【摘要】：目的：探討臍血來源間充質干細胞在纖維蛋白凝塊上分化為膀胱尿路上皮細胞的可能性。 內容：實驗組,將臍血間充質干細胞接種于人工制備的臍血來源的纖維蛋白凝塊上與胎兒膀胱尿路上皮細胞共培養(yǎng)。對照組,臍血間充質干細胞接種于人工制備的臍血來源的纖維蛋白凝膠上僅用DMEM/F12培養(yǎng)基培養(yǎng)。倒置相差顯微鏡觀察臍血間充質干細胞、胎兒膀胱尿路上皮細胞的形態(tài),透射電子顯微鏡下觀察鑒定尿路上皮,免疫組織化學染色觀察細胞廣譜角蛋白胞漿表達。 方法：1.采用密度梯度離心從胎兒的臍帶血中分離提取間充質干細胞,采用貼壁篩選培養(yǎng)法進行培養(yǎng)和純化UCB-MSCS。 2.取生長良好的第三代UCB-MSCs,通過流式細胞儀檢測細胞表面抗原標記(CD44, CD90CD34, CD45)進行細胞檢測。 3.利用膠原酶消化法分離膀胱尿路上皮細胞,用透射電鏡和免疫組織化學染色細胞廣譜角蛋白鑒定細胞。 4.采用加入氯化鈣溶液的方法用臍血的血漿,制備纖維蛋白凝塊。 5.將-定密度的臍血間充質干細胞接種于纖維蛋白凝塊表面,使其生長。 6.應用共培養(yǎng)技術將接種有UCB-MSCs的纖維蛋白凝塊與膀胱尿路上皮細胞共培養(yǎng),通過體外微環(huán)境誘導UCB-MSCs,收集誘導14天后的纖維蛋白凝塊上的UCB-MSCs進行透射電鏡和免疫組織化學染色細胞廣譜角蛋白鑒定細胞。 結果：1.采用密度梯度離心從胎兒的臍帶血中分離提取間充質干細胞,經過傳代培養(yǎng)得到純化,第三代細胞大部分呈長梭形,排列規(guī)則,呈水草樣狀。流式細胞儀檢測細胞表面標志物CD44, CD90表達陽性,而造血干細胞和內皮細胞的標記CD34, CD45表達陰性,這表明細胞為間充質干細胞。 2.利用膠原酶消化法分離得到的膀胱尿路上皮細胞,可以進行傳代培養(yǎng),通過免疫細胞化學染色細胞廣譜角蛋白陽性和透射電鏡兩種方法來鑒定,結果表明細胞為膀胱尿路上皮細胞。 3.應用共培養(yǎng)技術將接種有UCB-MSCs的纖維蛋白凝塊與膀胱尿路上皮細胞共培養(yǎng)：纖維蛋白凝塊上臍血間充質干細胞逐漸伸展呈多角形,細胞扁平變大,透射電子顯微鏡下具有膀胱尿路上皮細胞的超微結構,抗廣譜角蛋白(AE1/AE3)免疫組織化學染色陽性。 結論：1.體外成功從胎兒臍帶血中分離培養(yǎng)得到純度較高的MSCs,且該細胞具有干細胞的自我更新和多向分化潛能的特點。 2.體外成功的從引產胎兒膀胱中分離、培養(yǎng)獲得完全純化的膀胱尿路上皮細胞,為共培養(yǎng)提供了細胞微環(huán)境。 3.體外利用共培養(yǎng)的方法將纖維蛋白凝塊上誘導臍血間充質干細胞分化為膀胱尿路上皮細胞,為膀胱組織工程提供了可以使用種子細胞和細胞支架。
[Abstract]:Objective: to investigate the possibility of umbilical cord blood derived mesenchymal stem cells (MSCs) differentiated into bladder urothelial cells on fibrin clot. Content: in the experimental group, umbilical cord blood mesenchymal stem cells were co-cultured with fetal bladder urothelial cells on the artificial cord blood-derived fibrin clot. In the control group, umbilical cord blood mesenchymal stem cells were inoculated on cord blood-derived fibrin gel and cultured only in DMEM/F12 medium. The morphology of umbilical cord blood mesenchymal stem cells and fetal bladder urothelial cells was observed by inverted phase contrast microscope. The urothelial cells were identified by transmission electron microscope. The cytoplasmic expression of broad-spectrum keratin was observed by immunohistochemical staining. Methods: 1. Mesenchymal stem cells were isolated from fetal umbilical cord blood by density gradient centrifugation. UCB-MSCS. was cultured and purified by adherent culture. 2. The cell surface antigen labeling (CD44, CD90CD34, CD45) of the third generation UCB-MSCs, was detected by flow cytometry. 3. Bladder urothelial cells were isolated by collagenase digestion and identified by transmission electron microscopy (TEM) and immunohistochemical staining for broad-spectrum keratin. 4. Fibrin clot was prepared by adding calcium chloride solution to cord blood plasma. 5. Umbilical cord blood mesenchymal stem cells of constant density were inoculated on the surface of fibrin clot to make it grow. 6. The fibrin clot inoculated with UCB-MSCs was co-cultured with bladder urothelial cells by co-culture technique, and UCB-MSCs, was induced by microenvironment in vitro. UCB-MSCs was collected from fibrin clot 14 days after induction and the cells were identified by transmission electron microscopy (TEM) and immunohistochemical staining for broad-spectrum keratin. Results: 1. Mesenchymal stem cells were isolated from fetal umbilical cord blood by density gradient centrifugation and purified by subculture. Most of the third generation cells were in the shape of long fusiform, regular arrangement and water grass-like. The positive expression of CD44, CD90 was detected by flow cytometry, but the expression of CD34, CD45 was negative in hematopoietic stem cells and endothelial cells, which indicated that the cells were mesenchymal stem cells. 2. The bladder urothelial cells isolated by collagenase digestion can be subcultured and identified by immunocytochemical staining for broad-spectrum keratin positive cells and transmission electron microscopy. The results showed that the cells were bladder urothelial cells. 3. Co-culture technique was used to co-culture the fibrin clot inoculated with UCB-MSCs with bladder urothelial cells. The mesenchymal stem cells from umbilical cord blood on fibrin clot gradually extended in polygonal shape, and the cells became flattened and enlarged. The ultrastructure of bladder urothelial cells was observed under transmission electron microscope (TEM), and anti-broad-spectrum keratin (AE1/AE3) immunohistochemical staining was positive. Conclusion: 1. MSCs, with high purity was successfully isolated and cultured from fetal umbilical cord blood in vitro, and the cells had the characteristics of self-renewal and multidirectional differentiation potential of stem cells. 2. The fully purified bladder urothelial cells were successfully isolated from fetal bladder induced labor in vitro, which provided a cell microenvironment for co-culture. 3. Human umbilical cord blood mesenchymal stem cells were induced to differentiate into bladder urothelial cells by co-culture in vitro, which provided seed cells and cell scaffolds for bladder tissue engineering.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R363

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