本文關(guān)鍵詞： 宮內(nèi)膜干細胞 成肝分化 肝損傷　出處：《浙江大學》2012年碩士論文　論文類型：學位論文

【摘要】：本研究目的為探討宮內(nèi)膜干細胞的體外成肝誘導分化及其在肝臟損傷中發(fā)揮的修復(fù)作用。首先,我們從招募的志愿者經(jīng)血中分離出單個核細胞進行體外傳代培養(yǎng)并對獲得的細胞進行間充質(zhì)干細胞鑒定。宮內(nèi)膜干細胞不具有致瘤性,可以呈單層貼壁生長,可以24小時倍增一次,為典型的棒狀或長梭狀的成纖維細胞樣細胞。流式細胞術(shù)分析這類細胞均表達CD44,CD73,CD90,和CD29,而不表達CD117,CD34, CD45,和HLA-DR。宮內(nèi)膜干細胞在體外可以被誘導分化為成骨細胞和成脂細胞。 其次,我們研究了宮內(nèi)膜干細胞的體外成肝誘導分化能力。宮內(nèi)膜干細胞在與HGF、FGF4、OSM以及DEX等共培養(yǎng)后,逐漸向上皮細胞樣細胞形態(tài)變化。誘導分化后的細胞能夠表達ALB和AFP蛋白,以及基因ALB、AFP、CK18、CK19、CYP1A1和CYP3A4等。表達譜芯片分析可見分化后的細胞能表達33個肝臟相關(guān)基因,這些基因在原代肝臟細胞中表達,而宮內(nèi)膜干細胞中則不表達。另外,誘導分化后細胞功能驗證顯示,其具有糖原合成、ICG排泌和尿素合成功能等。這些數(shù)據(jù)可以初步證實,所誘導的細胞是具有肝細胞表型且能發(fā)揮部分肝臟細胞功能的較成熟的細胞。 最后,為了驗證移植細胞能否參與肝損傷修復(fù)過程,小鼠肝臟部分切除后,分別移植宮內(nèi)膜干細胞和誘導分化后細胞,移植后10天,移植分化組小鼠肝功能較移植宮內(nèi)膜干細胞組恢復(fù)明顯,具有顯著性差異。移植8周后,在小鼠肝臟中可以檢測到移植細胞的存在,并且移植宮內(nèi)膜干細胞在受體肝臟內(nèi)發(fā)生分化。Fah-/小鼠作為移植受體,移植細胞后8周,發(fā)現(xiàn)誘導分化組小鼠肝臟可見大量Fah陽性細胞,而移植宮內(nèi)膜干細胞組則僅有少量散在分布的Fah陽性細胞存在。這些數(shù)據(jù)說明,分化后細胞可以部分重建小鼠肝臟,未分化細胞可以發(fā)生分化但整體水平較低。結(jié)果顯示,移植細胞可以隨血流遷移至小鼠肝臟,并在小鼠肝臟內(nèi)達到一定程度的重建。 綜上所述,宮內(nèi)膜干細胞及其來源的細胞在肝臟疾病治療中可以發(fā)揮一定作用。
[Abstract]:The purpose of this study was to investigate the differentiation of endometrial stem cells in vitro and its role in the repair of liver injury. We isolated mononuclear cells from the blood of recruited volunteers and cultured them in vitro and identified the obtained cells by mesenchymal stem cells. The endometrial stem cells were not tumorigenic and could be monolayer adherent. It can be doubled once in 24 hours and is a typical rod-like or long-fusiform fibroblast-like cell. All of these cells express CD44-CD73 CD90 and CD29 by flow cytometry. However, the endometrial stem cells could be induced to differentiate into osteoblasts and adipoblasts without expression of CD117, CD45, and HLA-DR.In vitro, endometrial stem cells could be induced to differentiate into osteoblasts and adipoblasts. Secondly, we studied the ability of endometrial stem cells to induce liver differentiation in vitro. Endometrial stem cells were co-cultured with HGFFGF4OSM and DEX. The cells induced by differentiation could express ALB and AFP proteins, as well as the gene ALBN-AFPCK18CK-19. CYP1A1 and CYP3A4. Microarray analysis showed that the differentiated cells could express 33 liver-associated genes, which were expressed in primary liver cells. In addition, after induction of differentiation, the cell function test showed that it had the function of glycogen synthesis, ICG excretion, urea synthesis and so on. These data can be preliminarily confirmed. The induced cells are mature cells with hepatocyte phenotype and function of some liver cells. Finally, in order to verify whether the transplanted cells can participate in the repair process of liver injury, after partial hepatectomy, endometrial stem cells and differentiated cells were transplanted, respectively, 10 days after transplantation. The liver function of the transplanted differentiation group was significantly different from that of the transplanted endometrial stem cell group. 8 weeks after transplantation, the presence of transplanted cells could be detected in the mouse liver. And transplanted endometrial stem cells in the recipient liver differentiation. Fah-/ mice as the transplantation receptor, 8 weeks after transplantation, we found that a large number of Fah positive cells were found in the liver of the induced differentiation group. However, there were only a few scattered Fah positive cells in the transplanted endometrial stem cells group. These data showed that the differentiated cells could partially reconstruct the liver of mice. The results showed that the transplanted cells could migrate to the mouse liver with the blood flow, and the transplanted cells could be reconstructed in the mouse liver to a certain extent. In conclusion, endometrial stem cells and their derived cells may play a role in the treatment of liver diseases.
【學位授予單位】：浙江大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R329

【共引文獻】

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1 檀家俊;白介素1受體拮抗劑納米緩釋顆粒聯(lián)合骨髓間充質(zhì)干細胞移植治療豬急性肝衰竭的實驗研究[D];南京大學;2012年

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本文編號：1449119