【摘要】： 目的:在體外分別采用有細胞因子的培養(yǎng)體系和無細胞因子的培養(yǎng)體系,探討人臍血干細胞是否可以在體外培養(yǎng)條件下誘導(dǎo)分化為肝細胞樣細胞,觀察肝細胞標志物的表達,以期為治療肝臟疾病提供了新的細胞來源。 方法:常規(guī)無菌采集新鮮臍血,采用6%羥乙基淀粉、淋巴細胞分離液分離臍血(umbilical cord blood,UCB)的單個核細胞(mononuclear cells,MNCs)。MNCs分組培養(yǎng),對照組不加細胞因子,實驗組加入重組人肝細胞生長因子(hepatocyte growth factor,HGF)、重組人成纖維生長因子4(fibroblast growth factor-4,FGF-4)和重組人制瘤素(oncostatin M,OSM),濃度分別為20μg/L、10μg/L、10μg/L。并于誘導(dǎo)前及誘導(dǎo)后的第7,14,21,28d采用免疫細胞化學(xué)染色檢測甲胎蛋白(alpha fetoprotein,AFP)、細胞角蛋白18(cytokeratin 18,CK18)、白蛋白(albumin ,ALB)和肝細胞抗原的表達,用PAS(periodic acid-schiff)法進行糖原染色,流式細胞儀(flow cytometry,FCM)檢測細胞中ALB+表達,采用間接免疫熒光方法分析各組細胞ALB的表達情況,并用放射免疫法(radioimmunoassay,RIA)檢測細胞培養(yǎng)液中AFP的分泌水平,分析是否誘導(dǎo)出肝細胞樣細胞。 結(jié)果: 1.實驗組誘導(dǎo)后7d的細胞免疫組化顯示AFP染色陽性,21d后基本為陰性;第14d檢測到CK18、ALB呈陽性表達,第21d檢測到肝細胞抗原表達,隨著誘導(dǎo)時間的延長,陽性率逐漸增高。對照組免疫組化未見陽性表達。 2.實驗組培養(yǎng)第21d時檢測到糖原染色陽性細胞,28d時呈強陽性表達。對照組培養(yǎng)的MNCs PAS染色呈陰性。 3.實驗組FCM檢測顯示第7d幾乎未檢測到ALB+細胞,隨著誘導(dǎo)培養(yǎng)時間的延長,ALB+細胞表達增加,呈上升趨勢;培養(yǎng)至第28d的ALB+細胞達80%以上。對照組未檢測到ALB+細胞。 4.免疫熒光結(jié)果顯示,實驗組誘導(dǎo)14d后檢測到ALB陽性表達,隨誘導(dǎo)時間的延長,ALB仍呈陽性表達;對照組未檢測到ALB的表達。 5.實驗組RIA檢測結(jié)果表明第7dAFP水平最高,隨著培養(yǎng)時間的延長呈逐步下降趨勢;對照組在0,7,14,21,28d可檢測到低水平的AFP表達,其AFP值無明顯差異。 結(jié)論: 1.在無細胞生長因子的作用下,臍血干細胞不能夠分化為肝細胞樣細胞。 2.在有細胞生長因子誘導(dǎo)下,臍血干細胞能夠分化為肝細胞樣細胞。 3.臍血有望成為治療重癥肝病的一種有效的干細胞來源。
[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 偽-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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R329

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