人原代肝细胞的分离、培养及可逆性永生化

发布时间：2018-05-30 09:52

本文选题：猪肝细胞 + 原代肝细胞　；参考：《华中科技大学》2010年博士论文

【摘要】： 第一部分：前期实验——猪原代肝细胞的分离、培养及可逆性永生化 [目的]建立大动物肝组织灌流分离原代肝细胞的方法；建立用携带永生化基因的逆转录病毒感染原代肝细胞实现肝细胞可逆性永生化的方法。 [方法]采用改良的四步循环灌流法分离乳猪肝细胞,然后用SSR#69包装细胞释放的携带Cre/LoxP系统／SV40T及潮霉素抗性基因的逆转录病毒感染猪原代肝细胞,潮霉素筛选阳性克隆细胞团,克隆环挑选目的克隆增生细胞团进行单独传代培养。最后添加Cre重组酶,切除两个LoxP位点间的永生化基因SV40T,实现永生化的逆转。 [结果]我们共获得6块大小合适的猪肝组织,成功分离获得活性很高的大量原代猪肝细胞(产量：9.4±7.5×106／g肝组织；活性：94.6±4.2%)。在此基础上利用SSR#69释放的含有SV40T的逆转录病毒转染猪肝细胞获得成功,永生化的肝细胞具有正常肝细胞的形态特点,能传代培养。加入Cre重组酶逆转永生化后,肝细胞停止增生。 [结论]我们建立了大动物肝组织四步循环灌流法分离原代肝细胞,并成功实现了用携带永生化基因SV40T的逆转录病毒转染猪原代肝细胞,获得了永生化的猪肝细胞。第二部分：人原代肝细胞的分离、培养 [背景及目的]药物代谢实验、生物人工肝及细胞移植等均需要大量的人肝细胞,而目前国际上利用外科手术切除后废弃的健康肝组织分离原代肝细胞的经验尚少。本部分实验旨在建立一种高效利用手术切除后废弃的健康肝组织分离人原代肝细胞的方法。 [方法]收集良性肝病患者外科手术切除后废弃的健康肝组织,采用我们自己建立的改良四步循环灌流法分离人原代肝细胞。同时评价获取肝组织的重量、冷热缺血时间等对肝细胞分离效果的影响。 [结果]我们共获得13块合适的健康肝组织,其中肝血管瘤患者10例,肝内胆管结石患者3例。所有患者查HCV-Ab、HIV-Ab及HBV-Ag均为阴性。患者手术前肝功能(ALT, ALB等)均在正常水平,平均年龄47岁(23—57岁),血型：O+型4例,A+型6例,AB+型1例,B+型2例。肝组织块重量为27.2±7.8g(15—42g)。热缺血时间(WIT) 10-35min,、所有13例组织块的冷缺血时间(CIT)均35 min。分离获得的细胞产量为4.8±2.1×106／g肝组织,台酚蓝染色后用血球计数板测肝细胞活率为78.1±10.4%(62%-93%)。 [结论]1,良性肝病(主要是肝血管瘤及肝内胆管结石)行部分肝切除后废弃的周边正常肝组织,可以采用改良的四步循环灌注法分离获得大量活性好的人肝细胞。2,对于存在中度以下纤维化的肝组织,如果切缘血管条件好,仍可以有很好的细胞分离效果。3,应尽量缩短获取肝组织的热缺血时间,提高肝细胞分离效果。4,对于较小的肝组织块,切面血管条件欠佳者,采用四步循环灌流法分离肝细胞效果欠佳。第三部分：人肝细胞的可逆性永生化 [背景及目的]人原代肝细胞体外培养时增殖困难,且存在培养时间短,不能及时获得等缺陷,使其应用受到很大的限制。具有正常肝细胞功能的永生化人源肝细胞,能够解决细胞来源紧缺的难题。本部分实验旨在利用逆转录病毒实现SV40T单个永生化基因的转染,构建人源肝细胞的可逆性永生化。 [方法]用SSR#69包装细胞释放的携带Cre/LoxP系统／SV40T及潮霉素抗性基因的逆转录病毒转染人原代肝细胞,7天后用潮霉素筛选阳性克隆细胞团,用合适大小的克隆环挑选目的克隆增生的细胞团进行单独传代培养。最后添加Cre重组酶,以切除两个LoxP位点间的永生化基因SV40T,实现永生化的逆转。RT-PCR及免疫荧光染色验证永生化基因SV40T的存在。 [结果]逆转录病毒转染7天,潮霉素进行筛选14天后,培养瓶中出现大量克隆增生的细胞团,形态类似肝细胞。克隆环挑选至新培养瓶中单独培养,细胞能传代培养并保持形态规则。添加Cre重组酶后逆转永生化后,细胞即停止生长。RT-PCR检测永生化细胞具有人原代肝细胞的标志基因,免疫荧光染色亦验证永生化细胞内SV40T的存在。 [结论]我们建立的可逆性永生化人源肝细胞,具有肝细胞的功能,体外能传代培养。更重要的是,我们成功实现了逆转录病毒单永生化基因转染人原代肝细胞的方法,为后继实验进行人肝细胞SV40T/hTERT双永生化基因转染,实现人源肝细胞的彻底永生化奠定方法学基础。
[Abstract]:Part one: previous experiments - isolation, culture and reversible immortalization of porcine primary hepatocytes
[Objective] to establish a method for isolating primary hepatocytes by perfusion of large animal liver tissue, and to establish a reversible immortalization method of hepatocyte by using retrovirus carrying immortalized gene to infect primary hepatocytes.
[Methods] a modified four step circulatory perfusion method was used to isolate pig hepatocytes, and then the porcine primary hepatocytes were infected with Cre/LoxP system / SV40T and hygromycin resistant retrovirus, and hygromycin was used to screen the positive cloned cell groups. Cloned cloned proliferative cell groups were selected to carry out a single generation culture. Finally, Cre recombinant enzyme was added to remove the immortalized gene SV40T between the two LoxP sites, so as to achieve immortalization reversal.
[results] a total of 6 suitable pig liver tissues were obtained, and a large number of primary porcine liver cells (9.4 + 7.5 * 106 / g liver tissue, 94.6 + 4.2%) were successfully isolated and obtained. On this basis, the transfected porcine hepatocytes, which were released by SSR#69, were successfully transfected into pig hepatocytes, and the immortalized hepatocytes were positive. The morphological characteristics of normal hepatocytes can be subcultured. After adding Cre recombinase to reverse immortalization, the hepatocytes cease to proliferate.
[Conclusion] we established a large animal liver tissue four step circulation perfusion method to separate the primary hepatocytes, and successfully transfected porcine primary hepatocytes with the retrovirus carrying immortalized gene SV40T, and obtained immortalized pig liver cells.
The second part: isolation and culture of human primary hepatocytes
[background and objective] drug metabolism experiments, biological artificial liver and cell transplantation all need a large number of human hepatocytes, but the international experience of separating primary hepatocytes from the abandoned healthy liver tissue after surgical excision is still less. Methods of primary hepatocyte generation.
