脐血干细胞体外分化为肝样细胞的实验研究

发布时间：2019-03-17 10:35

【摘要】： 目的:在体外分别采用有细胞因子的培养体系和无细胞因子的培养体系,探讨人脐血干细胞是否可以在体外培养条件下诱导分化为肝细胞样细胞,观察肝细胞标志物的表达,以期为治疗肝脏疾病提供了新的细胞来源。方法:常规无菌采集新鲜脐血,采用6%羟乙基淀粉、淋巴细胞分离液分离脐血(umbilical cord blood,UCB)的单个核细胞(mononuclear cells,MNCs)。MNCs分组培养,对照组不加细胞因子,实验组加入重组人肝细胞生长因子(hepatocyte growth factor,HGF)、重组人成纤维生长因子4(fibroblast growth factor-4,FGF-4)和重组人制瘤素(oncostatin M,OSM),浓度分别为20μg/L、10μg/L、10μg/L。并于诱导前及诱导后的第7,14,21,28d采用免疫细胞化学染色检测甲胎蛋白(alpha fetoprotein,AFP)、细胞角蛋白18(cytokeratin 18,CK18)、白蛋白(albumin ,ALB)和肝细胞抗原的表达,用PAS(periodic acid-schiff)法进行糖原染色,流式细胞仪(flow cytometry,FCM)检测细胞中ALB+表达,采用间接免疫荧光方法分析各组细胞ALB的表达情况,并用放射免疫法(radioimmunoassay,RIA)检测细胞培养液中AFP的分泌水平,分析是否诱导出肝细胞样细胞。结果: 1.实验组诱导后7d的细胞免疫组化显示AFP染色阳性,21d后基本为阴性;第14d检测到CK18、ALB呈阳性表达,第21d检测到肝细胞抗原表达,随着诱导时间的延长,阳性率逐渐增高。对照组免疫组化未见阳性表达。 2.实验组培养第21d时检测到糖原染色阳性细胞,28d时呈强阳性表达。对照组培养的MNCs PAS染色呈阴性。 3.实验组FCM检测显示第7d几乎未检测到ALB+细胞,随着诱导培养时间的延长,ALB+细胞表达增加,呈上升趋势;培养至第28d的ALB+细胞达80%以上。对照组未检测到ALB+细胞。 4.免疫荧光结果显示,实验组诱导14d后检测到ALB阳性表达,随诱导时间的延长,ALB仍呈阳性表达;对照组未检测到ALB的表达。 5.实验组RIA检测结果表明第7dAFP水平最高,随着培养时间的延长呈逐步下降趋势;对照组在0,7,14,21,28d可检测到低水平的AFP表达,其AFP值无明显差异。结论: 1.在无细胞生长因子的作用下,脐血干细胞不能够分化为肝细胞样细胞。 2.在有细胞生长因子诱导下,脐血干细胞能够分化为肝细胞样细胞。 3.脐血有望成为治疗重症肝病的一种有效的干细胞来源。
[Abstract]:Objective: to investigate whether human umbilical cord blood stem cells can be induced to differentiate into hepatocyte-like cells in vitro and observe the expression of hepatocyte markers by using cytokine-containing culture system and non-cytokine-free culture system, respectively, and explore whether human umbilical cord blood stem cells can be induced to differentiate into hepatocyte-like cells under the condition of in vitro culture. In order to provide a new source of cells for the treatment of liver diseases. Methods: fresh cord blood was collected by routine aseptic method. Mononuclear cells (mononuclear cells,MNCs) of cord blood (umbilical cord blood,UCB were separated by 6% hydroxyethyl starch and lymphocyte separation solution, and no cytokines were added to the control group. In the experimental group, recombinant human hepatocyte growth factor (hepatocyte growth factor,HGF), recombinant human fibroblast growth factor (4 (fibroblast growth factor-4,FGF-4) and recombinant human tumorigenin (oncostatin M) were added at the concentrations of 20 渭 g / L, 10 渭 g / L and 10 渭 g / L, respectively. The expressions of alpha-fetoprotein (alpha fetoprotein,AFP), cytokeratin 18 (cytokeratin-18, CK18), albumin (albumin, ALB) and hepatocyte antigen were detected by immunocytochemical staining before and 7,14,21 and 28 days after induction, and the expression of 伪-fetoprotein (AFP), cytokeratin 18 (cytokeratin-18), albumin (albumin, ALB) and hepatocyte antigen were detected by immunocytochemical staining. Glycogen staining was performed by PAS (periodic acid-schiff method, ALB expression was detected by flow cytometry (flow cytometry,FCM), ALB expression was analyzed by indirect immunofluorescence method, and radioimmunoassay (radioimmunoassay,) was used to detect the expression of ALB. RIA) was used to detect the secretion of AFP in the culture medium and to analyze whether hepatocyte-like cells were induced. Results: 1. The expression of AFP was positive on the 7th day after induction in the experimental group, and was negative on the 21st day after induction, and the expression of CK18,ALB was positive on the 14th day and the 21st day, and the positive rate gradually increased with the prolongation of the induction time. No positive expression was found in the control group by immunohistochemistry. 2. Glycogen staining positive cells were detected on the 21st day of culture in the experimental group, and strongly positive on the 28th day. In the control group, the MNCs PAS staining was negative. 3. The results of FCM showed that ALB cells were almost not detected on the 7th day in the experimental group, and the expression of ALB cells increased with the prolongation of the induction time, and the number of ALB cells cultured on the 28th day was more than 80%. No ALB cells were detected in the control group. 4. The results of immunofluorescence showed that the positive expression of ALB was detected in the experimental group after 14 days of induction, and the expression of ALB was still positive with the prolongation of the induction time, while the expression of ALB was not detected in the control group. 5. The level of RIA in the experimental group was the highest on the 7th day, and decreased gradually with the prolongation of the culture time, while the low level of AFP expression was detected in the control group at 0,7,14,21,28 days, and there was no significant difference in the AFP value between the control group and the control group at 0, 7, 14, 21, 28 days. Conclusions: 1. Umbilical cord blood stem cells could not differentiate into hepatocyte-like cells under the effect of no cell growth factor. 2. Umbilical cord blood stem cells can differentiate into hepatocyte-like cells under the induction of cell growth factor. 3. Umbilical cord blood is expected to be an effective stem cell source for the treatment of severe liver diseases.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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