【作者】 李君；

【导师】 李兰娟；

【作者基本信息】 浙江大学 ， 内科学， 2003， 博士

【摘要】 研究背景 随着肝细胞分离、高密度培养以及生物反应器等技术的日臻成熟，新一代以培养肝细胞为基础的现代生物人工肝(bioartificial liver，BAL)——体外人工肝支持系统(extracorporeal bioartificial liver support system，EBLSS)被认为是治疗因各种原因所致暴发性肝衰竭或急性肝衰竭(fulminant hepatic failure，FHF，acute hepatic failure，AHF)最有前途的，并已在动物实验及临床应用中取得了确切疗效。肝细胞作为BAL的主要生物成分，在该系统中扮演着举足轻重的角色。正常人肝组织和培养人肝细胞是理想的BAL细胞源和体外药物代谢模型，但由于全世界范围内的供肝短缺以及原代培养时存活时间短和难以传代等缺点，使得利用正常人肝脏分离肝细胞来作为BAL的细胞源显得非常困难。胎肝细胞尤其是从较大月龄胎肝分离到的肝细胞，虽能在体外继续分化增殖，且免疫原性弱，较少引起异种蛋白反应，但同样受制于来源问题和伦理学原因，使得难以在临床推广应用。 为此有学者试度采用永生化肝细胞来克服这些不足，如C3A、HepG2、CL—1等细胞系。在美国采用C3A和HepG2细胞构建EBLSS治疗FHF动物和少量临床试验已获肯定结果，其中以C3A细胞构建的EBLSS已被批准进行二期临床试验。但上述细胞系均来源于肿瘤细胞，研究显示无论在P450活性、氨清除功能以及尿素合成等方面均不如猪肝细胞，加之以肿瘤源性肝细胞作为治疗工具，用于处于免疫抑制状态下的FHF患者，尤其是将接受肝脏移植、需终生使用免疫抑制剂的患者身上，总觉令人担忧。本实验采用脂质体转染法，将猿猴病毒40大T抗原(SV40LTag)基因转染至来源于正常成人原位肝移植的供肝细胞，建立一永生化肝细胞系。m03浙江人学博士论文 第一部分摘婴 新型人源性肝细胞系的构廷材料和方法 SV40 DNA（strain 776）、月 质体转染试剂盒、IV型胶原酶、hepatoZYME－SFM培养基、DMEM培养基、G418等购自美国Invitrogen 公司；真核表达载体 pCDNA3．1（－）为本研究所保存载体；胎牛血清＊BS）购自美国Hyclone公司；PCR产物纯化试剂盒。凝胶电泳切胶回收试剂盒、质粒抽提试剂盒等购自美国 QIAGEN公司；限制性内切酶 BamH、限制性内切酶 BstXl、T4连接酶、DNA Marker等购自美国 Promega公司；DHS u大肠杆菌山本实验室提供。实验所需主要试仪器设备有：PCR扩增仪、蛋白核酸测定仪、纯水仪、低温高速离心机、水平电泳仪、低温冰箱，凝胶电泳成相系统、自行设计的体外灌流装置。成人肝细胞来自一位O型血、年龄25岁的健康男性原位肝移植供肝。 首选采用 PCR方法，以 SV40全序列 DNA为模板，扩增 SV40LTag DNA；T4 DNA连接酶连接经 BamH酶切 SV40LT8g DNA牙 pcDNA3．1（十 构建SV40LTag—pCDNA3．1卜）重组载体并转化至 DHS Q大肠杆菌感受态细胞，通过BStX酶切鉴定和 DNA序列测定以确定重组载体；最后利用脂质体转染法将SVp0LTag—pCDNA3．1（－）重组载体转染至正常成人肝细胞，经G418筛选，直至获得阳性克隆细胞并观察转染细胞的形态特征。结果 经DNA序列测定和比只结合GenBank网站公布的SV40LTag DNA序列，SV40LT8g基因共 2607hp正确插入真核表达载体 pCDNA3．1（－卜利用脂质体转染法将 SV40LTag—pcDNA3．1（－）重组载体转染至正常成人肝细胞，经700S G418筛选 42天，出现一明显的上皮细胞样抗 G418的阳性克隆细胞，空载体组和空白对照组细胞大多死亡。形态学观察表明，转染肝细胞呈上皮细胞样、单层贴壁生长，在含 15％FBS的低糖 DMEM培养基中，传代一次只需3＊天时间：相比在怕p扒。＊＊MB8「M无血清培养基中细胞增殖时间明显延 22皿3iM江大学博士论文 柬一部分摘婴 新型人源性肝细胞床的构延长，传代一次需5天时间：但转染肝细胞在两种培养基中不予传代存活时间相差无几，约12天左右，之后便出现迅速死亡。结论1 在国内首次利用正常成人肝移植供肝进行肝细胞分离和原代培养。2在国内首次利用 SV40LTag 基因和真核表达载体 pCDNA3．1个卜 构建SV40LTSP—PCDNA3．1（－）重组载体，经脂质体转染至成人肝细胞建立一永生化肝细胞。3新建人源性肝细胞系在体外可无限传代。更多还原

