永生化肝前体细胞株的构建及维甲酸在肝前体细胞分化中的作用研究

发布时间：2018-05-10 08:23

本文选题：肝前体细胞 + 永生化细胞株　；参考：《重庆医科大学》2009年博士论文

【摘要】： 肝脏疾病最终常常发生功能的紊乱和脏器的衰竭。肝移植为肝脏疾病的治疗提供了无限前景。但供体肝源不足、排斥反应及长期免疫抑制剂的治疗限制了肝移植的发展。肝干细胞移植成为一种有潜力的替代治疗方法。同时对肝干细胞增殖、分化的研究还有助于了解肝脏的发生、发育机制,为进一步研究肝癌的发病机制及治疗提供基础。近年来,随着分子生物学、细胞生物学、细胞培养技术及免疫学的发展,肝干细胞的分离鉴定和培养取得了一些进展。但如何分离、筛选获得高纯度、高特异性的肝干细胞,如何建立肝干细胞稳定的体外培养系统,如何调节肝干细胞特异分化,仍是急需解决的问题。在该课题的第一部分中,我们首先制备了含LoxP位点特异修饰的SV40大T抗原基因的SSR#69逆转录病毒液,并感染从孕14.5d的胎鼠肝脏中分离出的肝细胞(此时的肝细胞被认为大部分为肝前体细胞),使其永生化。再用有限稀释法培养获得单克隆永生化细胞株,并进一步筛选鉴定。首先取不同阶段的肝组织:孕12.5d、14.5d、16.5d、18.5d及出生后1d、7d、14d、28d,利用real time PCR观察常见的早期指标和晚期指标的表达趋势。结果发现其早期标志基因CD34、Pou5f1随组织的发育成熟逐渐降低;AFP早期逐渐上升,至出生前达到最高,出生后开始迅速降低;DLK早期逐渐上升,至16.5d达到最高,后逐渐降低。晚期标志基因Alb、CK18、TAT、ApoB随组织的发育成熟逐渐上升,TAT上升较晚,约出生后第二周开始。在此基础上我们以相同方法获得的出生后14d的永生化肝细胞株LC14d及肝肿瘤细胞株Hepa1-6为对照,筛选出高表达早期标志基因的单克隆细胞株5个,并进一步通过形态学观察、地塞米松和二甲亚枫诱导分化等进行鉴定。结果显示筛选获得的单克隆细胞株细胞增殖旺盛、成集落样生长,体积较小、多边形、核浆比例高。经诱导分化后,ICG摄取试验证实筛选出的单克隆细胞株可诱导分化为成熟的肝细胞,为具有分化潜能的肝前体细胞。连续传代50代后检测细胞无明显差异。第二步我们检测导入的外源基因SV40大T抗原是否可逆。首先我们用western blot检测证实获得的永生化肝前体细胞株有大T抗原的表达,说明SV40大T抗原的导入成功。而加入腺病毒Ad-Cre处理后,细胞无大T抗原的表达,说明在Cre重组酶的作用下,大T抗原可以被敲除。而经腺病毒Ad-Cre处理的单克隆细胞株生长曲线降低,白蛋白荧光素酶报告质粒检测显示白蛋白表达上升,即分化增加。也证实SV40大T抗原可促进细胞的增殖,抑制细胞的分化,而Cre重组酶可敲除LoxP位点特异修饰的SV40大T抗原基因。第三步体内成瘤实验。我们于皮下注入荧光素酶标记的永生化肝前体细胞株,对侧注入同样标记的经Cre重组酶处理的永生化肝前体细胞株,并以肝癌细胞株为对照,活组织成像随访。结果显示随着时间的延长,植入细胞荧光素酶的表达降低,说明皮下存活的细胞数减少。与肝癌细胞株相比,皮下植入永生化肝前体细胞株荧光素酶的表达在10天内基本消失,没有成瘤的趋势。而经Cre重组酶处理后的永生化肝前体细胞株,荧光素酶的表达消失提前,说明细胞增殖降低,存活时间缩短。这同样也说明导入的大T抗原可以被敲除,是可逆的。因此,在第一部分实验中,我们获得了可逆的永生化的肝前体细胞株,为我们第二部分的实验打下良好的基础。在该课题的第二部分中,我们检测了维甲酸(Retinoic acid,RA)对肝前体细胞分化的影响。维甲酸是维生素A的体内衍生物。和其它类视黄醇一样,它在脊椎动物的胚胎发育、内环境的稳定及细胞的增殖分化中都发挥着重要作用。但维甲酸在组织特异性前体细胞分化中的作用报道却很少。因此,本课题在第一部分构建永生化肝前体细胞株的前提下,进一步研究了维甲酸对胎肝前体细胞分化的影响,以期获得一种诱导肝前体细胞分化的新方法,并进一步研究维甲酸诱导肝前体细胞分化的机制。