the antigen through the stimulation of antigen-specific immune protection obtained, that is no separation of viruses, bacteria system of seedlings, but rather through the application of liniment levamisole administration from time to time quantitative skin by transdermal absorption, improvement, rehabilitation, control the body's immune function, of foreign antigens to inhibit or even eliminate the role of foreign antigen, so that after invasion of pathogens to kill, inhibit, to lose their pathogenicity without causing disease, and the use of foreign antigen-specific immune stimulation to obtain the protection of vaccine coverage can be achieved without the system access to protective immunity against infectious diseases, completely changed the procedures for vaccination against infectious diseases. Non-antigen-specific immune drugs increases the immune prevention of infectious diseases in this new method, known as the twenty-first century, the human immune engineering research.
Results of exploration and practice
Levamisole can improve the body of non-specific immune response of immune cells, such as increasing medium-sized role of granulocyte chemotaxis, increasing the phagocytic function of phagocytes, when the antigen into the body, the immune group of animals have more immune cells more quickly to play a non-specific immunity should play a role in anti-infection without disease.
Followed by levamisole can enhance the activity of lymphocytes, increasing the number of lymphocytes, antigen invasion, can produce a stronger specific immune response, so the immune group is almost no incidence of mice and horses. Research suggests that by improving the body's non-specific immune response, in antigen-stimulated conditions, can enhance the body against the antigen-specific immune response, to gain protective immunity. Through the liniment levamisole enhance immune cell activity can enhance the body's immune response to hepatitis B vaccine-specific, it is possible to reach liniment levamisole synergy with the hepatitis B vaccine.
Results of exploration and practice
The multi-party discussions Kaihua Pharmaceutical Science Alliance, chairman of the world's productivity, the productivity of the world Branch of China Academy of Sciences Academician David
Liver disease - the invisible killer can not be ignored
The liver is an important human immune organs, known as the body's "life tower", which in human metabolism, digestion, detoxification, blood coagulation, immune regulation, and these play a very important role in the body's essential "Chemical plant. "
The liver is an important human immune organs, known as the body's "life tower", which in human metabolism, digestion, detoxification, blood coagulation, immune regulation, and these play a very important role in the body's essential "Chemical plant. " If the liver are damaged, the blood glucose concentration will be an imbalance, the body of toxic substances in a timely manner can not be ruled out, the body's normal metabolism would therefore rise to confusion.
With the improvement of living standards in recent years, diet change and the growing consumption of alcoholic beverages, alcoholic liver disease and fatty liver incidence is growing rapidly. According to reports, the
Fatty Liver threat posed to the human body is significant. Many clinical studies have shown that fatty liver and liver fibrosis and cirrhosis of the liver often have occurred or in combination. Clinically, no matter what kind of fatty liver can be induced liver fibrosis in patients with fatty liver due to lipid metabolism disorder, so that excessive intake of liver cells has a strong cytotoxicity of free fatty acids, not only damage the liver cell membrane, but also accelerated triglyceride and other lipids in the liver cells of the synthesis and accumulation, so that the excessive fatty infiltration of liver cell degeneration and necrosis. Over time, it led to the occurrence of liver fibrosis and ultimately can lead to cirrhosis and liver failure. Cirrhosis secondary to have a higher risk of hepatocellular carcinoma.
In addition, about 1 / 4 of patients with fatty liver will also suffer from other liver diseases (viral hepatitis and drug-induced liver disease, etc.), the patient is undoubtedly worse.
Selenium - magic Liver factor
Primary liver disease, including alcohol treatment, treatment of liver injury, prevention and treatment in combination with other liver disease, halting or reversing the progress of liver fibrosis and promote regeneration, reduce complications and improve quality of life.
The choice of drug in the treatment of liver disease must be very careful. Scientific research shows that selenium is an essential trace element. In the human body, the liver is the largest organ of selenium content, while the majority of patients with liver disease exist in the body selenium deficiency phenomenon, and the more severe disease, selenium deficiency more apparent.
1. The lack of selenium will not only result in reduced immune function, but also cause the body anti-oxidation system were damaged. At this point, the daily metabolism of the body of harmful substances "free radicals" by the removal of obstacles, excessive free radicals can cause liver damage, which can cause the deterioration of liver disease. The selenium as a strong antioxidant, glutathione peroxidase can be played by antioxidant, protecting the structural integrity of liver cells, removing free radicals, accelerate the decomposition of lipid peroxides, reducing its blood vessels attached to possibility wall to avoid the occurrence and development of fatty liver.
2. Selenium can block the process of development of hepatitis B to cirrhosis of the liver, so that white blood cells and red blood cells in the blood increase in the number hepatoprotective effect of drug treatment be improved in a relatively short period of time, greatly improve the patient's appetite, fatigue, gloomy face and other symptoms improved.
