半月板碎片来源间充质干细胞样细胞的分离培养及多向分化潜能

发布时间：2019-05-16 23:46

【摘要】：目的:探讨半月板碎片来源间充质干细胞(MSC)样细胞的分离培养和多向分化潜能。方法:通过酶消化的方法分离半月板碎片来源细胞,体外培养至第3代,显微镜下观察其形态和生长状态。取第3代细胞并对其进行向成骨、成脂和成软骨诱导分化。结果:经酶消化的方法分离半月板碎片来源细胞4~6天可见少量的长梭形贴壁,10~14天细胞成簇生长,克隆集落形成。第3代细胞形态较均一,生长增殖良好。成骨诱导14天后ALP和茜素红染色阳性,有矿化结节形成。成脂诱导14天后油红O染色阳性提示细胞内有脂滴形成。采用细胞微团无血清成软骨诱导21天后甲苯胺蓝染色呈阳性,细胞外有蛋白多糖形成;免疫组织化学染色示特异软骨基质Ⅱ型胶原表达阳性。结论:采用混合胶原酶消化的方法分离获得的半月板碎片来源的细胞贴壁生长,呈典型的长梭形的MSC样细胞形态,在体外适当的诱导条件下具有分化成骨、成脂、成软骨的潜能,有望成为半月板再生的种子细胞来源。
[Abstract]:Aim: to investigate the isolation, culture and multidirectional differentiation potential of (MSC)-like cells derived from meniscus fragments. Methods: the cells from meniscus fragments were isolated by enzyme digestion and cultured to the third generation in vitro. the morphology and growth state of meniscus fragments were observed under microscope. The third passage cells were taken and induced to osteogenic, adipogenic and cartilage-forming. Results: a small amount of long fusiform adherent cells were isolated from meniscus fragments by enzyme digestion for 6 days, and the cells grew in clusters at 10 to 14 days, and clone colonies were formed. The morphology of the third passage cells was uniform and the growth and proliferation of the cells were good. After 14 days of osteogenic induction, ALP and alizalin red staining were positive and mineralization nodules were formed. After 14 days of adipogenic induction, the positive staining of oil red O suggested the formation of lipid droplets in the cells. After 21 days of induction with serum-free cartilage formation, toluene blue staining was positive and proteoglycan was formed outside the cells, and the expression of type II collagen in specific cartilage matrix was positive by immunohistochemical staining. Conclusion: the cells from meniscus fragments isolated by mixed collagenase digestion showed typical long fusiform MSC-like cell morphology and differentiated into bone and fat under appropriate induction conditions in vitro. The potential of cartilage formation is expected to be a source of seed cells for meniscus regeneration.
【作者单位】：四川大学华西医院骨科;
【基金】：国家自然科学基金(81301560) 中国博士后科学基金(2012M521698)
【分类号】：R87

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