复制型HBV转基因小鼠的建立、生物学特性、应用及无免疫耐受研究

发布时间：2018-05-23 09:40

本文选题：乙型肝炎病毒 + 转基因小鼠　；参考：《第一军医大学》2007年博士论文

【摘要】： 人类乙型肝炎病毒具有严格的种属特异性，一直缺乏理想的动物模型；．通过转基因技术建立HBV转基因小鼠模型，在国内外已被广泛用于乙型肝炎发病机理、抗乙肝治疗和药物筛选评价研究。作者建立的复制型HBV全基因组转基因小鼠，经不断选育现已稳定遗传20代以上，且可用常规ELISA和荧光定量PCR检测到乙肝病毒在血清中的复制和表达，与国际上普遍认同的由Guidott建立的复制型HBV转基因小鼠相当。该复制型HBV转基因小鼠模型为研究HBV的发病机制研究以及抗乙肝药物开发研究提供了良好的实验动物模型。目前已被国内近百家科研单位应用。本论文的研究内容包括：(1) HBV转基因小鼠模型的建立；(2) HBV转基因小鼠生物学特性；(3) HBV转基因小鼠在抗乙肝药物药效评价中的应用；(4)利用siRNA短暂基因沉默技术制备无免疫耐受HBV转基因小鼠；(5)总结与展望。第一章复制型1.3copy D基因型HBV转基因小鼠的建立。本实验将1.3copy的D基因型HBV全基因组作为目的基因，采用显微注射技术将其注入小鼠受精卵细胞雄原核，然后将受精卵移植于受体假孕母鼠输卵管内，发育产生子代小鼠。仔鼠尾组织提取DNA进行HBV基因PCR整合检测，再用酶联免疫吸附法(ELISA)检测血清乙型肝炎表面抗原及e抗原，荧光定量PCR检测血清HBV DNA。结果：共获卵数1210枚，注射882枚，存活397枚，成活率45％；移植卵数337枚，受体数14只，怀孕鼠9只，产仔54只，移植成功率为16.02％(54／337)，用PCR法检测54只仔鼠尾组织，15只为整合阳性。G0代经血清检测HBVDNA和HBsAg阳性4只；G1代检测61只仔鼠，血清HBsAg阳性13只。可遗传子代Founder2只。定量检测血清中HBsAg表达量为80～930μg／L。取转基因小鼠肝、脾、肾、小肠及股动脉等组织，进行HBsAg、HBcAg免疫组化检测，仅发现肝及肾组织有HBsAg、HBcAg散在不均匀表达。第二章对已制备获得的稳定传代复制型1.3copy D基因型HBV转基因小鼠的F14-F20代小鼠进行了生物学特性研究。所观察的主要内容包括以下几个方面：(1)乙肝病毒指标复制表达水平及稳定性；(2)免疫学特性及病理观察；(3)乙肝病毒基因在各组织的转录水平；(4)传代培育。首先，，本研究应用ELISA和荧光定量PCR的方法对血清乙肝相关指标进行了分析，发现转基因小鼠血清中存在HBsAg、HBeAg、HBcAg、pre S1及HBV DNA，其中HBsAg的表达水平较高，达5107.5±4144.8IU／ml，个体间有明显的差异但性别之间无显著差异；而HBeAg表达水平较低，仅为(1.94±1.66s／co)，血清中存在HBV DNA含量达到10~4～10~6copy／ml。通过超高速离心的方法，在透射电镜下观察到HBV转基因小鼠血清中存在HBsAg颗粒和Dane氏类颗粒，此结果证实了该转基因小鼠中整合的HBV基因组DNA可复制包装成病毒颗粒并分泌入血，本转基因鼠为复制型。免疫组织化学结果显示：转基因小鼠肝细胞中表达HBsAg和HBcAg蛋白两种病毒蛋白，但并不是所有的肝细胞同时表达HBsAg、HBcAg蛋白。HBsAg分布于肝细胞质中，HBcAg蛋白分布于肝细胞核中。肝组织H-E染色分析发现，转基因小鼠肝组织中未见肝细胞病变。采用流式细胞术和Elispot法对该品系转基因小鼠脾细胞悬液DC的特异性标志CD11c及其上TLR2或其胞内TLR9的表达检测发现，HBV转基因小鼠自身除对HBV病毒存在免疫耐受外，其天然和获得性免疫功能未见异常。利用real-time RT-PCR荧光定量技术对转基因小鼠肝、肾、脾等十余多种组织中的总RNA进行定量检测，分析了HBV基因在转基因小鼠中各组织的转录特征，结果表明转基因小鼠基因组中整合的HBV基因主要在肝、肾组织能够转录出乙肝的mRNA，并以肝组织转录水平最高。在早期培育阶段，将两个首建鼠(Founder)分别与正常鼠杂交进行传代，在每一世代仔鼠中进行检测发现其阳性率始终保持在40％—50％之间。这说明1.3copyD型HBV已稳定整合表达，并能可靠地遗传给下一代。为增加外源基因的拷贝数，提高阳性率和表达水平，本研究把两个首建鼠分别传代至F14代进行互交并检测互交后生产仔鼠，其阳性率达到80％左右，这说明不同的首建鼠基因的整合位点不同且呈多位点整合，互交后可显著提高阳性表达率。上述实验结果表明我们建立的复制型1.3copy D基因型HBV转基因小鼠是国内比较理想的乙肝转基因动物模型。第三章利用HBV转基因小鼠对多种抗乙肝药物进行药效评价研究。