3. Selenium as a potent immune regulator, is capable of regulating humoral and cellular immune system, enhance the body's immune function. As the general immune function in patients with liver disease, body identification, and ability to inhibit the virus will be weakened. The virus was often difficult to obtain clear and repeated attack, resulting in long-term damage to the body. Selenium in enhancing the immune function of the liver while improving its resistance to diseases, helps prevent liver disease keeps returning.
4. Selenium also has the function of detoxification, can antagonize a variety of toxic heavy metals (such as: mercury, lead, arsenic, etc.) and some harmful compounds, thereby reducing the environment of toxic substances in the liver damage.
Cancer Institute of the Chinese Academy of Medical Sciences Professor Yu Shuyu experts, who after 16 years of studies have found that Se can decrease the incidence of hepatitis, 35%, so that a family history of hepatitis, liver cancer incidence rate has dropped 50%.
In 138 cases in
Therefore, selenium because of its excellent liver protection, protecting liver functions by experts known as the "protective factor against liver necrosis." However, selenium has one characteristic, namely, intake must reach a certain level, will produce significant results. Safety of different selenium compounds also vary. Demand for a safe, low toxicity of selenium products became the focus of the majority of scientists. After nearly 40 years of research, the emergence of nano-selenium finally this vision into reality, the preparation of zero valent elemental selenium into the nano-particles (Nano-Se) to produce changes the nature of its activity and safety of other selenium compounds that can not be match.
The treatment of chronic hepatitis B
Principle of treatment of chronic hepatitis B can be broadly grouped into the following points:
(1) reasonable rest, reasonable nutrition, good mentality is to rehabilitate an important factor in the disease: For patients with chronic hepatitis can not stress the absolute bed rest, can be taken to the activities of combining static and dynamic manner, fluctuations in the condition is not large, For example, no jaundice, you can engage in a general household chores, you can walk, but not to do a very intense physical activity. Face reality, the correct treatment of the disease, is an important prerequisite for the swift return of health.
(2) suitable, appropriate anti-viral therapy: the so-called suitable, appropriate anti-viral therapy is: ① viral replication in patients with clear indicators, here to point out that, HBV-DNA positive by dot-blot hybridization to be positive as the Instead, they can not be confined to polymerase chain reaction-positive as an indicator; ② Select the timing of antiretroviral therapy should be appropriate for patients with the disease should choose the most active period in the inflammation, ALT should be higher than the obvious inference in liver biopsy in chronic hepatitis light-or medium-sized, so it easy to achieve a higher efficacy; ③ types of antiviral drugs of choice for patients with appropriate doses of appropriate course of treatment suitable for; ④ close observation, to avoid the toxic side effects of antiviral drugs occur; ⑤ If it is combined medication, Drug mix should be reasonable; ⑥ treatment of choice should be the patient's economic means to tolerance.
(3) carefully selected patients with 12 kinds of convenient, easy to get the immune enhancer, the choice of drugs may be a result of land vary. Species should not be too much.
(4) With regard to the choice of drugs should be followed in simple not complex, should not refined the principles of chaos. A total of not more than five kinds of drugs available from the anti-virus, immune regulation, anti-fibrosis, to improve the symptoms of 34 aspects. According to different conditions, different stage of disease, different ages, different symptoms vary.
(5) In considering the various joint drug program, should pay attention to the different roles of the joint mechanism of drugs such as interferon plus thymosin, interferon plus Zadaxin, interleukin -2 plus interferon, interleukin -2 gala lamivudine and so on, do not select the same mechanism of action of the drug re-union, such as acyclovir plus lamivudine.
(6) should increase the incentive to avoid the disease, such as fatigue, upper respiratory infection, diarrhea, emotional volatility, to liver damage drug applications.
(7) regular follow-up, track time due to people, and the illness to another. Follow-up, in addition to check the liver function, it is important to dynamically track the changes in B-ultrasound to observe the size of the liver, with or without thickening of the spleen, portal and splenic vein widened and so on.
Gene therapy for viral hepatitis
Is mainly caused by viral pathogens, hepatitis viruses, including hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), hepatitis E virus (HEV ), recent studies have shown that, GB virus (GBV) / hepatitis G virus (HGV) and transfusion transmitted virus (TTV). Reported recently discovered SEN virus abroad (SENV), may be explained, there are still 10 to 15% of the viral hepatitis patients with unclear etiology of the problem. No matter what kind of hepatitis virus infection in acute, chronic viral hepatitis, the root cause is the presence of viral replication and expression, the virus antigen-induced immune response principles, resulting in inflammation of the liver damage. Therefore, in order to fundamentally solve the problem of viral hepatitis, from the genetic level, seeking to block, inhibit, or even clear the hepatitis virus, which is a very important way of thinking. Gene therapy is the field of biomedical research a hot spot.
