本文選題：乙型肝炎病毒 + 轉(zhuǎn)基因小鼠��； 參考：《第一軍醫(yī)大學(xué)》2007年博士論文

【摘要】： 人類乙型肝炎病毒具有嚴(yán)格的種屬特異性，一直缺乏理想的動(dòng)物模型；．通過(guò)轉(zhuǎn)基因技術(shù)建立HBV轉(zhuǎn)基因小鼠模型，在國(guó)內(nèi)外已被廣泛用于乙型肝炎發(fā)病機(jī)理、抗乙肝治療和藥物篩選評(píng)價(jià)研究。 作者建立的復(fù)制型HBV全基因組轉(zhuǎn)基因小鼠，經(jīng)不斷選育現(xiàn)已穩(wěn)定遺傳20代以上，且可用常規(guī)ELISA和熒光定量PCR檢測(cè)到乙肝病毒在血清中的復(fù)制和表達(dá)，與國(guó)際上普遍認(rèn)同的由Guidott建立的復(fù)制型HBV轉(zhuǎn)基因小鼠相當(dāng)。該復(fù)制型HBV轉(zhuǎn)基因小鼠模型為研究HBV的發(fā)病機(jī)制研究以及抗乙肝藥物開發(fā)研究提供了良好的實(shí)驗(yàn)動(dòng)物模型。目前已被國(guó)內(nèi)近百家科研單位應(yīng)用。本論文的研究?jī)?nèi)容包括：(1) HBV轉(zhuǎn)基因小鼠模型的建立；(2) HBV轉(zhuǎn)基因小鼠生物學(xué)特性；(3) HBV轉(zhuǎn)基因小鼠在抗乙肝藥物藥效評(píng)價(jià)中的應(yīng)用；(4)利用siRNA短暫基因沉默技術(shù)制備無(wú)免疫耐受HBV轉(zhuǎn)基因小鼠；(5)總結(jié)與展望。 第一章復(fù)制型1.3copy D基因型HBV轉(zhuǎn)基因小鼠的建立。本實(shí)驗(yàn)將1.3copy的D基因型HBV全基因組作為目的基因，采用顯微注射技術(shù)將其注入小鼠受精卵細(xì)胞雄原核，然后將受精卵移植于受體假孕母鼠輸卵管內(nèi)，發(fā)育產(chǎn)生子代小鼠。仔鼠尾組織提取DNA進(jìn)行HBV基因PCR整合檢測(cè)，再用酶聯(lián)免疫吸附法(ELISA)檢測(cè)血清乙型肝炎表面抗原及e抗原，熒光定量PCR檢測(cè)血清HBV DNA。結(jié)果：共獲卵數(shù)1210枚，注射882枚，存活397枚，成活率45％；移植卵數(shù)337枚，受體數(shù)14只，懷孕鼠9只，產(chǎn)仔54只，移植成功率為16.02％(54／337)，用PCR法檢測(cè)54只仔鼠尾組織，15只為整合陽(yáng)性。G0代經(jīng)血清檢測(cè)HBVDNA和HBsAg陽(yáng)性4只；G1代檢測(cè)61只仔鼠，血清HBsAg陽(yáng)性13只。可遺傳子代Founder2只。定量檢測(cè)血清中HBsAg表達(dá)量為80～930μg／L。取轉(zhuǎn)基因小鼠肝、脾、腎、小腸及股動(dòng)脈等組織，進(jìn)行HBsAg、HBcAg免疫組化檢測(cè)，僅發(fā)現(xiàn)肝及腎組織有HBsAg、HBcAg散在不均勻表達(dá)。 第二章對(duì)已制備獲得的穩(wěn)定傳代復(fù)制型1.3copy D基因型HBV轉(zhuǎn)基因小鼠的F14-F20代小鼠進(jìn)行了生物學(xué)特性研究。所觀察的主要內(nèi)容包括以下幾個(gè)方面：(1)乙肝病毒指標(biāo)復(fù)制表達(dá)水平及穩(wěn)定性；(2)免疫學(xué)特性及病理觀察；(3)乙肝病毒基因在各組織的轉(zhuǎn)錄水平；(4)傳代培育。 首先，，本研究應(yīng)用ELISA和熒光定量PCR的方法對(duì)血清乙肝相關(guān)指標(biāo)進(jìn)行了分析，發(fā)現(xiàn)轉(zhuǎn)基因小鼠血清中存在HBsAg、HBeAg、HBcAg、pre S1及HBV DNA，其中HBsAg的表達(dá)水平較高，達(dá)5107.5±4144.8IU／ml，個(gè)體間有明顯的差異但性別之間無(wú)顯著差異；而HBeAg表達(dá)水平較低，僅為(1.94±1.66s／co)，血清中存在HBV DNA含量達(dá)到10~4～10~6copy／ml。