(1)抗乙肝病毒复制药物拉咪呋啶或阿德福韦酯可显著降低转基因小鼠血清HBV DNA滴度，停药后HBV DNA又恢复至原来水平，该结果与临床应用药效及国外同类研究结果一致；(2)大剂量乙肝治疗性疫苗的应用可抑制HBV DNA的复制，动物体内IL-2和IFN-γ水平升高；(3)IFN-α1b可显著降低HBV转基因小鼠体内HBV DNA的复制，但5周后药效开始减弱；(4)人源化HBsAg单抗在4小时内可使血清HBsAg滴度迅速下降。上述实验结果与此类药物临床治疗效果相符，说明我们制备HBV转基因小鼠模型不仅可通过临床上经典有效的抗乙肝药物得到验证，而且可用于抗乙肝药物药效评价。这充分证明该转基因动物模型在乙肝发病机制、抗乙肝药物药效评价和新药开发研究中的科学意义及应用价值。第四章无免疫耐受HBV转基因小鼠的建立研究。由于HBV基因在转基因小鼠胚胎期即表达，导致小鼠对HBV的免疫耐受，不能模拟肝炎患者的肝损伤症状，因而在一定程度上限制其应用。本文利用siRNA暂短基因沉默技术，通过胚胎腹腔或孕鼠尾静脉注射质粒pU6-siHBV11，以期制备无免疫耐受HBV转基因小鼠。该质粒根据siRNA靶点选择的原则，以2．2．15细胞中HBV的序列为基准，在保守区域选出12条序列作为抑制靶点。通过2．2．15细胞和HBV转基因小鼠模型体内抑制实验，从中筛选出一条高效抑制作用的siRNA质粒即pU6-siHBV11。本研究对14天—18天胚胎时期转基因仔鼠RNA干扰，使HBV基因转录启动延迟和抑制。我们对所获实验仔鼠在6月龄进行肝组织病理切片检查，观察到有轻度～中度肝浊肿病变，这为制备无免疫耐受HBV转基因小鼠及乙肝发病机制研究提供新的途径。第五章总结和展望我们培育出复制型HBV转基因小鼠模型，现已稳定传代20代以上，有多种乙肝指标表达已达到实用阶段，可与国外公认Guidotti建立的HBV转基因小鼠模型相媲美。利用我们制备的HBV转基因小鼠模型现已初步建立抗乙肝药物评价体系，可从DNA、mRNA水平、细胞水平、机体免疫水平对抗乙肝药物进行药效评价和开发抗乙肝新药研究。经典有效的抗乙肝药物对HBV转基因小鼠疗效反证了该动物模型可真实的模拟表现人类乙型肝炎疾病许多生物学特征。通过提高目的基因的拷贝数的育种手段可有效提高转基因仔代鼠的阳性率、表达水平。应用RNA干扰短期基因沉默技术制备无免疫耐受有肝损伤的HBV转基因小鼠，可模拟乙肝患者肝损伤的病理变化。 HBV转基因鼠基础与应用研究结果对慢性乙肝治疗研究具有指导和验证作用。抗乙肝病毒药物结合提高免疫力是慢性乙肝治疗的重要原则。单纯应用抗病毒药物治疗有效率为30％左右，药物只在少数患者体内对病毒有持续抑制作用。慢性乙肝治疗多数采用抗病毒药物联合免疫干预剂。这种免疫干预包括增强特异性免疫和机体固有免疫两个方面。HBV转基因小鼠自身除对HBV病毒存在免疫耐受外，其天然和获得性免疫功能未见异常，这为上述治疗提供很好的理论依据；另一治疗思路，与上述传统慢性乙型肝炎治疗原则相反，不是提高机体免疫，清除病毒，而是努力保持慢性乙型肝炎患者免疫耐受状态，避免机体免疫反应引起肝损伤肝炎。HBV转基因小鼠免疫耐受状态不出现肝损伤，对这一新的治疗思路形成具有借鉴意义。综上所述，本实验应用HBV转基因小鼠进行乙肝发病机理和药效评价研究，其结果支持临床上应用抗病毒药物联合增强免疫剂的治疗原则。但对那些通过垂直传播感染的患者则与通常的临床治疗思路相反，而是积极维持慢性乙型肝炎患者免疫耐受状态，以避免机体免疫反应引起肝损伤肝炎。
[Abstract]:Human hepatitis B virus (HBV) has strict species specificity and lacks ideal animal models. The establishment of HBV transgenic mice model by transgenic technology has been widely used in the pathogenesis of hepatitis B, anti HBV treatment and drug screening evaluation.
The replicative HBV genome transgenic mice established by the authors have been steadily inherited for more than 20 generations, and the replication and expression of hepatitis B virus in serum can be detected by conventional ELISA and fluorescence quantitative PCR, which is equivalent to that of the replicative HBV transgenic mice established by Guidott, which