First, the concepts and strategies for gene therapy
Gene therapy is to use a normal or wild-type gene correction or conversion of a defect or disease-causing genes molecular biology level of treatment. In the traditional sense of gene therapy (gene therapy), is the target gene into the target cells within the host cells after the gene integrated into the main camp as part of genetic material of cells, the expression of target gene products play a role in the treatment of the disease. In recent years, the use of certain gene-transfer technology, even if the purpose of the primary cells, genes and camping integration of non-occurrence of genes, gene expression and can be temporary. The expression of target gene product also has a certain therapeutic effect. Such gene therapy target gene should be, as we normally use in clinical drug like. To the traditional sense of the relative difference between gene therapy, this gene does not occur with the host cell genome integration, the temporary expression products play a therapeutic role of gene therapy is called gene therapy (gene therapeutics).
Gene Therapy for the host disease, in accordance with the measures taken by the different cell gene, is divided into gene conversion (gene replacement), gene amendment (gene correction), genetically modified (gene augmentation), gene inactivated (gene inactivation) and genetic vaccines (gene vaccine) and other five major strategies. Genes and gene inactivated vaccine is blocking and inhibiting hepatitis B virus gene replication and expression of important gene therapy. Conditions of gene therapy include: the purpose of gene acquisition, the choice of target cells to gene nest principal cells into efficient means of gene transfer. Gene therapy steps include: preparation of target genes, the receptor cells in culture, the choice of carrier will be the target gene into the target cells and so on.
Second, viral gene therapy
In the anti-tumor gene therapy, genetic diseases and the treatment of infectious diseases, there is a very important application prospects in recent years, studies have shown that gene therapy in the treatment of viral hepatitis also can play an important impact. Although the viral gene therapy research has made a series of progress, but, due to the pathogenesis of viral hepatitis have not yet fully understood clearly that the current gene therapy in the treatment of viral hepatitis are far from the highest level. Viral gene therapy depends on the one hand, gene therapy technology itself is the speed of development and status, it also depends on the study of viral pathogenesis of the depth of their own.
(A) Transgenic expression of lymphokine
Lymphokines in the treatment of communicable diseases that have an important role and broad application prospects. One of the interferon (IFN) has become the only recognized treatment of anti-hepatitis B virus genetic engineering of effective drugs. Lymphoid gene expression using gene therapy research of infectious diseases, is gene therapy in infectious diseases is an important application and important research direction.
1, interferon gene transfer and expression of
Seif and other mice of interferon β (IFNβ) gene placed in major histocompatibility complex (MHC) promoter sequence under the control of recombinant expression vector transfected Babl / c mouse fibroblast cell line NIH3T3, has been continued IFNβ expression. IFNβ expression cell lines, for follicular stomatitis virus (VSV, vesicular stomatitis virus), encephalomyocarditis virus (EMCV, encephalomy-ocarditis virus), and
2, interleukin -2 transgenic expression of
Guidotti applications such as HBV DNA transgenic mice confirmed that IL-2 Dui HBV DNA transcription of the mRNA of 2.1kb has apparently abated. That the IL-2 Dui HBV DNA transcription promoter activity of the 2.1kb mRNA has obvious negative regulation. At the same time, through tumor necrosis factor (TNF) and interferon-γ (IFNγ) to achieve the induced anti-viral effect. Because there is no liver cell membrane IL-2 receptor, so that the IL-2 of the antiviral activity of its promoter activity of HBV DNA used to direct inhibition achieved. And that the IL-2 inhibited the expression of HBV DNA in the post-transcriptional level occurred.
(B) oligoadenylate synthetase transgene expression
Chebath such as 2-5AS constructed the eukaryotic expression vector, and as marker genes dihydrofolate reductase (dhfr) gene expression vector in order to co-transfection of the way into Chinese hamster ovary (CHO), receiving The expression of 2-5AS. The transduction of cell lines and their parental cells, compared with a significant anti-ability of small RNA viruses, such as the door fruit virus (mengovirus) and other infections. The study results showed that the expression of 2-5 AS cell lines can bypass the induced IFN, the expression of 2-5 AS alone can have a significant anti-viral effects. Such 2-5 AS in IFN inducible anti-viral therapy is of universal significance. Therefore, it is a valuable exploration of the direction.
(C) virus antigen expression in transgenic gene encoding
Felgner and other first HIV glycoprotein gp120 gene with the cytomegalovirus (CMV) of the immediate-early (IF, immediate early) promoter sequence re-constructed the recombinant expression vector expressing gp120. Such recombinant expression vector DNA for intramuscular injection of quality chisel, some cells carried out the availability of such DNA to express gp120. As an antigen, the body can produce immune protective antibodies. This specific immune response is an important way to combat HIV. Morin, also obtained similar results.