而维甲酸可诱导与肝脏具有共同起源的胰腺祖细胞的增殖和进一步分化为β细胞,也提示维甲酸可能在肝脏的发育中有重要作用。在该部分研究中我们首先检测了不同阶段的肝组织中内源性维甲酸合成代谢相关基因及维甲酸受体和辅助因子的表达水平。结果显示维甲酸受体RARα、RXRα和RXRγ在所有样品中均有表达;而RARβ和RXRβ在出生前表达较低而出生后逐渐升高;RARγ在出生前无表达,出生后逐渐上升。维甲酸合成酶Raldh1和Raldh2在所有组织中均有表达,Raldh3在出生前表达很低,出生后稳定表达。核受体共抑制剂在所有样品中均高表达,而共激动剂在出生前逐渐降低,出生后逐渐上升。提示维甲酸信号系统可能和肝前体细胞的分化密切相关。我们进一步利用白蛋白荧光素酶报告质粒筛选获得了全反式维甲酸和9-顺式维甲酸最适药物浓度,通过最适药物浓度的全反式维甲酸和9-顺式维甲酸诱导,RT-PCR检测可见其早期标志基因表达降低,晚期标志基因表达上升,免疫荧光检测得出一致的结论。糖原沉积试验结果显示诱导后糖原沉积增加。以上结果说明全反式维甲酸和9-顺式维甲酸能促进肝前体细胞向前肝细胞的分化。为进一步了解参与该途径的维甲酸受体,我们构建了腺病毒RARα和RXRα,感染细胞后通过白蛋白荧光素酶报告质粒检测其分化的改变。结果显示感染腺病毒RARα或RXRα后,肝前体细胞株HP14.5中白蛋白的表达无明显改变,说明全反式维甲酸和9-顺式维甲酸在参与调节肝前体细胞分化的过程中,可能并不是通过受体RARα或RXRα发挥作用。结论:成功分离和构建了可逆的永生化的肝前体细胞株;维甲酸能促进肝前体细胞的分化,但可能不是通过受体RARα或RXRα发挥作用。
[Abstract]:Liver diseases often often have functional disorders and organ failure. Liver transplantation provides an unlimited prospect for the treatment of liver diseases. However, donor liver insufficiency, rejection and long-term immunosuppressive therapy restrict the development of liver transplantation. Liver stem cell transplantation has become a potential alternative treatment. The study of colonization and differentiation also helps to understand the occurrence and development mechanism of the liver and provide the basis for further research on the pathogenesis and treatment of liver cancer. In recent years, some progress has been made with the development of molecular biology, cell biology, cell culture and immunology, and the isolation and culture of liver stem cells have been made. It is still an urgent problem to get high purity, highly specific liver stem cells, how to establish a stable culture system for liver stem cells, and how to regulate the specific differentiation of liver stem cells.