is generally recognized by Guidott. This replicative HBV transgene is small The rat model provides a good experimental animal model for the study of the pathogenesis of HBV and the development of anti HBV drug development. It has been used by nearly 100 research institutes in China. The research contents of this paper include: (1) the establishment of HBV transgenic mice model; (2) the biological characteristics of HBV transgenic mice; (3) HBV transgenic mice are resistant to them. Application of hepatitis B drug efficacy evaluation; (4) using siRNA transient gene silencing technology to prepare HBV transgenic mice without immune tolerance; (5) summary and prospect.
The first chapter is the establishment of the replicative 1.3copy D genotype HBV transgenic mice. This experiment took the whole genome of 1.3copy D genotype HBV as the target gene and injected it into the male prokaryotic cells of the fertilized egg cells of mice, then transplanted the fertilized eggs into the fallopian tube of the recipient mouse, and developed the offspring mice. DNA was used to detect HBV gene PCR integration, and then enzyme linked immunosorbent assay (ELISA) was used to detect the serum hepatitis B surface antigen and e antigen, and the results of serum HBV DNA. were detected by fluorescence quantitative PCR: 1210 eggs were obtained, 882 were injected, 397 survived and 45%; the number of transplanted eggs was 337, the number of recipients was 14, 9 pregnant rats and 54 offspring were successfully transplanted. The rate was 16.02% (54 / 337), 54 rat tail tissues were detected by PCR, 15 were positive for positive.G0 generation and 4 were detected by serum HBVDNA and HBsAg, 61 offspring were detected in the G1 generation, and 13 of serum HBsAg positive. The quantitative detection of HBsAg expression in serum was 80~930 u g / L. for transgenic mice liver, spleen, kidney, small intestine and femur. Arterial and other tissues were detected by HBsAg and HBcAg immunohistochemistry. Only HBsAg was found in liver and kidney tissues, and HBcAg was scattered inhomogeneously.
