人脐带非酶解法培养MSCs的方法建立

发布时间：2018-05-25 23:33

本文选题：间充质干细胞 + 人脐带　；参考：《中国输血杂志》2017年08期

【摘要】：目的建立从人脐带Wharton's jelly组织中直接培养间充质干细胞(MSCs)的方法。方法将人脐带切成5 cm左右的片段,纵向剖开,剔除血管,把富含Wharton's jelly组织的脐带面置于10 cm2塑料培养皿的培养面上,加入含20%FBS的LG-DMEM培养基连续培养10 d;移去脐带组织后,培养皿中的细胞继续培养至80%-90%融合。传代和扩增3代(次)后,以倒置光学显微镜观察细胞形态,流式细胞仪检测细胞免疫表型及细胞周期,用含有成骨细胞诱导剂、脂肪细胞诱导剂对传代培养至第3代的细胞分别定向诱导分化为成骨细胞、脂肪细胞,并对诱导后细胞用碱性磷酸酶检测试剂、Von-Kossa染色检测试剂和茜苏红染色试剂作细胞生物学检测。结果脐带组织在贴壁培养5 d,组织周围可见有少量细胞贴壁生长,主要呈"成纤维样",形状不规则,移去脐带组织后继续培养至14-15 d时,细胞达到80%-90%汇合;细胞表面表达CD73、CD105、CD90、CD44、CD29、CD71及CD13,不表达CD34、CD14、CD133、CD45、HLA-DR;培养至第4代时约72.724%的细胞处在G1期,S期细胞占18.069%,第6代时,G1期细胞约为83.875%、S期细胞仅为9.606%左右。细胞经成骨细胞诱导分化后,碱性磷酸酶染色显示呈强阳性,茜苏红染色和Von-Kossa染色显示细胞有钙盐沉积并形成钙结节;细胞经成脂细胞诱导分化后,油红O染色呈红色,显示胞浆内有大量甘油三酯的聚集。结论采用非酶解法从人脐带Wharton's jelly中分离及培养扩增的细胞具备MSCs的基本特性,具有分化为成骨细胞和脂肪细胞的能力。
[Abstract]:Objective to establish a direct culture method of mesenchymal stem cells (MSCs) from human umbilical cord Wharton's jelly tissue. Methods the human umbilical cord was cut into about 5 cm segment, cut out longitudinally, removed the blood vessels, placed the cord surface rich in Wharton's jelly tissue on the culture surface of 10 cm2 plastic petri dish, added 20s LG-DMEM medium for 10 days, and removed the umbilical cord tissue for 10 days. The cells in the dish continued to grow to 80-90% fusion. Cell morphology was observed by inverted optical microscope, immunophenotype and cell cycle were detected by flow cytometry and osteoblast inducer was used. Adipocytes were induced to differentiate into osteoblasts and adipocytes respectively. After induction, the cells were detected by Von-Kossa staining reagent and hematoxylin staining reagent by alkaline phosphatase assay. Results after 5 days of culture, there were a few cells around the cord tissue, mainly "fibroblast" and irregular shape. When the umbilical cord tissue was removed and cultured for 14-15 days, the cells converged to 80-90%. The cell surface expressed CD73T, CD105CD90, CD44, CD29, CD71 and CD13, and did not express CD34, CD14, CD133, CD45 and HLA-DR.At the fourth passage, about 72.724% of the cells were in the G 1 phase of S phase, and the number of cells in the G 1 phase was about 9.606% in the sixth generation. After differentiation induced by osteoblasts, alkaline phosphatase staining showed strong positive, hematoxylin staining and Von-Kossa staining showed that the cells had calcium salt deposition and formed calcium nodules, oil red O staining was red after differentiation induced by adipogenic cells. There is a large accumulation of triglycerides in the cytoplasm. Conclusion the cells isolated and cultured from human umbilical cord Wharton's jelly by non-enzymatic hydrolysis have the basic characteristics of MSCs and the ability to differentiate into osteoblasts and adipocytes.
【作者单位】：安徽省血液中心;解放军第105医院;Global
【基金】：合肥市卫生计生委2016年应用医学研究项目(hwk2016zc020)
【分类号】：R329.2

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