(D) protective antibody gene transfer and expression of
HIV infection of CD4 + cells and their destruction, resulting in reduction in the number of CD4 + cells, or even totally lost. So associated with defects in immune function. CD4 + cells to protect against infection, in which a strategy is to use gene therapy into HIV-1 antibody gene expression, and in HIV-infected, Marasco, etc. imported against HIV-1 single-chain antibody genes, can be specific sex with HIV-1 envelope glycoprotein in combination, to inhibit or block HIV's ability to infect. With HIV, cDNA encoding a single-chain antibody genes were co-transfected T lymphocytes, which express their HIV core protein (gag) level changed little, but the ability to produce infectious amount of HIV virus particles was significantly reduced. Recently, Pomerantz and other research data shows that anti-Rev single-chain antibody expression in the cytoplasm can be captured Rev protein, and can inhibit HIV expression. Tips to HIV antibody encoding genes as target genes for anti-viral gene therapy is a promising approach. However, HIV can encode a variety of structural and regulatory proteins as antigens can trigger the body produce different antibodies, in order to optimize the expression of intracellular antibodies, anti-HIV gene therapy strategy for infection must be possible inhibition or blocking antibodies compare the effect of HIV infection in order to obtain a more satisfactory results of gene therapy.
(E) the process of blocking the virus to enter cells
HIV infection and enter the host cell, the necessary envelope glycoprotein pg120 with its host cell membrane with the combination of CD4 antigen, which is undermining HIV choose the next important reason for CD4 + cells. So to use an excessive amount of CD4 antigen and HIV-envelope glycoprotein gp120, can be blocked and the ability to inhibit HIV infection. Thus to protect uninfected CD4 + cells. As long as the right to ensure the number of CD4 + cells, HIV infected persons will not develop into a serious immune deficiency, lead to serious opportunistic infections. Fischer Deng Jun soluble CD4 + (sCD4) molecules encoding genes into cultured T-lymphocytes, sCD4 molecules can indeed block the HIV-1 infection. CD4 molecules in the immune system's normal function and major histocompatibility complex (MHC Ⅱ)-type molecules are closely related to immune recognition. Therefore, the application of this sCD4 interfering HIV-1 and the combination of CD4 + cells with anti-viral gene therapy for fear of interfering with MHC Ⅱ-specific T-cell function. Transgenic mice results show that in fact this fear is unfounded. Weber, etc. to express sCD4 molecules was established transgenic mouse lines, continued to express sCD4 molecules up to 100ug/ml, but such a high level of sCD4 molecule expression and no effect on CD4 + cells in mice with different antigen or anti-CD3 antibody to stimulate the immune recognition and immune response.
In addition, the helper T cell-mediated in vivo antibody response mechanism, are not subject to the impact of over-expression of sCD4 molecules. Morgan has applications such as retroviral expression vector - packaging cell line gene transfer system, the gene encoding sCD4 into human T lymphocytes, express, can inhibit HIV-1 infection. Recently, sCD4 and immunoglobulin fusion gene expression, but also to achieve a significant anti-HIV-1 effect. However, the sCD4 gene as the purpose of anti-HIV-1 gene in gene therapy phase I clinical trials failed to achieve the expected success. Isolated from clinical specimens to the resistance of anti-sCD4 molecule HIV-1 virus strains, from one side of one of the reasons explained. Experiments confirmed that the resistant strains, and sCD4 molecules sCD required number is 200-2700 times the sCD4-sensitive strains. Therefore, taking into account the use of sCD4 as a target gene for the gene encoding anti-viral gene therapy, we must take into account the regulation of gene transfer and expression technology, but also taking into account the sCD4 molecule itself and some properties and characteristics.
In recent years, HIV-1 infection in the pathogenesis of the most exciting areas of research findings is the HIV-1 infection, have to rely on auxiliary molecule (cofactor) fusion elements (fusin), namely, that CC CKR5 chemokine by body involvement. Because HIV-1 can not only efficiently infected with CD4-cell lines, such as the central nervous system infections, and that the gene encoding the CD4 molecule CD4-transfected cell lines to the phenotype by CD4-into CD4 +, are also can not lead to its sensitivity to HIV-1 infection increases. Finally thought of the possibility of supporting elements. To this end, scientists from various countries fought a full eight spring and autumn, before the candidate molecules from a number of confirmed HIV infection is the integration of elements must be integrated with the host cell auxiliary molecules. This important discovery, must be able to lead to new therapies against HIV infection appear. Su-antibody fusion molecule for gene therapy as well as the fusion gene-specific ribozyme (ribozyme) of molecular design, it will be inhibit or block HIV infection of target cells in gene therapy.