通過(guò)超高速離心的方法，在透射電鏡下觀察到HBV轉(zhuǎn)基因小鼠血清中存在HBsAg顆粒和Dane氏類顆粒，此結(jié)果證實(shí)了該轉(zhuǎn)基因小鼠中整合的HBV基因組DNA可復(fù)制包裝成病毒顆粒并分泌入血，本轉(zhuǎn)基因鼠為復(fù)制型。 免疫組織化學(xué)結(jié)果顯示：轉(zhuǎn)基因小鼠肝細(xì)胞中表達(dá)HBsAg和HBcAg蛋白兩種病毒蛋白，但并不是所有的肝細(xì)胞同時(shí)表達(dá)HBsAg、HBcAg蛋白。HBsAg分布于肝細(xì)胞質(zhì)中，HBcAg蛋白分布于肝細(xì)胞核中。肝組織H-E染色分析發(fā)現(xiàn)，轉(zhuǎn)基因小鼠肝組織中未見(jiàn)肝細(xì)胞病變。采用流式細(xì)胞術(shù)和Elispot法對(duì)該品系轉(zhuǎn)基因小鼠脾細(xì)胞懸液DC的特異性標(biāo)志CD11c及其上TLR2或其胞內(nèi)TLR9的表達(dá)檢測(cè)發(fā)現(xiàn)，HBV轉(zhuǎn)基因小鼠自身除對(duì)HBV病毒存在免疫耐受外，其天然和獲得性免疫功能未見(jiàn)異常。利用real-time RT-PCR熒光定量技術(shù)對(duì)轉(zhuǎn)基因小鼠肝、腎、脾等十余多種組織中的總RNA進(jìn)行定量檢測(cè)，分析了HBV基因在轉(zhuǎn)基因小鼠中各組織的轉(zhuǎn)錄特征，結(jié)果表明轉(zhuǎn)基因小鼠基因組中整合的HBV基因主要在肝、腎組織能夠轉(zhuǎn)錄出乙肝的mRNA，并以肝組織轉(zhuǎn)錄水平最高。 在早期培育階段，將兩個(gè)首建鼠(Founder)分別與正常鼠雜交進(jìn)行傳代，在每一世代仔鼠中進(jìn)行檢測(cè)發(fā)現(xiàn)其陽(yáng)性率始終保持在40％—50％之間。這說(shuō)明1.3copyD型HBV已穩(wěn)定整合表達(dá)，并能可靠地遺傳給下一代。為增加外源基因的拷貝數(shù)，提高陽(yáng)性率和表達(dá)水平，本研究把兩個(gè)首建鼠分別傳代至F14代進(jìn)行互交并檢測(cè)互交后生產(chǎn)仔鼠，其陽(yáng)性率達(dá)到80％左右，這說(shuō)明不同的首建鼠基因的整合位點(diǎn)不同且呈多位點(diǎn)整合，互交后可顯著提高陽(yáng)性表達(dá)率。 上述實(shí)驗(yàn)結(jié)果表明我們建立的復(fù)制型1.3copy D基因型HBV轉(zhuǎn)基因小鼠是國(guó)內(nèi)比較理想的乙肝轉(zhuǎn)基因動(dòng)物模型。 第三章利用HBV轉(zhuǎn)基因小鼠對(duì)多種抗乙肝藥物進(jìn)行藥效評(píng)價(jià)研究。(1)抗乙肝病毒復(fù)制藥物拉咪呋啶或阿德福韋酯可顯著降低轉(zhuǎn)基因小鼠血清HBV DNA滴度，停藥后HBV DNA又恢復(fù)至原來(lái)水平，該結(jié)果與臨床應(yīng)用藥效及國(guó)外同類研究結(jié)果一致；(2)大劑量乙肝治療性疫苗的應(yīng)用可抑制HBV DNA的復(fù)制，動(dòng)物體內(nèi)IL-2和IFN-γ水平升高；(3)IFN-α1b可顯著降低HBV轉(zhuǎn)基因小鼠體內(nèi)HBV DNA的復(fù)制，但5周后藥效開始減弱；(4)人源化HBsAg單抗在4小時(shí)內(nèi)可使血清HBsAg滴度迅速下降。上述實(shí)驗(yàn)結(jié)果與此類藥物臨床治療效果相符，說(shuō)明我們制備HBV轉(zhuǎn)基因小鼠模型不僅可通過(guò)臨床上經(jīng)典有效的抗乙肝藥物得到驗(yàn)證，而且可用于抗乙肝藥物藥效評(píng)價(jià)。這充分證明該轉(zhuǎn)基因動(dòng)物模型在乙肝發(fā)病機(jī)制、抗乙肝藥物藥效評(píng)價(jià)和新藥開發(fā)研究中的科學(xué)意義及應(yīng)用價(jià)值。 第四章無(wú)免疫耐受HBV轉(zhuǎn)基因小鼠的建立研究。由于HBV基因在轉(zhuǎn)基因小鼠胚胎期即表達(dá)，導(dǎo)致小鼠對(duì)HBV的免疫耐受，不能模擬肝炎患者的肝損傷癥狀，因而在一定程度上限制其應(yīng)用。