In the second chapter, the biological characteristics of the F14-F20 mice of the stable 1.3copy D genotype HBV transgenic mice had been studied. The main contents were as follows: (1) the level and stability of the replication and expression of hepatitis B virus index; (2) immunological characteristics and pathological observation; (3) HBV gene The transcriptional level of each organization; (4) subculture.
First, this study used ELISA and fluorescence quantitative PCR to analyze the serum hepatitis B related indexes, and found that there were HBsAg, HBeAg, HBcAg, pre S1 and HBV DNA in the serum of transgenic mice, and the expression level of HBsAg was high, up to 5107.5 + 4144.8IU / ml, there were obvious differences among the individuals but there was no significant difference between the sexes. The level of Da was only (1.94 + 1.66s / CO), and the content of HBV DNA in serum reached 10~4 to 10~6copy / ml. by ultra high speed centrifugation. The HBsAg particles and Dane's particles in the serum of HBV transgenic mice were observed under transmission electron microscope. The results confirmed that the integrated HBV genomic DNA in the transgenic mice could be copied and packaged. The virus particles were secreted into the blood, and the transgenic mice were replicated.
Immunohistochemical results showed that two viral proteins were expressed as HBsAg and HBcAg protein in the hepatocytes of transgenic mice, but not all liver cells expressed HBsAg at the same time. The HBcAg protein.HBsAg was distributed in the cytoplasm of the liver, and the HBcAg protein was distributed in the nucleus of the liver. The liver tissue H-E staining analysis found that the liver tissues of the transgenic mice had no liver. Flow cytometry and Elispot method were used to detect the specific marker of DC in the splenocytes suspension of the strain of the strain of the strain of the strain CD11c and the expression of its TLR2 or its intracellular TLR9. It was found that the natural and acquired immune functions of the HBV transgenic mice were not abnormal except for the immune tolerance to the HBV virus. Real-time RT-PCR fluoreme was used. Quantitative detection of total RNA in more than ten tissues such as liver, kidney, spleen and other tissues of transgenic mice was quantified. The transcriptional characteristics of the HBV gene in the transgenic mice were analyzed. The results showed that the integrated HBV gene in the genome of transgenic mice was mainly in the liver, and the renal tissue could transcribe the mRNA of hepatitis B, and the transcriptional level of the liver tissue was the most. High.
In the early breeding stage, two first mice (Founder) were hybridized with normal mice respectively. The positive rate of 1.3copyD was kept between 40% and 50% in each generation of offspring, which indicated that the HBV was stable and integrated, and could be reliably inherited to the next generation. The sex rate and expression level of the two first mice were passed to the F14 generation to produce each other, and the positive rate of the offspring was about 80%. This showed that the integration sites of the different first mice genes were different and multipoint integration, and the positive rate could be significantly increased after intercross.
These results indicate that our replicated 1.3copy D genotype HBV transgenic mice are ideal hepatitis B transgenic animal models in China.
In the third chapter, HBV transgenic mice were used to evaluate the efficacy of a variety of anti hepatitis B drugs. (1) the anti HBV replicating drug lamifuridine or adefovir ester could significantly reduce the serum HBV DNA titer of transgenic mice, and the HBV DNA was restored to the original level after stopping the drug, and the results were consistent with the clinical application and the results of the same kind in foreign countries. (2) the application of high-dose HBV therapeutic vaccine could inhibit the replication of HBV DNA and increase the level of IL-2 and IFN- gamma in animals; (3) IFN- alpha 1b could significantly reduce the replication of HBV DNA in HBV transgenic mice, but the effect began to weaken after 5 weeks; (4) the serum HBsAg titer could decrease rapidly in 4 hours. The results of the above experimental results were as follows: The clinical effect of this kind of drug is consistent, which indicates that our HBV transgenic mice model can not only be verified by the classic effective anti HBV drugs in clinical, but also can be used to evaluate the efficacy of anti hepatitis B drugs. This fully proves that the transgenic animal model is in the pathogenesis of hepatitis B, the evaluation of anti HBV drug efficacy and the development of new drugs. The scientific significance and the application value of the medium.