With regard to HBV receptors have always thought that HBV infection of PHSA-R is the target cell receptor molecules, but this point of view there are also serious deficiencies. Recent research results indicate that annexin V (annexin) may be another target cells infected with HBV receptor molecules. HCV receptors on the findings of a study of this transmembrane molecule CD81 may be target cells infected with HCV receptor molecules. Hepatitis virus receptor molecules according to the nature of anti-hepatitis B virus may be to explore treatment methods important way of thinking, but there is no substantive progress.
(Vi), the cellular immune
Malim, etc. According to HIV-1 trans-activator protein Rev structural features, designed anti-HIV-1 gene therapy, intracellular immunization strategies. Rev protein, leucine-rich carboxyl terminal is not the role of its absolute dependence trans-zone. This area of mutants as the wild-type Rev protein competitive inhibitor, for failure to cut a single shear stability of HIV RNA expression has a significant impact. Amino acid sequences of the HIV Rev in 78 (LD) and 79 (EL) for site-directed mutagenesis, then the formation of mutant Rev M10. This mutant maintained the wild-type Rev and the Rev responsive element (RRE) combination of capacity, but there is no activation. With retroviral vectors - packaging cell line gene transfer system, the gene encoding Rev M10 into human T cell lines after stable expression of Rev M10 transfected cell lines can significantly resistant HIV-1 infection, and in human peripheral blood T lymphocytes to be repeated. In addition, the virus-specific ribozyme gene encoding genes into T cells among the transduced cells can also produce the cells against the virus within the immune status. Was screened using phage display technology, hepatitis virus-specific single-chain antibody variable region may be that anti-hepatitis B virus immune cell gene therapy is an important research direction.
(G), bait Design
Virus gene molecular biology studies of anti-viral gene therapy provides a new design ideas. Such as human immunodeficiency virus (HIV) gene regulation there is a very complex cis-regulatory mechanisms and anti-workers, these regulatory mechanisms have become the design of anti-virus program an important basis for gene therapy. HIV trans-conditioning (trans regulation), that some trans-activator (transactivator) and HIV RNA with the corresponding target site with the HIV genome replication and expression has a very close relationship. Therefore, we can design a gene, its transcripts and HIV RNA competitive manner, then a combination of trans-stimulated, HIV RNA is not enough trans-stimulated, then the combination of agent and stimulation, it can not be effective replication and expression. Competitive with the HIV RNA and trans-activator cited combination of RNA fragments, known as the bait (decoy) molecules.
Decoy RNA molecule expression vector design and gene therapy is to prevent HIV replication and expression of an important strategy. Program design and application of bait for virus nucleic acid with the trans-activator of the separation, which requires elements to be in the amount of bait advantage. That must be in the "over-expression (overexpression)" status. Only in this way in order to have enough bait molecules and competitive combination of HIV RNA trans-activator. Against HIV, the bait most commonly used gene fragment designed for Tat and Rev and so on. Gilboa, etc. will be HIV-1 in Tat and Rev of the two genes were broken into retroviral vectors, including the endogenous pol Ⅲ (tRNAmet) under the control of the promoter family, express, can inhibit HIV replication in CEM cells, and expression. In order to improve the expression level of bait and the anti-viral effect can be more than the gene encoding the bait head and tail tandem together, would enhance the number of molecules expressed by the bait and enhance its anti-viral effect.
Viral gene trans-regulatory mechanism, not only is the design of anti-viral gene therapy directly to the theoretical basis, but also to improve the expression level of target gene provides an effective means. Bednarick so the human recombinant IFN in HIV-1 LTR promoter in the series of downstream, into Vero cells after its secretion level of IFN only in between 50 ~ 150u/ml. If to this expression system to provide LTR promoter in the anti-trial activator tst protein by trans-activated, allows the expression level of IFN to 103u/ml. At present the field of gene therapy research, the gene expression level is not high, impeding gene therapy technologies to play a better role, this mechanism is of great significance. Not only the expression levels of cytokines in this strategy can be applied to improve the expression level of bait molecules can also take advantage of this trans-activation mechanism has been upgraded.
(8) virus-infected cells of the suicide mechanism of
Anti-viral gene therapy research, in most cases is based on molecular biological characteristics of the virus, according to the principles of the immune cell design. Design of these programs to a large extent dependent on protein and RNA, including infection of factors, including continuing high levels of expression, which is why the immune cells is difficult to obtain a thorough treatment of an important reason. Therefore, can be designed to remove the virus-infected cells in gene therapy programs, rather than simply interfering with the process of viral replication and expression. Among them, suicide gene and virus-infected cells into cleared of gene therapy strategies in this area occupies an important position.
