【作者】 吕丽萍；

【导师】 周虹； 许金波；

【作者基本信息】 中国人民解放军军事医学科学院 ， 生物化学与分子生物学， 2009， 博士

【摘要】 由乙型肝炎病毒(hepatitis B virus,HBV)和丙型肝炎病毒(hepatitis C virus,HCV)等人嗜肝病毒感染所导致的急、慢性病毒性肝炎,以及由此发展成的肝硬化和肝癌是严重危害人类健康的重大传染性疾病。目前全球至少有3.6亿HBV慢性感染者和1.7亿HCV感染者,每年由于HBV和HCV感染导致死亡的人数达1500万。过去的十多年间,虽然在预防和治疗急、慢性病毒性肝炎方面取得了一定进展,但也带来了许多问题,其中比较突出的是缺乏特效治疗药物以及耐药性病毒株的出现,而制约这一工作进程的难题之一是没有适用于药物筛选的肝炎病毒体外感染细胞模型和实验动物评价模型。用肝炎病毒阳性血清直接感染原代培养的人肝细胞或胎肝细胞是研究嗜肝性病毒自然感染和复制的最佳方式,但由于胎肝细胞的分化程度影响着其对肝炎病毒的易感性,因此用原代人肝细胞研究肝炎病毒进入细胞的早期感染过程以及由此引起的细胞先天免疫等方面具有不可比拟的优势。人肝细胞属于高度分化的类上皮细胞。体外培养的人肝细胞生存期短,很快失去增值分化能力,细胞衰老、死亡。细胞的生存寿命与细胞内端粒的长度密切相关。端粒位于染色体末端,由核蛋白复合体构成,它起着稳定细胞染色体的重要作用。随着细胞分裂,端粒长度逐渐缩短,当缩短到一定长度时,细胞就会出现衰老或凋亡。端粒酶是一种能维持端粒长度的核酸蛋白酶,在胚胎干细胞、生殖细胞、永生化细胞和肿瘤细胞中有表达。端粒酶逆转录酶(telomerase reverse transcriptase,TERT)是端粒酶的一种核心亚基单位,其表达量的高低对端粒酶活性起决定作用,与端粒酶活性高度一致。用人TERT (human TERT,hTERT)修饰人体细胞可激活端粒酶活性,抑制端粒的缩短,从而延长细胞的体外生长时间。鉴于人肝细胞来源的有限性和体外培养的困难性,本研究将编码hTERT的基因转入成人肝细胞,筛选出了经hTERT修饰后的成人肝细胞,可传代培养,并在此基础上研究了HBV对该细胞和移植鼠的自然感染性,为建立抗HBV药物筛选和疫苗评价平台奠定了一定基础,也为建立HCV自然感染细胞模型提供了新的思路。具体研究内容及结果如下:一、原代成人肝细胞的分离和培养。运用改进的两步灌流技术从成人正常小块肝组织分离肝细胞并进行体外培养,观察细胞形态特征并对其代谢特点进行评价。肝细胞培养7天时出现细胞克隆,细胞核浆比例大,色浅,具有单、双甚至多个细胞核,胞浆丰富,扩大培养后肝细胞形态未见明显改变。通过测定肝细胞特异性基因发现,分离的肝细胞中具有成熟肝细胞(即肝细胞晚期基因)的mRNA,未检测到甲胎蛋白(α-fetoprotein,AFP)的mRNA,提示所分离培养的原代肝细胞是成熟、正常的肝细胞。肝细胞培养1至9天,其白蛋白的分泌量呈递增趋势,第9天的分泌量达到峰值,然后呈现下降趋势,这些结果与肝细胞的生长状态相一致。二、hTERT修饰成人肝细胞株的建立。用脂质体介导法包装hTERT cDNA的重组逆转录病毒载体。将包装的重组逆转录病毒载体转入上述原代肝细胞,经G418加压筛选得到表达hTERT的原代肝细胞,即hTERT修饰的原代成人肝细胞,命名为HC-T(human hepatocytes transducted with gene encoding hTERT)。修饰后的肝细胞具有白蛋白(albumin,Alb),谷氨酰胺合成酶(glutamine synthetase,GS)和转铁蛋白(transferrin,TF)的mRNA,未检测到原癌基因AFP的mRNA;免疫荧光结果显示,细胞角蛋白18(cytokeratin 18,CK18)均匀分布于细胞质内,而核内未见;糖原染色显示,hTERT修饰后的原代成人肝细胞糖原染色呈阳性。上述结果表明,经hTERT修饰的原代成人肝细胞仍具有正常肝细胞的形态和生理功能。该细胞株可连续传代培养。三、hTERT修饰的成人肝细胞对肝炎病毒的易感性。用HBV阳性血清直接感染HC-T细胞并采用ELISA法和Real-time PCR技术检测原代细胞上清、传代细胞上清和上清再感染细胞后的HBsAg和HBV DNA。与原代成人肝细胞相比,HC-T对HBV的易感性未见明显改变。HBV感染HC-T细胞2天后可在其上清液中检测到HBsAg和HBV DNA,这说明可能有病毒释放到细胞外。随着培养时间的延长,细胞上清液中的病毒量呈现波动,这可能是释放的病毒再次感染细胞所致,再感染的细胞包括分裂增殖子代细胞和尚未感染病毒的细胞。被HBV感染的HC-T经过传代,仍可在传代细胞上清液中检测到HBsAg和HBV DNA,但其含量较低,推测可能与HBV的慢性感染有关,病毒编码的某些蛋白可能抑制了细胞的凋亡,实现了HBV与细胞的共存状态。感染HBV的细胞上清液仍具有感染性,感染特点与HBV阳性血清相似,也呈现一定的波动性。上清中的病毒DNA水平和蛋白水平基本一致,HBcAg主要定位在胞浆内,核内偶见,这些都与HBV阳性血清感染原代肝细胞的结果相一致。四、hTERT修饰成人肝细胞移植鼠的探索。感染HBV的HC-T细胞经鼠的尾静脉输入裸鼠体内,观测其在裸鼠体内的生长状况,同时用ELISA和Real-time PCR技术测定裸鼠血清中HBsAg和HBV DNA的表达水平,用免疫组化技术分析鼠肝脏中人CK18定位和HBcAg的表达。结果表明,在移植HC-T细胞的裸鼠血清中可检测到HBsAg和HBV DNA,虽然含量较低,但两者出现和降低的时间相似,均在移植2周时达到高峰,随后逐渐下降,到第4周时含量最低。鼠的肝脏可见HBcAg表达,主要定位于胞浆和胞核内。通过尾静脉移植,HC-T细胞主要分布于肝脏的中央区和汇管区。上述实验为建立HC-T细胞移植的人肝嵌合鼠模型奠定了基础。总之,本研究建立了hTERT修饰的成人肝细胞,它可连续传代培养,且与正常肝细胞的形态和生理功能相似,可以自然感染HBV,这为建立和完善抗HBV和HCV药物以及疫苗筛选技术平台奠定了基础。更多还原

【Abstract】 The hepatitis B virus (HBV) and hepatitis C virus (HCV), which cause acute or chronic viral hepatitis and other liver diseases including cirrhosis and hepatocellular carcinoma (HCC), are hepatotropic viruses that represent a serious global health problem. The number of chronically infected subjects is estimated at 360 million for HBV and 170 million for HCV and annually these pathogens kill more than 1.5 million people worldwide.In the past decade substantial progress has been made in treating these chronic infections as well as bring many problems.The side effects of treatment and emergence of variants defying therapeutic treatment are major problems.Therefore developing new antiviral