体外诱导猪骨髓间充质干细胞向膀胱平滑肌细胞和尿路上皮细胞分化的实验研究
发布时间：2018-11-05 14:26
【摘要】：[目的]体外获得培养、扩增及鉴定滇南小耳猪骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)的方法,为组织工程膀胱的构建提供一种新的种子细胞来源。 [方法]用骨穿针抽取滇南小耳猪的骨髓液,采用骨髓液离心加贴壁培养法培养。观察细胞形态、生长及扩增情况,并采用MTT法绘制生长曲线,流式细胞仪检测第一代、第二代BMSCs的表面标志物CD29、CD44、CD45。 [结果]细胞形态均为梭形生长,呈成纤维细胞样形态。流式细胞检测结果：P1CD29阳性率为99.44%,P1CD44阳性率为99.96%,P1CD45阴性率为99.55%。P2CD29阳性率为100%,P2CD44阳性率为100%,P2CD45阴性率为99.89%。 [结论]采用骨髓液离心加贴壁法能有效地培养及扩增出滇南小耳猪BMSCs。通过流式细胞检测,对第一代及第二代BMSCs阳性率的比较,第二代的BMSCs纯度高,可作为种子细胞的来源。 [目的]探讨猪膀胱平滑肌组织和尿路上皮组织在体外原代培养、扩增及鉴定的方法,为构建组织工程膀胱提供种子细胞来源。 [方法]在无菌条件下通过外科手术切取滇南小耳猪膀胱组织,采用机械分离法分离出膀胱粘膜层和膀胱平滑肌层,用胰蛋白酶和胶原酶I联合消化膀胱平滑肌层获取膀胱平滑肌细胞,用中性蛋白酶4℃冷消化膀胱组织,机械分离法获取获取膀胱粘膜层,酶消化法获取尿路上皮细胞。用含10%FBS的DMEM/F12培养基培养膀胱平滑肌细胞,DK-SFM(含表皮生长因子)培养基培养尿路上皮细胞,再分别观察两者细胞的形态变化,生长及增殖过程,并通过H-E染色和免疫组织化学染色鉴定细胞。 [结果]平滑肌细胞原代培养12h后可见大量细胞贴壁,培养2-3天后细胞融合达90%以上。尿路上皮细胞原代培养18h后可见大量细胞贴壁并发生形态改变,原代细胞培养4-5天后,融合至80%-90%。在倒置相差显微镜下观察平滑肌细胞为长梭形,并且表现出典型的“谷和峰”形态,尿路上皮细胞表现出典型的“铺路石”样结构。通过苏木素—伊红染色观察其符合平滑肌细胞和尿路上皮细胞形态特征,分别用平滑肌肌动蛋白抗体和角蛋白AE1/AE3单克隆抗体鉴定两者细胞结果都呈阳性。 [结论]采用该方法能够有效地建立起体外培养猪膀胱平滑肌细胞和尿路上皮细胞的培养体系：所培养出来的细胞纯度高、活力好,可作为构建组织工程膀胱的种子细胞。 [目的]探讨体外诱导骨髓间充质干细胞(BMSCs)向膀胱平滑肌细胞和尿路上皮细胞分化的方法,为组织工程膀胱的建立提供一种新的种子细胞来源。 [方法]选取稳定培养系中的第三或四代BMSCs、尿路上皮细胞和平滑肌细胞。按照模拟机体内环境的方法,将实验分成实验组和对照组。实验组：A组：BMSCs+尿路上皮细胞+表皮细胞生长因子B：BMSCs+尿路上皮细胞条件培养液+表皮细胞生长因子.C组:BMSCs+平滑肌细胞+碱性成纤维生长因子.D组：BMSCs+平滑肌细胞条件培养液+碱性成纤维生长因子.对照组：E:BMSCs+表皮细胞生长因子.F组：BMSCs+碱性成纤维生长因子.G组：BMSCs2ml。共培养诱导的时间为10天左右,分别于3、5、7、10天观察细胞形态的变化并记录下来。用平滑肌肌动蛋白抗体和角蛋白AE1/AE3单克隆抗体鉴定实验组和对照组。 [结果]实验组于5天左右开始出现细胞形态的变化,7天后细胞形态明显改变,10天后90%以上的BMSCs向诱导分化的目的细胞形态转变。对照组细胞形态变化不明显。角蛋白AE1/AE3单克隆抗体鉴定实验组A、B组和对照组E、G组,平滑肌肌动蛋白抗体鉴定实验组C、D组和对照组F、G组,实验组结果均为阳性,对照组结果均为阴性。 [结论]骨髓间充质千细胞具有多向分化的能力,在模拟体内的微环境的条件下能诱导BMSCs向尿路上皮细胞和平滑肌细胞的分化.可为组织工程膀胱的建立提供一种新的种子细胞来源。
[Abstract]:[Objective] To obtain the method of culture, amplification and identification of bone marrow mesenchymal stem cells (BMSCs) in small ear pigs in southern Yunnan to provide a new source of seed cells for the construction of bladder.[Methods] Bone marrow fluid from the small ear pig of southern Yunnan was extracted with bone penetrating needle, and the bone marrow liquid was used to centrifuge the bone marrow liquid. The cell morphology, growth and amplification were observed, and the growth curve was drawn by MTT method. The surface markers CD29, CD44, CD of the first generation and the second generation BMSCs were detected by flow cytometry. 45.[Results] The morphology of the cells was shuttle-like growth, which was in the form of fibers. The positive rate of PCD29 was 99. 96%, the positive rate of PCD45 was 99. 55%, the positive rate of P2CD29 was 100%, the positive rate of P2CD44 was 100%, and the negative rate of P2CD45 was 9. 9. 89%.