In the first part of the project, we first prepared the SSR#69 retrovirus fluid containing the LoxP site specific modified SV40 T antigen gene, and infected the liver cells isolated from the fetal rat liver of pregnant 14.5d (the liver cells at this time are considered as the most of the liver precursor cells) to make it immortalized. 12.5d, 14.5d, 16.5d, 18.5d and postnatal 1D, 7d, 14d, 28d, and the expression trends of common early indicators and late indexes were observed by real time PCR. The results showed that the early marker gene CD34 was gradually reduced with the development of tissue. The period is rising gradually, reaching the highest before birth and rapidly decreasing after birth; the early stage of DLK gradually rises, to the highest 16.5d, and then gradually decreasing. The late marker gene Alb, CK18, TAT, ApoB are gradually rising with the development of tissue, TAT rises later, about second weeks after birth. On this basis, we obtained the same method of birth 1. 4D's immortalized hepatocyte strain LC14d and liver tumor cell line Hepa1-6 were compared, and 5 monoclonal cell lines with high expression of early marker genes were screened and further identified by morphological observation, dexamethasone and two Maple Maple induced differentiation. Long, small size, polygon and high ratio of nuclear plasma. After differentiation, the ICG uptake test confirmed that the screened monoclonal cell line could induce the differentiation into mature liver cells, which had differentiation potential of the liver precursor cells. There was no significant difference in the detection of cells after 50 generations of continuous generation. The second step was to detect the exogenous gene SV40 T antigen introduced. No reversible. First we confirmed the expression of large T antigen in the immortalized liver progenitor cells obtained by Western blot detection, indicating that the SV40 T antigen was introduced successfully. After adding adenovirus Ad-Cre treatment, the cells had no large T antigen expression, indicating that the large T antigen could be knocked out under the action of Cre recombinant enzyme, and the adenovirus Ad-Cre was treated by Ad-Cre. The growth curve of the monoclonal cell line decreased, and the albumin luciferase reporter plasmid detection showed that the expression of albumin increased, that is, the differentiation increased. It was also proved that SV40 T antigen could promote cell proliferation and inhibit cell differentiation, and Cre recombinant enzyme could knock out SV40 large T antigen gene specifically modified by LoxP site. The third step in vivo tumorigenesis experiment. An immortalized hepatic precursor cell line labeled with fluorescein was subcutaneously injected into an immortalized hepatic precursor cell line labeled with a Cre recombinant enzyme, and the live tissue was followed up with the liver cancer cell line. The results showed that the expression of the implanted cell fluorescent enzyme decreased with the prolongation of time, indicating the number of subcutaneously surviving cells. Decrease. The expression of luciferase of immortalized liver precursor cells subcutaneously disappeared in 10 days and did not become a tumor, while the expression of luciferase, after Cre recombinant enzyme treatment, disappeared ahead of time, indicating that cell proliferation and survival time were reduced. The large T antigen can be knocked out and reversible. Therefore, in the first part of the experiment, we obtained a reversible immortalized liver precursor cell strain, which laid a good foundation for our second part experiment.
In the second part of the subject, we detected the effect of Retinoic acid (RA) on the differentiation of hepatic precursor cells. Retinoic acid is a derivative of vitamin A in vivo. Like other retinoids, it plays an important role in the embryonic development of vertebrates, the stability of the internal environment and the proliferation and differentiation of the cells. There are few reports on the role of the differentiation of the heterosexual precursor cells. Therefore, in the first part, the effect of retinoic acid on the differentiation of fetal liver precursor cells was further studied in the first part of the construction of the immortalized preliver cell line, in order to obtain a new method to induce the differentiation of the hepatic precursor cells and to further study the induction of hepatic precursors by retinoic acid. The mechanism of cell differentiation, and retinoic acid can induce the proliferation and further differentiation of pancreatic progenitor cells with the common origin of the liver into beta cells, suggesting that retinoic acid may play an important role in the development of the liver.
In this part of the study, we first detected the expression level of endogenous retinoic acid synthesis related genes and retinoic acid receptors and cofactors in different stages of liver tissue. The results showed that retinoic acid receptor RAR alpha, RXR alpha and RXR gamma were expressed in all samples, while RAR beta and RXR beta were lower before birth and gradually increased after birth. RAR gamma is not expressed before birth and gradually rises after birth. Retinoic acid synthetase Raldh1 and Raldh2 are expressed in all tissues. Raldh3 is expressed very low before birth and stable after birth. The co inhibitor of nuclear receptor is highly expressed in all samples, and the co agonists gradually decrease before birth and gradually rise after birth. The signal system may be closely related to the differentiation of the hepatic precursor cells. We further screened the optimum drug concentration of all trans retinoic acid and 9- CIS retinoic acid by using the albumin luciferase reporter plasmid, and the early marker gene table can be detected by RT-PCR detection through the optimal drug concentration of all trans retinoic acid and 9- CIS retinoic acid. The results showed that all trans retinoic acid and 9- CIS retinoic acid could promote the differentiation of anterior hepatocytes in the hepatic precursor cells. It was a further understanding of the retinoic acid receptors involved in this pathway. We constructed the adenovirus RAR alpha and RXR alpha, and detected the differentiation by the albumin luciferase reporter plasmid after the infection. The results showed that the expression of albumin in the HP14.5 of the hepatic precursor cell strain HP14.5 was not significantly changed after the infection of RAR alpha or RXR a, indicating that all trans retinoic acid and 9- CIS retinoic acid were involved in regulating the differentiation of the liver precursor cells. It may not play a role in the process of receptor RAR alpha or RXR alpha.
Conclusion: a reversible and immortalized hepatic precursor cell line has been successfully isolated and constructed, and retinoic acid can promote the differentiation of the hepatic precursor cells, but it may not play a role through the receptor RAR alpha or RXR alpha.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R329

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