Causes cells to commit suicide so-called "suicide gene (suicide gene)" many different types, such as certain viral genes are known to diphtheria toxin (DTA) in the case of very low level can cause cell death, the promotion of drug metabolism and conversion of genes, such as varicella - zoster virus (VSV) thymidine kinase (TK), allows non-toxic ganciclovir (GAN, ganciclovir) is converted to highly toxic metabolites, killing cells, HSV's TK gene has a similar effect. Also included are caused by programmed cell death (PCD, programmmed cell death) of the genes.
The suicide gene into to be effective in anti-transduced cells are no doubt, but how can they ensure that cell death is limited to virus-infected cells, while no side-effects on normal cells, the design is the key. Such gene therapy program also can take advantage of the characteristics of the virus-infected cells design. France Klatzman other HIV and the infected cells according to the characteristics of the design of the suicide mechanism of HIV-infected cells to remove virus-infected cells made to take a more satisfactory therapeutic effect. First, the DTA gene was recombined into deletion mutant HIV LTR promoter downstream of, `If the cells do not increase HIV infection and to provide trans-activator protein, such DTA will not expression; but if the cells are infected with HIV, HIV replication and expression of the process at the same time the expression of DTA provides a trans-activator, by stimulating expression of later, can cause the death of HIV-infected cells. The existence of cells not infected with HIV trans-activator protein, DTA did not express, you can survive intact. This mechanism can be used to selectively remove the virus-infected cells. Therefore, a mechanism is also anti-drug suicide gene therapy is an important component.
(9) antisense oligonucleotides
Antisense oligonucleotides, including antisense oligodeoxynucleotide (ODN, oigodeoxynucleotide), and two kinds of antisense RNA. Antisense RNA in the study of gene regulation in prokaryotic cells, found in a negative regulation factor. But later proved that the genetic regulation of eukaryotic cells also have the involvement of antisense RNA. That the antisense RNA molecule in the regulation of gene expression of universal significance. Antisense RNA sequences complementary to its target RNA to acid-base combination of methods. Block-based intracellular transport, shear processing, and ribosome binding. And the activation of endogenous nuclear ribozymes (eg, RNaseH, etc.) will be its decomposition. In order to inhibit or block a particular gene expression and function. The gene expression regulation mechanism, soon applied the anti-viral gene therapy research.
Chatterjee applications such as adeno-associated virus (AAV) vector of HIV RNA antisense RNA expression vectors, such recombinant expression vector into T lymphocytes, expression of antisense RNA may be with the HIV LTR TARA section of 63bp sequence fill. This expression of antisense RNA against HIV-1 inhibition rate of 70-90%. Antisense RNA, not only in controlling HIV-1 infection plays an important role, but also in control of HBV, HSV human papillomavirus (HPV), Lloyd's sarcoma toxicity (RSV) infection, etc. have important applications.
(+) Ribozyme
Nucleic acids (ribozyme) is a kind of enzyme catalysis, can such a specific nucleotide sequence GUC downstream cutting a class of small RNA molecules RNA molecules. The discovery of RNA nucleic acid molecules, breaking the protein in the enzyme dominance of the field, the concept of the enzyme from the protein extended to the field of nucleic acids. RNA nucleic acid molecules and the substrate RNA molecule base combination of acid on the way to ensure a high degree of specificity of the ribozyme effect. At the same time, the nuclear DNA and the formation of the enzyme substrate-binding active site, substrate can be cut, destroy its structure. In this way, by cutting the RNA chain can no longer be a template for translation of the protein, but also can not serve as a template for RT-nucleic acid replication. Ribozyme RNA lysate highly specific and efficient cleavage of the nature of the substrate RNA, as a new type of anti-viral gene therapy molecules, has been widely appreciated. That the nucleic acid technology is the design of antiviral gene therapy, an important direction to explore. First, nucleic acids in plant virus, satellite viruses and some protozoa, such as Tetrahymena (tetrahynena) such as the study found. Summing up the basic structure of nucleic acid RNA molecules, generally have hammerhead (hannerhead motif). Hairpin (hairpin motif), as well as an ax-shaped (axed motif) structure type. The basic structure of nucleic acids based on these types of relatively conservative design of the active center of the structure, according to the substrate RNA chain cleavage site nucleotide sequence of the structure and its annexes, design ribozyme flanking sequence (flanking sequence). Theoretically speaking, as long as the understanding of the substrate RNA and the structural properties of the series, we can design RNA molecules directed against any specific ribozyme. Ribozyme RNA molecules in the flanking sequence 17nt long ridiculed as long as the guarantee above, the design of ribozyme and substrate specificity of RNA molecules would be a normal human cell line RNA solution reaction occurs. Because even if there is a nucleotide can not be correctly paired active site can not be formed, the column solution reaction does not occur.
