本文選題：肝前體細胞 + 永生化細胞株　； 參考：《重慶醫(yī)科大學》2009年博士論文

【摘要】： 肝臟疾病最終常常發(fā)生功能的紊亂和臟器的衰竭。肝移植為肝臟疾病的治療提供了無限前景。但供體肝源不足、排斥反應及長期免疫抑制劑的治療限制了肝移植的發(fā)展。肝干細胞移植成為一種有潛力的替代治療方法。同時對肝干細胞增殖、分化的研究還有助于了解肝臟的發(fā)生、發(fā)育機制,為進一步研究肝癌的發(fā)病機制及治療提供基礎。近年來,隨著分子生物學、細胞生物學、細胞培養(yǎng)技術(shù)及免疫學的發(fā)展,肝干細胞的分離鑒定和培養(yǎng)取得了一些進展。但如何分離、篩選獲得高純度、高特異性的肝干細胞,如何建立肝干細胞穩(wěn)定的體外培養(yǎng)系統(tǒng),如何調(diào)節(jié)肝干細胞特異分化,仍是急需解決的問題。 在該課題的第一部分中,我們首先制備了含LoxP位點特異修飾的SV40大T抗原基因的SSR#69逆轉(zhuǎn)錄病毒液,并感染從孕14.5d的胎鼠肝臟中分離出的肝細胞(此時的肝細胞被認為大部分為肝前體細胞),使其永生化。再用有限稀釋法培養(yǎng)獲得單克隆永生化細胞株,并進一步篩選鑒定。首先取不同階段的肝組織:孕12.5d、14.5d、16.5d、18.5d及出生后1d、7d、14d、28d,利用real time PCR觀察常見的早期指標和晚期指標的表達趨勢。結(jié)果發(fā)現(xiàn)其早期標志基因CD34、Pou5f1隨組織的發(fā)育成熟逐漸降低;AFP早期逐漸上升,至出生前達到最高,出生后開始迅速降低;DLK早期逐漸上升,至16.5d達到最高,后逐漸降低。晚期標志基因Alb、CK18、TAT、ApoB隨組織的發(fā)育成熟逐漸上升,TAT上升較晚,約出生后第二周開始。在此基礎上我們以相同方法獲得的出生后14d的永生化肝細胞株LC14d及肝腫瘤細胞株Hepa1-6為對照,篩選出高表達早期標志基因的單克隆細胞株5個,并進一步通過形態(tài)學觀察、地塞米松和二甲亞楓誘導分化等進行鑒定。結(jié)果顯示篩選獲得的單克隆細胞株細胞增殖旺盛、成集落樣生長,體積較小、多邊形、核漿比例高。經(jīng)誘導分化后,ICG攝取試驗證實篩選出的單克隆細胞株可誘導分化為成熟的肝細胞,為具有分化潛能的肝前體細胞。連續(xù)傳代50代后檢測細胞無明顯差異。第二步我們檢測導入的外源基因SV40大T抗原是否可逆。首先我們用western blot檢測證實獲得的永生化肝前體細胞株有大T抗原的表達,說明SV40大T抗原的導入成功。而加入腺病毒Ad-Cre處理后,細胞無大T抗原的表達,說明在Cre重組酶的作用下,大T抗原可以被敲除。而經(jīng)腺病毒Ad-Cre處理的單克隆細胞株生長曲線降低,白蛋白熒光素酶報告質(zhì)粒檢測顯示白蛋白表達上升,即分化增加。也證實SV40大T抗原可促進細胞的增殖,抑制細胞的分化,而Cre重組酶可敲除LoxP位點特異修飾的SV40大T抗原基因。第三步體內(nèi)成瘤實驗。我們于皮下注入熒光素酶標記的永生化肝前體細胞株,對側(cè)注入同樣標記的經(jīng)Cre重組酶處理的永生化肝前體細胞株,并以肝癌細胞株為對照,活組織成像隨訪。結(jié)果顯示隨著時間的延長,植入細胞熒光素酶的表達降低,說明皮下存活的細胞數(shù)減少。與肝癌細胞株相比,皮下植入永生化肝前體細胞株熒光素酶的表達在10天內(nèi)基本消失,沒有成瘤的趨勢。而經(jīng)Cre重組酶處理后的永生化肝前體細胞株,熒光素酶的表達消失提前,說明細胞增殖降低,存活時間縮短。這同樣也說明導入的大T抗原可以被敲除,是可逆的。因此,在第一部分實驗中,我們獲得了可逆的永生化的肝前體細胞株,為我們第二部分的實驗打下良好的基礎。 在該課題的第二部分中,我們檢測了維甲酸(Retinoic acid,RA)對肝前體細胞分化的影響。維甲酸是維生素A的體內(nèi)衍生物。和其它類視黃醇一樣,它在脊椎動物的胚胎發(fā)育、內(nèi)環(huán)境的穩(wěn)定及細胞的增殖分化中都發(fā)揮著重要作用。但維甲酸在組織特異性前體細胞分化中的作用報道卻很少。因此,本課題在第一部分構(gòu)建永生化肝前體細胞株的前提下,進一步研究了維甲酸對胎肝前體細胞分化的影響,以期獲得一種誘導肝前體細胞分化的新方法,并進一步研究維甲酸誘導肝前體細胞分化的機制。而維甲酸可誘導與肝臟具有共同起源的胰腺祖細胞的增殖和進一步分化為β細胞,也提示維甲酸可能在肝臟的發(fā)育中有重要作用。 在該部分研究中我們首先檢測了不同階段的肝組織中內(nèi)源性維甲酸合成代謝相關(guān)基因及維甲酸受體和輔助因子的表達水平。結(jié)果顯示維甲酸受體RARα、RXRα和RXRγ在所有樣品中均有表達;而RARβ和RXRβ在出生前表達較低而出生后逐漸升高;RARγ在出生前無表達,出生后逐漸上升。維甲酸合成酶Raldh1和Raldh2在所有組織中均有表達,Raldh3在出生前表達很低,出生后穩(wěn)定表達。核受體共抑制劑在所有樣品中均高表達,而共激動劑在出生前逐漸降低,出生后逐漸上升。提示維甲酸信號系統(tǒng)可能和肝前體細胞的分化密切相關(guān)。我們進一步利用白蛋白熒光素酶報告質(zhì)粒篩選獲得了全反式維甲酸和9-順式維甲酸最適藥物濃度,通過最適藥物濃度的全反式維甲酸和9-順式維甲酸誘導,RT-PCR檢測可見其早期標志基因表達降低,晚期標志基因表達上升,免疫熒光檢測得出一致的結(jié)論。糖原沉積試驗結(jié)果顯示誘導后糖原沉積增加。以上結(jié)果說明全反式維甲酸和9-順式維甲酸能促進肝前體細胞向前肝細胞的分化。為進一步了解參與該途徑的維甲酸受體,我們構(gòu)建了腺病毒RARα和RXRα,感染細胞后通過白蛋白熒光素酶報告質(zhì)粒檢測其分化的改變。結(jié)果顯示感染腺病毒RARα或RXRα后,肝前體細胞株HP14.5中白蛋白的表達無明顯改變,說明全反式維甲酸和9-順式維甲酸在參與調(diào)節(jié)肝前體細胞分化的過程中,可能并不是通過受體RARα或RXRα發(fā)揮作用。 結(jié)論:成功分離和構(gòu)建了可逆的永生化的肝前體細胞株;維甲酸能促進肝前體細胞的分化,但可能不是通過受體RARα或RXRα發(fā)揮作用。
[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.

【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R329

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本文編號：1868567