【Abstract】 The bioartificial liver support system (BALs) is a promising therapy for the treatment of acute hepatic failure(AHF) and fulminant hepatic failure(FHF) with the developments in isolation of hepatocyte, culture of high density and bioreactor. The promising effects have been confirmed in animal experiments and clinical tests. As a main biomaterial of BALs, the hepatocytes perform an important role. Although primary human hepatoyctes are most suitable to BALs and the extracorporeal model of drug metabolism, they have many disadvantages such as limited lifespan, functional activities decline rapidly after several days in culture and the organ shortage all over the world. So it is too difficult to isolate normal human hepatocyte applicated in BALs. As another ideal hepatocyte resource for BALs and hepatocyte transplantation, the fetal heaptocytes not only maintain the functions of proliferation and differentiation in extracorporeal culture, but also have a lower immunogenicity. Because of ethnical problems, the fetal hepatocyte can’t applicated in clinical therapy.Many researcher around the world want to use immortal hepatocyte line such as C3A, HepG2 and CL-1 to overcome above difficulties. Extracorporeal bioartificial liver support system(EBLSS) has been established with immortal C3A and HepG2 cell line for treatment of FHF animal models and the II clinical test of EBLSS with C3A cell has been approved in USA. In order to Wang’s report, the immortalized human hepatoma cell lineC3A has lower viability, metabolic capacity and activities in cytochrome P450 compared toprimary porcine hepatocytes. For these reasons, hepatocytes isolated from the liver of a normal 25-year old male donor were transfected with pcDNA3.1(-) recombined vector containing the genes encoding Simian Virus 40 large T antigen(SV40LTag ). One of the resulting hepatocyte clone, has shown highly differentiated liver function and immortalized characteristics. Materials and MethodsThe reagents were purchased as follows: SV40 viral DNA(strain 776), lipofectAMINE 2000, collagenase IV, G418, hepatoZYME-SFM, pcDNA3.1(-) vector and DMEM medium from Invitrogen company(USA); fetal bovine serum from Hyclone company(USA ); QIAquick gel extraction kit, QIAprep spin miniprep kit and QIAquick DNA purify kit from QIAGEN company(USA); restriction endonuclease BamH, BsfX I and T4 DNA ligase from Promega company(USA); cell culture flasks and plates from Nunc Medos Company (Australia). The hepatocytes were isolated from the liver of 25-year old normal male donor by the standard two-step collagenase perfusion technique.SV40LTag DNA was obtained from the complete sequence of SV40 viral DNA (strain 776) by the method of polymerase chain reaction (PCR). The recombined vector containing pcDNA3.1(-) vector and SV40LTag DNA was constructed with T4 DNA ligase and expressed in the competence cells of E. coli DH5a. The SV40LTag-pcDNA3.1(-) vector was characterized by DNA sequencing with Ganger’s dideoxy chain termination composition method and compared with nucleic acid sequences in the GenBank. Hepatocytes from human liver were transfected by a lipofection method with the recombined vector containing both SV40LTag and the bacterial neomycin phosphotransferase gene, which confers resistance to geneticin G418.ResultsThe sequence of SV40LTag DNA of the recombined vector extracted from positive bacteria is similar to shown in GenBank. One line of surviving cells after 42 day’ selection of 700-300Mg/mL G418, grew steadily in the chemically defined serum-free hapatoZYME-SFM medium. The immortalized hepatocyte appeared epithelial and displayed morphologic characteristics of liver parenchymal cells, such as a large round nucleus with a few nucleoli and many cytoplasmic granules. The speed of cell proliferation of hepatocyte cultured in the lower sugar DMEM medium containing 15% FBS is faster than in hepatozYME-SFM medium. Similarly, the time of 3 to 4 days of cell passage cultured in DMEM medium is shorter than cultured in hepatoZYME-SFM medium. The new establ更多还原