Is the essence of nucleic acid RNA, extracted from the natural purification of the base calendar for the specific fragment of the ribozyme RNA molecules will not work. Artificial synthesis of RNA is a way, but expensive. No practical future. RNA gene has no pharmaceutical breakthrough. Therefore, the practical application of ribozyme gene therapy approach needs to go. Namely, the gene encoding ribozyme in vitro designed to retroviral vector into the target cells express them. This gene therapy strategy has been a more thorough level of cells and animals, systematic study of the
In 1990, Sarver and so on for the first time HIV-specific ribozyme was constructed recombinant expression vector, and in cells, extracellular successfully HIV RNA was cut, and anti-HIV gene therapy experiments. Anti-HBV ribozyme gene therapy are also well under way. In 1992, Chen designed the nine target site such as anti-HIV ribozyme and Weizsacker has designed three other anti-HBV ribozyme target site, so that ribozyme anti-virus technology day was ripe to become anti-viral gene therapy in another major research directions. Do not believe anti-virus ribozyme gene therapy approach in the future anti-viral gene therapy research to play a greater role.
3, viral hepatitis, genetic vaccines
The recombinant expression vector directly into the body organs and tissues, and obtain the expression of target genes, which had achieved more than 10 years ago, and as a gene therapy program of some exogenous gene into the body of a way that has been widely attention. But the use of this technology will be a protective immune response induced by antigen protein recombinant expression vector into the body, prevention and treatment of infectious diseases as a technology, but in recent years thing. Ulmer, etc. First, in order to influenza virus (influenza) nucleocapsid protein (NP) of the recombinant expression vector DNA vaccinated healthy mice to obtain an effective humoral and cellular immune response and can protect the immunized animals against a variety of influenza virus infection attacks, and thus created a DNA vaccine against infectious diseases, a new era.
(A) the development of DNA vaccine
Influenza virus gene vaccine to create a genetic vaccine prevention and treatment of infectious diseases, a new era. First of all influenza virus gene vaccine using highly conserved influenza virus nucleocapsid protein coding genes, obtained with different strains of influenza viruses cross-immune protection. In particular, which in turn has a more important practical application value. Because, such as human immunodeficiency virus (HIV), hepatitis C virus (HCV) virus genome such as the existence of different genotypes, but also under the pressure of immune selection continues to mutate, such as the HCV quasi-species sex is a typical example. For influenza viruses, viral genetic variability of this antigen vaccine's success, its meaning is broad and profound.
Since 1993, influenza virus genome to construct gene expression vector and the success of the vaccine after a short period of 2 years, genetic vaccine technology has been rapid development, not only from the influenza virus extended to HIV, HCV, hepatitis B virus (HBV ) and other areas of viral infection, but also extended to bacteria, parasites, mycoplasma, and other areas of infectious diseases. Moreover, the anti-cancer, autoimmune diseases, the genetic vaccine research has progressed rapidly. By 1995, the anti-tumor, anti-HIV genetic vaccines in the completion of laboratory and animal models to study and achieve good results, based on, has begun Phase I clinical trials. This is a biological treatment methods, concepts and techniques from the proposed route, to the Phase I clinical trials, is the shortest one. Of high-tech modern biology is growing faster than is evident.
(B) of infectious disease DNA vaccine Research
With regard to infectious diseases, genetic vaccine research, on the one hand to find an effective immune protection of vulnerable populations, taking another gene vaccine for the treatment of refractory diseases has provided new opportunities.
1, viral infectious diseases, genetic vaccine
HBV gene vaccine research, using hepatitis B virus surface antigen (HBsAg) and hepatitis B virus core protein (HBcAg) gene were used to construct expression vector, or construct two kinds of protein fusion gene can be induced by immunization of mice appear specific humoral and cellular immune responses. HCV genetic vaccine research is focused on core-shell protein, as well as non-structural protein 3 (NS3) and other coding gene region, but there are few studies using area E1 and E2 envelope gene immunization areas. HIV genetic vaccine is mainly concentrated in the HIV envelope glycoprotein encoding gene region, such as gp160, gp120 and gp41 coding gene region, etc.. HIV various protein components will now be assembled into non-nucleic acid components of non-infectious viral protein particles, the immune effect of a single viral protein content than the much better. In addition to influenza virus gene vaccine antigen varied hemagglutinin antigen (HA) coding genes, it also uses a highly conserved
2, bacterial infection of sexually transmitted diseases, the genetic vaccine
Designed to target the cells infected with the gene vaccine made remarkable progress. Mycobacterium tuberculosis infection is currently on a global scale also showed an upward trend. The immune effect of BCG vaccination is also worthy of further study, but also in many countries there is no universal BCG vaccination carried out. According to some characteristics of Mycobacterium tuberculosis immunization designed Mycobacterium tuberculosis 65kDa heat shock protein (HSP65) gene vaccine can induce specific humoral and cellular immune response, for the subsequent infection of Mycobacterium tuberculosis vaccine has significant resistance to diseases. To HSP65 gene vaccine-induced immune protective effect of live BCG vaccine immune protection similar effect, but HSP65 protein as a subunit vaccine for immunization had no protective effect of this immunity. At the same time, to come from Mycobacterium tuberculosis 36kDa proline-rich protein coding genes genetic immunization can also obtain the immune protective effect of Mycobacterium tuberculosis infection. Huygen applications such as Mycobacterium tuberculosis antigen 85 (Ag85) of the gene encoding genetic immunization, also received a good immune effect.