compounds,which are more efficacious,little side effect and better tolerated, are emergent.The key is to find cell culture systems besides animal models for antivirus compounds selected and vaccines evaluated.Infection normal human hepatocytes or fetal hepatocytes with serum-derived HBV or HCV are the ideal system in which to study hepatotropic virus infectivity. Differentiated phase affected the susceptibility of cells to virus infection. Accordingly, it was found that only human hepatocyte primary cultures were susceptible to both HBV and HCV infection,which facilitated systematic investigations of virus early event and consequently innate immune response.Human hepatocytes belong to well-differentiated epithelioid cells. When cultured in vitro, however, they proliferate poorly and divide only a few times, eventually result in senescence or death. Continuous proliferation even immortalized could be achieved however by introducing oncogenes. Telomeres are nucleoprotein complexes at the chromosome ends which play a critical role in the maintenance of chromosomal integrity.The telomere lengths is relevant to cell survival.Progressive telomere shortening with each cell division eventually triggers an alteration in telomere structure, which cause senescence or apoptosis from the perspective. Telomerase is a specialized RNA-protein complex that which responsible for the de novo synthesis and maintenance of telomere repeats.Telomerase expression is low or absent in most human somatic tissues, except for tissue stem cells, germ cells, immortalized cells and tumor cells. The telomerase reverse transcriptase(TERT) is core subunit, which regulate telomerase activity strictly.The human somatic cells transducted with gene encoding human TERT(hTERT) can escape p53-mediated senescence or other checkpoint responses,thus prolong in vitro culture time.In order to answer this question, the aim of the study was established. In view of limited availability and difficult culture, human hepatocyte clones were developed by transduction with the gene encoding the hTERT. The transgenic cells infectivity was assayed by exposing HBV-positive serum to both cell and animal level. The transgenic cells were useful tool for selecting antivirus compounds and evaluating vaccines against HBV, which lay the foundation for further study on developing models of HCV infection too.The main contents and results of the study are discussed below.1. Isolation and culturing of human hepatocytes from rejected livers. The human hepatocytes were isolated from liver fragment using modified“two-step”collagenase procedure.The cells were assessed for morphological characters as well as metabolism traits when culture in vitro. After seven days in culture, the human hepatocyte clones were appeared. The cells morphology was same as adult human hepatocytes. The hepatocyte-specific markers were analyzed by RT-PCR.The isolated primary hepatocytes displayed expression of ALB, AAT, HGF, CK18, TF, GS except for AFP.The isolated primary hepatocytes were the same as the freshly isolated primary hepatocytes. Trace albumin detection showed the albumin secretion increased from day 1 to day 9,which reach a peak on day 9 followed by descent corresponding to cell growth.2. Screening and identification of