本文關鍵詞：人臍帶血間充質(zhì)干細胞體外定向分化為肝樣細胞的研究　出處：《廣西醫(yī)科大學》2008年碩士論文　論文類型：學位論文

  更多相關文章： 臍帶血 間充質(zhì)干細胞 誘導 肝樣細胞

【摘要】： 目的將來源于健康產(chǎn)婦的臍帶血進行體外分離、培養(yǎng)出間充質(zhì)干細胞(mesenchymal stem cells,MSCS),誘導成肝樣細胞并進行鑒別,探討間充質(zhì)干細胞體外培養(yǎng)和定向分化為肝樣細胞的可行性,為終末期肝病的肝細胞移植治療和生物型人工肝提供一種新的細胞來源。 方法采集正常產(chǎn)婦臍帶血,密度梯度離心法和貼壁法分離,培養(yǎng)純化臍血MSCS,使用肝細胞生長因子(hepatocyte growth factor,HGF)誘導MSCS。采用RT-PCR和免疫細胞化學法檢測肝樣細胞標志物的表達情況并進行糖原染色檢測細胞糖原的表達;采用透射電鏡觀察間充質(zhì)干細胞和誘導肝樣細胞的超微結(jié)構;流式細胞儀檢測間充質(zhì)干細胞表面抗原CD34,CD44表達情況。 結(jié)果1.采用密度梯度離心法和貼壁法分離接種的MSCs形態(tài)大小均一透亮度好,24小時內(nèi)貼壁,以后逐漸增多,呈克隆性生長,3天左右可見梭形、三角形細胞,10天左右達到對數(shù)生長期。細胞傳至第三代時,細胞融合成單層,梭形突起變長,排列有明顯方向性,集落呈旋渦狀生長,臺盼藍染色細胞存活率＞95%。2.經(jīng)過傳代,純化,誘導的細胞由長梭形慢慢開始變圓,呈放射狀的細胞排列結(jié)構逐步消失,形態(tài)變短,變類圓形或多角形,胞漿出現(xiàn)顆粒,部分出現(xiàn)雙核,折光性強,分裂相多。非誘導組仍保留梭型的細胞形態(tài)。3.標志物檢測:非誘導組始終沒有見到肝細胞的標志物白蛋白(albium,ALB)、糖原表達,甲胎蛋白(alpha fetoprotein,AFP)第0天有少量表達此后不再表達;誘導組可以檢測到肝細胞的標志物AFP、ALB和糖原的明顯的表達。4.培養(yǎng)至第三代的細胞表面抗原CD44陽性表達,不表達CD34。 結(jié)論1.采用密度梯度離心法和貼壁培養(yǎng)法可以在體外成功分離培養(yǎng)出臍帶血MSCS。2.經(jīng)HGF誘導的細胞初步確定為肝樣細胞。
[Abstract]:To come from healthy maternal umbilical cord blood were isolated, cultured mesenchymal stem cells (mesenchymal stem cells, MSCS), and induced to differentiate into hepatocyte like cells were identified on mesenchymal stem cells in vitro and differentiate into hepatocyte like cells is feasible, end-stage liver disease liver cell transplantation and bioartificial liver provides a new cell source.
Methods normal maternal umbilical cord blood by density gradient centrifugation and adherent culture, purification of MSCS in umbilical cord blood, the use of hepatocyte growth factor (hepatocyte growth factor, HGF MSCS.) induced by RT-PCR and immunocytochemical method to detect hepatocyte like cells marked expression of glycogen staining cells and expression of glycogen by transmission; electron microscopic observation of the ultrastructure of mesenchymal stem cells and induced hepatocyte like cells; detection of mesenchymal stem cell surface antigen CD34 by flow cytometry, the expression of CD44.
Results 1. by density gradient centrifugation and adhering to the MSCs form wall isolation with uniform brightness, within 24 hours after the adherent was gradually increased, the clonal growth of about 3 days, visible fusiform, triangle cells, about 10 days to reach the logarithmic growth phase. When cells spread to the third generation, cell fusion single spindle, elongate, arranged in a clear direction, the colony was swirling growth, trypan blue staining and cell survival rate was 95%.2. after passage, purification, induced cells from spindle slowly began to turn round, radial arrangement of cells structure gradually disappear, form shorter, variable type round or polygonal, cytoplasm granules, part of the nucleus, strong refraction, split phase. Non induced group still retain markers of cell morphology of.3. spindle type: non induced group has not seen the hepatocyte marker albumin (albium, ALB), the expression of glycogen, AFP Protein (alpha fetoprotein, AFP) was expressed on a small scale for zeroth days, and then no longer expressed. The expression of AFP, ALB and glycogen in hepatocyte markers could be detected in the induction group,.4. was positive in CD44 cells cultured on the third generation, and CD34. was not expressed.
Conclusion 1.. Cord blood MSCS.2. can be successfully isolated and cultured in vitro by density gradient centrifugation and adherent culture. The cells induced by HGF are initially identified as hepatocyte like cells.

【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R329

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