在纤维蛋白凝块上诱导脐血间充质干细胞向膀胱尿路上皮细胞分化的研究
发布时间：2019-03-20 16:34
【摘要】：目的：探讨脐血来源间充质干细胞在纤维蛋白凝块上分化为膀胱尿路上皮细胞的可能性。 内容：实验组,将脐血间充质干细胞接种于人工制备的脐血来源的纤维蛋白凝块上与胎儿膀胱尿路上皮细胞共培养。对照组,脐血间充质干细胞接种于人工制备的脐血来源的纤维蛋白凝胶上仅用DMEM/F12培养基培养。倒置相差显微镜观察脐血间充质干细胞、胎儿膀胱尿路上皮细胞的形态,透射电子显微镜下观察鉴定尿路上皮,免疫组织化学染色观察细胞广谱角蛋白胞浆表达。 方法：1.采用密度梯度离心从胎儿的脐带血中分离提取间充质干细胞,采用贴壁筛选培养法进行培养和纯化UCB-MSCS。 2.取生长良好的第三代UCB-MSCs,通过流式细胞仪检测细胞表面抗原标记(CD44, CD90CD34, CD45)进行细胞检测。 3.利用胶原酶消化法分离膀胱尿路上皮细胞,用透射电镜和免疫组织化学染色细胞广谱角蛋白鉴定细胞。 4.采用加入氯化钙溶液的方法用脐血的血浆,制备纤维蛋白凝块。 5.将-定密度的脐血间充质干细胞接种于纤维蛋白凝块表面,使其生长。 6.应用共培养技术将接种有UCB-MSCs的纤维蛋白凝块与膀胱尿路上皮细胞共培养,通过体外微环境诱导UCB-MSCs,收集诱导14天后的纤维蛋白凝块上的UCB-MSCs进行透射电镜和免疫组织化学染色细胞广谱角蛋白鉴定细胞。 结果：1.采用密度梯度离心从胎儿的脐带血中分离提取间充质干细胞,经过传代培养得到纯化,第三代细胞大部分呈长梭形,排列规则,呈水草样状。流式细胞仪检测细胞表面标志物CD44, CD90表达阳性,而造血干细胞和内皮细胞的标记CD34, CD45表达阴性,这表明细胞为间充质干细胞。 2.利用胶原酶消化法分离得到的膀胱尿路上皮细胞,可以进行传代培养,通过免疫细胞化学染色细胞广谱角蛋白阳性和透射电镜两种方法来鉴定,结果表明细胞为膀胱尿路上皮细胞。 3.应用共培养技术将接种有UCB-MSCs的纤维蛋白凝块与膀胱尿路上皮细胞共培养：纤维蛋白凝块上脐血间充质干细胞逐渐伸展呈多角形,细胞扁平变大,透射电子显微镜下具有膀胱尿路上皮细胞的超微结构,抗广谱角蛋白(AE1/AE3)免疫组织化学染色阳性。 结论：1.体外成功从胎儿脐带血中分离培养得到纯度较高的MSCs,且该细胞具有干细胞的自我更新和多向分化潜能的特点。 2.体外成功的从引产胎儿膀胱中分离、培养获得完全纯化的膀胱尿路上皮细胞,为共培养提供了细胞微环境。 3.体外利用共培养的方法将纤维蛋白凝块上诱导脐血间充质干细胞分化为膀胱尿路上皮细胞,为膀胱组织工程提供了可以使用种子细胞和细胞支架。
[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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363
[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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363
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相关期刊论文 前10条
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