[Methods] to collect the discarded healthy liver tissues after surgical excision of the patients with benign liver disease, and to separate the human primary hepatocytes by the improved four step circulatory perfusion method established by ourselves. The effects of the weight of liver tissue, the cold and hot ischemic time on the isolation of liver cells were evaluated.
[results] 13 healthy liver tissues were obtained, including 10 cases of hepatic hemangioma and 3 cases of intrahepatic bile duct stones. All patients were examined with HCV-Ab, HIV-Ab and HBV-Ag were negative. The liver function (ALT, ALB, etc.) before operation were at normal level, the average age was 47 years (23 to 57 years), the blood group was 4 cases of O+, 6 cases of A+, 1 cases of AB+, and 2 B+ type. The weight of the liver tissue block was 27.2 + 7.8G (15 - 42g). Hot ischemic time (WIT) 10-35min. The yield of all 13 cases of tissue block cold ischemic time (CIT) 35 min. separation was 4.8 + 2.1 x 106 / g liver tissue, and the rate of liver cell viability was 78.1 + 10.4% (62%-93%) after trypan blue staining.
[conclusion]1, benign liver disease (mainly hepatic hemangioma and intrahepatic bile duct stone) can be separated from the abandoned normal liver tissue after partial hepatectomy. The improved four step perfusion method can be used to separate a large number of active human hepatocytes,.2. For the liver tissue with moderate fibrosis, it can still be good if the marginal vascular conditions are good. The effect of cell separation,.3, should be shortened as far as possible to obtain the thermal ischemia time of the liver tissue, and to improve the effect of liver cell separation,.4. For the smaller liver tissue blocks and the poor cutting conditions, the four step circulation perfusion method is not effective.
The third part: reversible immortalization of human hepatocytes
[background and objective] the proliferation of human primary hepatocytes in vitro is difficult, and there is a short culture time, which can not be obtained in time. The application is greatly restricted. The immortalized human hepatocyte with normal hepatocyte function can solve the problem of cell source shortage. This part of the experiment aims to make use of retrovirus to realize SV40T Transfection of single immortalized gene constructs reversible immortalization of human hepatocytes.
[Methods] human primary hepatocytes were transfected with Cre/LoxP system / SV40T and hygromycin resistance gene released by SSR#69 packaging cells. The positive cloned cell groups were screened by hygromycin 7 days later. The cells were selected by the appropriate size cloned ring to clone the proliferating cell group. Finally, the Cre recombinant enzyme was added to the cell. In addition to the immortalized gene SV40T between the two LoxP loci, the immortalized reverse.RT-PCR and immunofluorescence staining were used to verify the existence of immortalized gene SV40T.
[results] 7 days after retrovirus transfection, a large number of cloned cell clusters appeared in the culture bottle for 14 days. The morphology was similar to hepatocytes. Clones were selected and cultured in the new culture bottle, and the cells could be cultured and kept in shape. After adding Cre recombinant enzyme, the cells stopped growing.RT-PCR detection. Immortalized cells possess the marker genes of human primary hepatocytes, and immunofluorescence staining also confirms the presence of SV40T in immortalized cells.
[Conclusion] the reversible immortalized human hepatocytes have the function of hepatocytes and can be cultured in vitro. More importantly, we have successfully transfected human hepatocytes into human hepatocytes by retrovirus single immortalization gene and transfection of human hepatocyte SV40T/ hTERT double immortalized gene for the successor experiment to realize human hepatocytes. The thorough immortalization lays the foundation of the methodology.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329

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