The fourth chapter is the establishment of HBV transgenic mice without immune tolerance. Due to the expression of HBV gene in the embryo period of transgenic mice, the immune tolerance of mice to HBV can not simulate the symptoms of liver injury in patients with hepatitis. Therefore, the application of siRNA temporary short gene silencing technique is used in this paper, through the embryo abdominal cavity or the pregnant rat tail. The plasmid pU6-siHBV11 was injected intravenously for the preparation of HBV transgenic mice without immune tolerance. According to the principle of siRNA target selection, the plasmid selected 12 sequences in the conservative region as the inhibition target based on the sequence of HBV in the 2.2.15 cells, and screened out one from the inhibition experiments in the model of 2.2.15 and HBV transgenic mice. The siRNA plasmid with high inhibitory effect was pU6-siHBV11. in the study of RNA interference in the 14 day to 18 day embryo transgenic mice, which made the HBV gene transcriptional start delay and inhibition. We examined the pathological section of the liver tissue in 6 month old of the experimental mice and observed the mild to moderate turbid swelling of the liver, which was to prepare no immune tolerance HBV turn. Gene mice and the pathogenesis of hepatitis B provide new ways.
The fifth chapter summarizes and looks forward to the development of a replicative HBV transgenic mouse model, which has been steadily passed over 20 generations. A variety of hepatitis B indicators have reached the practical stage, which can be compared to the HBV transgenic mice model established by the foreign recognized Guidotti. The anti HBV drug has been initially established by using the HBV transgenic mice model prepared by us. The evaluation system can be used to evaluate the efficacy of DNA, mRNA level, cell level, immune level against hepatitis B drugs and to develop new anti hepatitis B drugs. The classic effective anti HBV effect on HBV transgenic mice proves that the animal model can truly simulate many biological characteristics of human hepatitis B disease. The breeding method of the copy number of gene can effectively improve the positive rate and expression level of transgenic offspring. RNA interfering short term gene silencing technique can be used to prepare HBV transgenic mice without immune tolerance and liver injury, which can simulate the pathological changes of liver injury in patients with hepatitis B.
The basic and application results of HBV transgenic mice have guiding and verifying effects on the study of chronic hepatitis B treatment. The combination of anti HBV drug combination to improve immunity is an important principle for the treatment of chronic hepatitis B. The effective rate of antiviral therapy alone is about 30%, and the drug has a sustained inhibition effect on the virus in a few patients. Most of the treatment of HBV is antiviral drugs combined with immune intervention agents. This immunization intervention includes the enhancement of specific immunity and the inherent immunity of the body in two aspects:.HBV transgenic mice have no immune tolerance to HBV virus, and their natural and acquired immune functions are not abnormal. This provides a good theoretical basis for the treatment of the above treatment. The other thought, contrary to the traditional treatment principle of the traditional chronic hepatitis B, is not to improve the immune system and remove the virus, but to keep the immune tolerance state of the patients with chronic hepatitis B, avoid the immune response of the body and prevent the liver injury of hepatitis.HBV transgenic mice from the immune tolerance and do not appear liver damage. The formation has reference significance.
To sum up, this experiment used HBV transgenic mice to study the pathogenesis and efficacy evaluation of hepatitis B. The results support the clinical application of antiviral drugs combined with enhanced immunotherapy. But for those who have passed vertical transmission, they are contrary to common clinical treatment, but actively maintain chronic hepatitis B patients. The immune tolerance state is to avoid liver damage caused by immune reaction.
【学位授予单位】：第一军医大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R-332

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