positive transfected cells. The recombinant plasmids were transfected into packaging PT67 cells using Lipofectamine 2000 according to provided protocol. Virus-producing cells were selected after two weeks G418-selection. The cultured human primary hepatocytes were infected with viral supernatants harvested. The transgenic human primary hepatocytes expressing hTERT were established, named HC-T. The transgenic human primary hepatocytes displayed expression of ALB, TF, GS except for AFP. Immunofluorescence staining showed CK18 was located in the cytoplasm.Glycogen storage detection indicated that plentiful glycogenic granules deposited in the cytoplasm.Morphologic, phenotypic and function analysis proved that the transgenic human primary hepatocytes had the same features as the freshly isolated primary hepatocytes.3. HBV infection of the transgenic human primary hepatocytes. As an infectious inoculum, the serum-derived HBV was selected to infect HC-T. HC-T cells were incubated with HBV positive serum. The HBsAg and HBV DNA in culture supernatant, passage supernatant and reinfection supernatant was analyzed by ELISA and Real-time PCR individually. HC-T cells did share many characteristics with normal human hepatocytes including morphologic, phenotypic, function aspects and HBV infectivity. HC-T infectivity to HBV was showed by the kinetics of HBsAg and HBV DNA .After entering into HC-T, HBV began to replicate. After a complete replicative cycle, the virus partical was released into supernatant at 2 days post inoculation ( pi). HBV was detected with cyclic fluctuations.The low level of HBV protein and DNA were relative to HBV persistent. The HBV certain protein may inhibit cells apoptosis, which need further study.The virion released into supernatant had infectivity and infective patter was similar to serum-derived HBV with cyclic fluctuations. Repetitive analyses indicated a strict correlation between the HBsAg secretion and HBV DNA level. The core antigen was detected in the cytoplasm of HC-T and was occasionally detected in the nucleus.These results revealed that at least 10 percent of HC-T cells were infected.The HC-T had the same HBV infectivity features as the freshly isolated primary hepatocytes.4. HBV infection of mice with chimeric HC-T. The HC-T cells exposed to HBV were transplanted to the liver of nude mice via caudal veins. HBV DNA and proteins were detected in the serum of the injected mice for 2 weeks by ELISA and Real-time PCR individually. The core antigen was detected in the cytoplasm of liver tissues.The results pave the way for mice model of other hepatotropic viruses, such as HCV.In conclusion, transgenic human hepatocytes were developed by transduction with the gene encoding the hTERT. The transgenic human primary hepatocytes had the same features as the human primary hepatocytes on the morphologic, phenotypic, molecular and functional levels.The most important is its unique susceptibility to HBV infection. The establishment of transgenic human primary hepatocytes provides the foundations to study new antiviral agents or vaccines. The transgenic human primary hepatocytes are very promising for many other applications especially in the development of types of antiviral drugs that interfere with early step of infection.更多还原