[Conclusion] Bone marrow fluid centrifugation and adherence method can be used to effectively cultivate and amplify the southern Yunnan province. BMSCs were detected by flow cytometry. Compared with the first generation and the second generation BMSCs, the second generation BMSCs were high in purity and could be used as the second generation BMSCs. Objective: To investigate the methods of primary culture, amplification and identification of porcine bladder smooth muscle tissue and upper urinary tract epithelial cells in vitro. The bladder tissue of the small ear pig of southern Yunnan was cut off by surgical technique under aseptic conditions. The bladder mucosa and bladder smooth muscle layer were separated by mechanical separation, and digested with trypsin and collagenase I. Bladder smooth muscle cells were obtained from smooth muscle layer, bladder tissue was digested with neutral protease at 4 鈩，
本文编号：2312361
[Abstract]:[Objective] To obtain the method of culture, amplification and identification of bone marrow mesenchymal stem cells (BMSCs) in small ear pigs in southern Yunnan to provide a new source of seed cells for the construction of bladder.[Methods] Bone marrow fluid from the small ear pig of southern Yunnan was extracted with bone penetrating needle, and the bone marrow liquid was used to centrifuge the bone marrow liquid. The cell morphology, growth and amplification were observed, and the growth curve was drawn by MTT method. The surface markers CD29, CD44, CD of the first generation and the second generation BMSCs were detected by flow cytometry. 45.[Results] The morphology of the cells was shuttle-like growth, which was in the form of fibers. The positive rate of PCD29 was 99. 96%, the positive rate of PCD45 was 99. 55%, the positive rate of P2CD29 was 100%, the positive rate of P2CD44 was 100%, and the negative rate of P2CD45 was 9. 9. 89%.[Conclusion] Bone marrow fluid centrifugation and adherence method can be used to effectively cultivate and amplify the southern Yunnan province. BMSCs were detected by flow cytometry. Compared with the first generation and the second generation BMSCs, the second generation BMSCs were high in purity and could be used as the second generation BMSCs. Objective: To investigate the methods of primary culture, amplification and identification of porcine bladder smooth muscle tissue and upper urinary tract epithelial cells in vitro. The bladder tissue of the small ear pig of southern Yunnan was cut off by surgical technique under aseptic conditions. The bladder mucosa and bladder smooth muscle layer were separated by mechanical separation, and digested with trypsin and collagenase I. Bladder smooth muscle cells were obtained from smooth muscle layer, bladder tissue was digested with neutral protease at 4 鈩，
本文编号：2312361
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