人髓核间充质干细胞的分离提纯方式及生物学活性鉴定

Purification and identification of human nucleus pulposus mesenchymal stem cells

中文关键词:  髓核间充质干细胞  流式细胞分选  分离提纯  生物学活性

英文关键词:Nucleus pulposus mesenchymal stem cells  Fluorescence activated cell sorting  Purification  Biological activity

中文摘要:

  【摘要】 目的：探索人髓核间充质干细胞（nucleus pulposus mesenchymal stem cells，NPMSCs）的提纯方法并鉴定其生物学活性。方法：收集3例腰椎间盘突出症患者的退变髓核组织（Pfirrmann分级均为Ⅳ级），利用酶消化法分离细胞。采用两种方法分离提纯NPMSCs，一组细胞采用贴壁法培养（贴壁组），另一组通过流式细胞分选技术利用NPMSCs表面阳性标志物CD73、CD90、CD105获得NPMSCs（流式组）。将两种方法获得的NPMSCs进行体外培养扩增，分别进行形态学观察，细胞计数试剂盒（Cell Counting Kit，CCK-8）检测增殖能力。贴壁组NPMSCs采用流式细胞分选仪在进行分选之前检测免疫表型，流式组NPMSCs在生长达80%~90%融合时进行免疫表型的检测。向成骨、成脂、成软骨诱导分化，诱导28d后分别进行茜素红染色观察其成骨能力、油红O染色观察其成脂能力、甲苯胺蓝染色观察其成软骨能力，利用Imag J软件计算染色区域所占的面积百分比。比较两组NPMSCs在形态学、免疫表型及增殖和分化能力的差异。结果：形态学观察发现，两组NPMSCs均呈漩涡状生长，贴壁组NPMSCs可见散在的单个细胞生长；流式组NPMSCs长梭形形态更长，排列更加紧密，少见散在的单个贴壁生长细胞。流式细胞分选后所得的NPMSCs占细胞总数的（89.67±2.52）%，可以进行体外培养扩增，细胞为典型的长梭形特征，漩涡状生长，在接种后12~15d达80%~90%融合，增殖能力在接种后5~13d明显高于贴壁组NPMSCs（P<0.05）。流式组NPMSCs的CD73、CD90、CD105的表达率明显高于贴壁组NPMSCs（P<0.05），并且低表达CD34、CD45及HLA-DR。两种方法获得的NPMSCs均能完成三系诱导分化，流式组成骨、成脂、成软骨染色区域百分比均明显高于贴壁组（P<0.05）。结论：利用流式细胞分选技术从人退变髓核组织中可获得较高纯度的NPMSCs，并能进行后续培养扩增。与贴壁法获得的NPMSCs相比，流式细胞分选的NPMSCs具有更强的增殖与分化能力。

英文摘要:

  【Abstract】 Objectives: To explore a method for purifying human nucleus pulposus mesenchymal stem cells(NPMSCs), and to identify its biological activity. Methods: Degenerative nucleus pulposus tissue(Pfirrmann Ⅳ) from 3 patients with lumbar disc heriniation was collected, cells were isolated from human nucleus pulposus tissue by using enzyme digestion. Adherent method and fluorescence activated cell sorting(FACS) method were used for purifying NPMSCs, NPMSCs were obtained by FACS accoding to the expression of surface markers including CD73, CD90 and CD105. Then, the obtained NPMSCs by two methods were cultured in vitro and the cellular morphology was observed under the microscope, cell proliferation tests were performed by using Cell Counting Kit-8(CCK-8). Immunophenotyping of NPMSCs of adherence group was detected by FACS, and NPMSCs of FACS group were detected after the fusion rate reaching 80%-90%. Differentiated potential of NPMSCs was investigated by multiple differentiation(Osteogenic, chondrogenic and adipogenic). Alizarin red staining was performed to observe the osteogenesis potential, oil red O staining was used to observe the adipogenic potential, toluidine blue staining was used to observe the chondrogenic potential. Imag J software was used to test the stained area percentage of differentiated NPMSCs under two methods. NPMSCs of both groups were compared. Results: NPMSCs of both groups showed spindle shape, single cell was observed in the adherence group. NPMSCs of FACS group showed longer shuttle and rare single adherent cell. After FACS, the ratio of CD73+, CD90+, CD105+ NPMSCs from NPMSCs reached(89.67±2.52)%, typically long spindle shape could be observed, 80%-90% confluence was reached after 12-15 days. Expressons of CD73, CD90 and CD105 of FACS were higher than those of adherent method(P<0.05), more than adherent method 5 days later(P<0.05). The multiple differentiation potentials(Osteogenic, chondrogenic and adipogenic) of NPMSCs by FACS were superior than those of adherent method(P<0.05). Conclusions: Large proportion of NPMSCs can be obtained through FACS, which provides a reliable cell separation and purification method. Compared with method of adherence, NPMSCs obtained by FACS show better proliferation and multiple differentiation potential compared with adherent method.