本文利用siRNA暫短基因沉默技術(shù)，通過(guò)胚胎腹腔或孕鼠尾靜脈注射質(zhì)粒pU6-siHBV11，以期制備無(wú)免疫耐受HBV轉(zhuǎn)基因小鼠。該質(zhì)粒根據(jù)siRNA靶點(diǎn)選擇的原則，以2．2．15細(xì)胞中HBV的序列為基準(zhǔn)，在保守區(qū)域選出12條序列作為抑制靶點(diǎn)。通過(guò)2．2．15細(xì)胞和HBV轉(zhuǎn)基因小鼠模型體內(nèi)抑制實(shí)驗(yàn)，從中篩選出一條高效抑制作用的siRNA質(zhì)粒即pU6-siHBV11。本研究對(duì)14天—18天胚胎時(shí)期轉(zhuǎn)基因仔鼠RNA干擾，使HBV基因轉(zhuǎn)錄啟動(dòng)延遲和抑制。我們對(duì)所獲實(shí)驗(yàn)仔鼠在6月齡進(jìn)行肝組織病理切片檢查，觀察到有輕度～中度肝濁腫病變，這為制備無(wú)免疫耐受HBV轉(zhuǎn)基因小鼠及乙肝發(fā)病機(jī)制研究提供新的途徑。 第五章總結(jié)和展望我們培育出復(fù)制型HBV轉(zhuǎn)基因小鼠模型，現(xiàn)已穩(wěn)定傳代20代以上，有多種乙肝指標(biāo)表達(dá)已達(dá)到實(shí)用階段，可與國(guó)外公認(rèn)Guidotti建立的HBV轉(zhuǎn)基因小鼠模型相媲美。利用我們制備的HBV轉(zhuǎn)基因小鼠模型現(xiàn)已初步建立抗乙肝藥物評(píng)價(jià)體系，可從DNA、mRNA水平、細(xì)胞水平、機(jī)體免疫水平對(duì)抗乙肝藥物進(jìn)行藥效評(píng)價(jià)和開發(fā)抗乙肝新藥研究。經(jīng)典有效的抗乙肝藥物對(duì)HBV轉(zhuǎn)基因小鼠療效反證了該動(dòng)物模型可真實(shí)的模擬表現(xiàn)人類乙型肝炎疾病許多生物學(xué)特征。通過(guò)提高目的基因的拷貝數(shù)的育種手段可有效提高轉(zhuǎn)基因仔代鼠的陽(yáng)性率、表達(dá)水平。應(yīng)用RNA干擾短期基因沉默技術(shù)制備無(wú)免疫耐受有肝損傷的HBV轉(zhuǎn)基因小鼠，可模擬乙肝患者肝損傷的病理變化。 HBV轉(zhuǎn)基因鼠基礎(chǔ)與應(yīng)用研究結(jié)果對(duì)慢性乙肝治療研究具有指導(dǎo)和驗(yàn)證作用�？挂腋尾《舅幬锝Y(jié)合提高免疫力是慢性乙肝治療的重要原則。單純應(yīng)用抗病毒藥物治療有效率為30％左右，藥物只在少數(shù)患者體內(nèi)對(duì)病毒有持續(xù)抑制作用。慢性乙肝治療多數(shù)采用抗病毒藥物聯(lián)合免疫干預(yù)劑。這種免疫干預(yù)包括增強(qiáng)特異性免疫和機(jī)體固有免疫兩個(gè)方面。HBV轉(zhuǎn)基因小鼠自身除對(duì)HBV病毒存在免疫耐受外，其天然和獲得性免疫功能未見(jiàn)異常，這為上述治療提供很好的理論依據(jù)；另一治療思路，與上述傳統(tǒng)慢性乙型肝炎治療原則相反，不是提高機(jī)體免疫，清除病毒，而是努力保持慢性乙型肝炎患者免疫耐受狀態(tài)，避免機(jī)體免疫反應(yīng)引起肝損傷肝炎。HBV轉(zhuǎn)基因小鼠免疫耐受狀態(tài)不出現(xiàn)肝損傷，對(duì)這一新的治療思路形成具有借鑒意義。 綜上所述，本實(shí)驗(yàn)應(yīng)用HBV轉(zhuǎn)基因小鼠進(jìn)行乙肝發(fā)病機(jī)理和藥效評(píng)價(jià)研究，其結(jié)果支持臨床上應(yīng)用抗病毒藥物聯(lián)合增強(qiáng)免疫劑的治療原則。但對(duì)那些通過(guò)垂直傳播感染的患者則與通常的臨床治療思路相反，而是積極維持慢性乙型肝炎患者免疫耐受狀態(tài)，以避免機(jī)體免疫反應(yīng)引起肝損傷肝炎。
[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.
【學(xué)位授予單位】：第一軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R-332

