本文選題：臍帶　切入點：間充質干細胞　出處：《大連醫(yī)科大學》2012年碩士論文　論文類型：學位論文

【摘要】：目的：通過分離人臍帶間充質干細胞(umbilical cord mesenchymal stem cells，UCMSCs)，采用系列細胞因子聯(lián)合誘導，探索高效可靠的體外誘導分化為肝樣細胞的技術方法。 方法：采用健康產婦臍帶，用酶消化法分離、培養(yǎng)臍帶間充質干細胞（UCMSC）；流式細胞儀檢測細胞抗原CD13、CD44、CD105、CD34、CD45、HLA-DR表達情況，鑒定分離的細胞是否為UCMSCs；用肝細胞生長因子(hepatocyte growth factor，HGF)、成纖維細胞生長因子-4(Fibroblast growth factor-4FGF-4)及白細胞介素6（Interleukin-6IL-6）進行組合分組：A組：僅基礎培養(yǎng)基，即為陰性對照組；B組：基礎培養(yǎng)基+HGF+FGF-4；C組：基礎培養(yǎng)基+HGF+IL-6；D組：基礎培養(yǎng)基+HGF+FGF-4+IL-6誘導培養(yǎng)第3代（P3）的臍帶MSCs，用免疫細胞化學法檢測細胞標志物AFP、ALB的表達情況，比較各個誘導組誘導成肝樣細胞的細胞比例。數(shù)據用均數(shù)±標準差（x±s）表示，，采用SPSS18.0統(tǒng)計分析軟件包進行分析，用單因素方差分析對資料進行統(tǒng)計分析。 結果： 1.UCMSC的細胞形態(tài)接種的UCMSC多于48小時后貼壁，5天后有短梭形細胞長出，后漸由短梭形變?yōu)榧氶L扁平的長梭形，15天后出現(xiàn)梭形細胞集落，呈漩渦狀或長條形生長，1月后細胞相互接觸,并鋪滿整個培養(yǎng)皿底部。 2.UCMSC鑒定取培養(yǎng)至第三代（P3）的臍帶間充質干細胞行流式細胞儀檢測細胞表面抗原表達，反映間充質干細胞的表面抗原CD13、CD44、CD105表達陽性，表達率分別為99.0%、98.4%、98.4%，而造血干細胞標志物CD34、CD45、HLA-DR表達陰性，表達率分別為4.6%、9.48%、5.04%。 3.UCMSC的誘導分化 3.1誘導成肝樣細胞的細胞形態(tài)取培養(yǎng)至第三代的UCMSCs加入各細胞因子組合向肝樣細胞誘導，經3~4周呈放射狀的細胞排列結構逐步消失，長梭形細胞變類圓形或多角形，存在離散現(xiàn)象，胞漿出現(xiàn)顆粒，部分出現(xiàn)雙核。 3.2肝樣細胞標志物表達對誘導出的肝樣細胞行其標志物甲胎蛋白(AFP)及白蛋白（ALB）檢測顯示：A組始終沒有見到肝樣細胞的標志物白蛋白(ALB)、甲胎蛋白(AFP)的表達。而B、C、D組可以檢測到肝細胞的標志物AFP、ALB的表達，AFP在誘導7天時，不同誘導組（B、C、D）比較，D組表達率最高；至15天時各組表達均減少，但D組仍最高；至誘導20天時各組表達均消失。而ALB在誘導7天、15天、20天都有強表達。在誘導第7天，B組表達率最高；誘導15天、20天， B、D組表達率高于C組。 結論： 1.使用酶消化法可以從人臍帶中成功分離、培養(yǎng)出UCMSC。 2.應用肝細胞生長因子(hepatocyte growth factor，HGF)、成纖維細胞生長因子-4(Fibroblast growth factor-4FGF-4)及白細胞介素6（Interleukin-6IL-6）聯(lián)合誘導UCMSCs可高效、穩(wěn)定培養(yǎng)出肝樣細胞。
[Abstract]:Aim: to isolate human umbilical cord mesenchymal stem cells (cord mesenchymal stem cells), and to explore a highly efficient and reliable method for inducing and differentiating into hepatoid cells in vitro by a series of cytokines. Methods: UCM SCC was cultured from umbilical cord of healthy pregnant women by enzyme digestion, and the expression of HLA-DR was detected by flow cytometry (FCM), and the expression of HLA-DR was detected by flow cytometry (FCM), and the expression of HLA-DR was detected by flow cytometry (FCM), and the expression of HLA-DR was detected by flow cytometry (FCM). To identify whether the isolated cells were UCMSCs or not, to classify the isolated cells into two groups: group A, combined with hepatocyte growth factor-HGF, fibroblast growth factor-4FGF-4, and interleukin 6 Interleukin-6IL-6). That is, the negative control group B: basic culture medium HGF FGF-4C group: basic medium HGF IL-6D group: basic medium HGF FGF-4 IL-6 induces and culture the third passage P3 umbilical cord MSCs. The expression of the cell marker AFPALB was detected by immunocytochemistry. The ratio of cells induced into hepatoid cells in each induction group was compared. The data were expressed by mean 鹵standard deviation (x 鹵s), analyzed by SPSS18.0 statistical analysis software package, and analyzed by single factor variance analysis (ANOVA). Results:. 1. After more than 48 hours of UCMSC inoculation, short fusiform cells of UCMSC grew after 5 days, and then gradually changed from short fusiform to slender fusiform cells. After 15 days, fusiform cell colonies appeared. After January, the cells contact each other and cover the bottom of the petri dish. 2. UCMSC identified the umbilical cord mesenchymal stem cells (UCMSC) cultured to the third generation of P3. Flow cytometry was used to detect the expression of surface antigen of mesenchymal stem cells, which indicated that the surface antigen of mesenchymal stem cells was positive. The expression rates of HLA-DR were 99.0 and 98.4, respectively, while the hematopoietic stem cell markers CD34, CD45, and HLA-DR were negative, and the expression rates were 4.69.48 and 5.04, respectively. 3. Differentiation of UCMSC. 3.1 the cell morphology of hepatoid cells induced to the third passage was induced by the addition of various cytokines to hepatoid cells. After 3 ~ 4 weeks, the radial cell arrangement gradually disappeared, and the long fusiform cells became round or polygonal. There is a discrete phenomenon, the cytoplasm appears granules, part of the binuclear. 3.2 expression of Hepatoid Cell markers in Hepatoid cells detected by AFP) and Albumin (ALB), the markers of Hepatoid cells (ALB) and Albumin (ALB) were not found in the liver like cells in group B: a, but the markers of ALB, AFP and AFP were not found in group B: C ~ (2 +), while those in group B ~ (2) C ~ (2) C ~ (3 +). The expression of AFPnALB, a marker of hepatocyte, was detected after 7 days of induction. The highest expression rate was observed in group D and decreased at day 15, but the expression rate in group D was still the highest in group D, but the expression rate in group D was higher than that in group C (P < 0.05). The expression rate of ALB was higher in group B than in group C on day 7 and day 20 after induction, and the expression rate of group B was higher than that of group C on day 7, and the expression rate of group B was higher than that of group C on the 7th day of induction, and the expression rate of ALB in group D was higher than that in group C on the 7th day of induction. Conclusion:. 1. UCMSCS could be successfully isolated from human umbilical cord by enzyme digestion. 2. The combination of hepatocyte growth factor-hGF-1, fibroblast growth factor-4FGF-4 and interleukin-6 Interleukin-6IL-6 can induce UCMSCs efficiently and stably.
【學位授予單位】：大連醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R329

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相關期刊論文 前2條

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