(C) prospects for viral gene vaccine
Infectious diseases, genetic vaccines, first of all infectious pathogens from the start of molecular biology. So far, the large number of infectious diseases, the genetic structure of pathogens, the nature of its encoded product has not yet clarified, it is hampered infectious disease DNA vaccine research a bottleneck. Only solve this premise, in order to choose the purpose of a good DNA vaccine genes.
Infectious diseases, genetic vaccine research, but also to study various components of pathogens antigen immune response mechanisms. Many studies have shown that not all pathogen gene construct into the DNA vaccine can induce protective immune response, and each kind of DNA vaccine immune response capabilities are vastly different. DNA vaccine can induce cell-mediated immunity to protective immunity-based immune response is to distinguish it from inactivated vaccine and subunit vaccine against an important symbol, but also generally agreed that DNA vaccine for treatment of refractory disease the most fundamental basis for the future. But the gene vaccine and its mechanism of immune response is not systematic.
Infectious diseases, genetic vaccine research, and its vitality lies in the new gene vaccine. Such as chronic hepatitis B patients with high titer of serum hepatitis B virus surface antigen (HBsAg), but the organism has developed into a multi-immune tolerance against HBsAg. Although studies have shown that genetic immunization because of their antigen processing and presenting a different path, resulting in a different protein antigen immune effect, but HBsAg gene vaccine, when vaccination is effective in the treatment of chronic hepatitis B is not necessarily. Because the majority of the current study focused on immune response in normal animals, but chronic hepatitis B virus HBsAg response to the fundamental changes that have taken place not only on the basis of the immune response of normal animals, suggesting individuals with chronic viral infection immune response. Anti-idiotypic DNA vaccine is probably an effective way to solve this problem. Design of a single T cell epitope of the DNA vaccine is a better option. In some cases, the design point for the inappropriate immune response in situ, but to promote the formation of pathogenic strains of immune escape or aggravated infection. Therefore, using T cells for a single locus gene vaccine immunization strategy is desirable. DNA vaccine studies have had great importance in antigen processing and antigen-presenting function of cofactors. In some cases, possibly because of the lack of strong immunogenic stimulus, but may also be due to antigen processing and presenting the auxiliary signal and obstacles, ultimately did not induce an effective immune response. At this time only considering the antigenicity of the pathogen is not enough, the immune response of co-stimulatory signals also have equal importance. Along with links to further address these technologies, I believe that genetic vaccine would be the first for clinical treatment of communicable diseases as a means of gene therapy.
Persistent infection in patients with chronic hepatitis B proved
Subject to understand the HBV core gene promoter mutation and the degree of liver damage or HBeAg status of the relationship research purposes. Experiments with serum collected from the major hospital in
Nested-PCR results showed that 59 cases of chronic hepatitis B patients 35 cases of HBV DNA positive, the positive rate was 59.3%. 16 patients with HBeAg-positive patients with HBV DNA positive, the positive rate of 57.1% (16/28), 19 patients with HBeAg-negative patients with HBV DNA positive, the positive rate of 61.3% (19/31). BCP sequencing results showed that without the usual sequence of samples, the most common type of mutation nt1762, 1764 pairs of sudden occurrence (A → T, G → A), accounting for 57.1% (20/35); followed by mutation types nt1799 sites mutation, from C → G, 54.4% (19/35), for non-sense mutation; nt1752 mutation, from the A → G, so that the password by isoleucine into valine, accounting for 37.1% (13 / 35); nt1753 T → C, accounting for 20.0% (7 / 35).
Core antigen and e antigen, some of the same sequence, this part of the sequence can be the host cytotoxic T lymphocytes recognize the core peptide of the changes in the immune attack may be caused by chronic liver disease. The results showed that samples of each district are base pairs in the Enh2/CP mutation, the most common mutation nt1762, 1764 pairs of sudden occurrence (A → T, G → A), meeting the common mutations are nt1799 mutation, from C → G, for the non-sense mutation; nt1752 mutation from A → G, so that the password by isoleucine into valine, nt1753 T → C. Core gene promoter to make hepatitis B virus mutations (HBV) can be persistent infection in the body, these mutations in patients with chronic active hepatitis related to.
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