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 王洪敏;劉光澤;賈彥征;熊一力;張宜俊;;adr型乙型肝炎病毒全基因組轉(zhuǎn)基因小鼠的制備[J];傳染病信息;2000年01期

2 熊一力;賈彥征;王洪敏;劉光澤;張宜俊;;高表達(dá)乙型肝炎病毒轉(zhuǎn)基因小鼠的制備[J];傳染病信息;2000年04期

3 張宜俊;;慢性乙型肝炎免疫治療若干問(wèn)題[J];傳染病信息;2005年03期

4 胡衛(wèi)江,李建秀,余宏宇,戴德順,王新民,孫偉,郝光榮,王肖鵬,胡以平;乙型肝炎病毒(adr亞型)轉(zhuǎn)基因小鼠的研究[J];第二軍醫(yī)大學(xué)學(xué)報(bào);1999年04期

5 蔣黎,毛青,王宇明,李俊剛,劉俊;HBV感染的人鼠嵌合肝動(dòng)物模型的建立[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2003年05期

6 耿士忠,鞠輝明,楊躍飛,成勇;乙肝病毒基因在轉(zhuǎn)基因小鼠中高效表達(dá)的研究[J];揚(yáng)州大學(xué)學(xué)報(bào);2002年04期

7 斯崇文;;慢性乙型肝炎的現(xiàn)狀和問(wèn)題[J];臨床藥物治療雜志;2006年04期

8 鄭曲波,張宜俊,劉惠萍,易學(xué)瑞,祖萍,章谷生;慢性HBV感染者外周血樹突狀細(xì)胞和T細(xì)胞體外應(yīng)答功能的檢測(cè)和分析[J];上海醫(yī)學(xué)檢驗(yàn)雜志;2003年02期

9 劉光澤,賈彥征,王洪敏,熊一力;乙型肝炎病毒轉(zhuǎn)基因小鼠外源基因的復(fù)制和傳代穩(wěn)定性觀察[J];實(shí)驗(yàn)動(dòng)物科學(xué)與管理;2001年01期

10 張靖溥,勞為德,張旭晨,魏影允,劉曄;外源基因在轉(zhuǎn)基因小鼠中遺傳和表達(dá)的穩(wěn)定性[J];遺傳學(xué)報(bào);1999年02期

本文編號(hào)：1924214