兔SMSCs的体外分离培养及细胞标记的实验研究

发布时间：2018-04-02 12:35

本文选题：滑膜间充质干细胞　切入点：组织块培养法　出处：《山西医科大学》2015年硕士论文

【摘要】：目的:探讨应用组织块培养法体外分离、纯化、扩增兔滑膜间充质干细胞(synovial mesenchymal stem cells,SMSCs)的可行性及其分离细胞的多向诱导分化潜能;建立增强型绿色荧光蛋白(enhanced Green Fluorescent Protein,e GFP)基因/超顺磁性氧化铁纳米颗粒(superparamagnetic iron oxide nanoparticles,SPIO)双标记兔滑膜间充质干细胞的技术,为解决SMSCs对关节内软骨损伤修复提供可靠的体内、外示踪方法。方法:无菌获取兔膝关节脂肪垫滑膜组织,通过组织块接种法分离原代SMSCs,联合有限稀释法体外纯化、扩增兔SMSCs;镜下观察SMSCs的细胞形态学及其生长特点;以最佳感染复数(Multiplicity Of Infection,MOI)的e GFP-慢病毒感染处于对数生长期的兔SMSCs,荧光显微镜下及流式细胞仪观察e GFP标记阳性率;e GFP标记SMSCs经嘌呤霉素充分筛选后行SPIO标记,透射电镜下及普鲁士蓝染色观察SPIO标记结果。采用Alcian Blue、碱性磷酸酶、油红 O‖染色检测双标记SMSCs分别在成软骨、成骨、成脂诱导液中的定向诱导分化情况。采用CCK8试剂检测e GFP/SPIO双标记后SMSCs的细胞增殖能力及其细胞活性。结果:1、兔脂肪垫表层滑膜组织经组织块接种法2~3天后,组织块周围即可开始长出原代滑膜细胞,10~12天便可获得大量滑膜细胞,经有限稀释法纯化扩增,SMSCs细胞形态更加均一。2、e GFP成功标记SMSCs后,荧光显微镜下可见细胞内出现绿色荧光,且荧光表达率随MOI值的增大而增高;当MOI值为100的慢病毒感染SMSCs时,细胞可获得(38.4±2.7)%的e GFP转染率,细胞仍保持良好的状态,经嘌呤霉素筛选后可达到95%以上的荧光表达率,标记30d之后,标记细胞荧光仍然稳定表达,当MOI100时,细胞形态出现改变。3、以50μg/ml的SPIO标记经筛选后的e GFP细胞,经普鲁士蓝染色,镜下可见细胞标记率达(98.4±1.2)%,透射电镜下观察显示SMSCs的细胞质和吞饮小泡内可见大量高电子致密度颗粒,而对照组细胞质内和吞饮小泡内未见明显高电子致密度颗粒。4、双标记SMSCs经成软骨细胞定向诱导21天后,诱导细胞Alcian Blue染色结果显示:细胞外基质明显染成蓝色;双标记SMSCs经成骨细胞定向诱导21天后,诱导细胞碱性磷酸酶染色显示:细胞内碱性磷酸酶表达阳性;双标记SMSCs经成脂肪细胞定向诱导14天后,诱导细胞油红 O‖染色结果显示:细胞内出现明显的红色颗粒状脂滴。5、CCK-8检测发现:SMSCs细胞双标记后仍保持良好的增殖状态,未标记组与标记组细胞比较无显著差异(P0.05),未产生明显细胞毒性。结论:1、组织块培养法能高效的获取原代SMSCs,并且操作简单,细胞污染几率小。2、分离、纯化后的SMSCs细胞具有良好的增殖能力,并具有明显的间充质干细胞的形态特性。3、e GFP/SPIO双标记SMSCs效率高,双标记后细胞仍具有多向诱导分化潜能,未产生明显细胞毒性,具有一定可行性,为SMSCs对关节内软骨损伤修复的示踪研究奠定了实验基础。
[Abstract]:Objective: to investigate the feasibility of isolating, purifying and amplifying synovial mesenchymal stem cells from rabbit synovial synovial cells by tissue mass culture in vitro and the potential of inducing differentiation of the cells. An enhanced Green Fluorescent protein (GFP) gene / superparamagnetic iron oxide nanoparticles-SPIOO (superparamagnetic iron oxide nanoparticles spIOO) double labeling technique for rabbit synovial mesenchymal stem cells was established to provide a reliable solution for repair of articular cartilage injury by SMSCs in vivo. Methods: the synovial tissue of rabbit knee joint fat pad was obtained by aseptic method. Primary SMSCs were isolated by tissue mass inoculation and purified in vitro with limited dilution method. The morphology and growth characteristics of SMSCs were observed under microscope. The positive rate of e GFP-lentivirus infection in logarithmic growth phase was observed by fluorescence microscope and flow cytometry. The positive rate of e GFP labeled SMSCs was detected by fluorescence microscope and flow cytometry. The SMSCs labeled with e GFP was fully screened by purine mycin and then labeled with SPIO, and the positive rate of e GFP-lentivirus infection was observed by fluorescence microscope and flow cytometry. The results of SPIO labeling were observed under transmission electron microscope and Prussian blue staining. The double labeled SMSCs was detected by Alcian Blue, alkaline phosphatase and oil red O O staining in cartilage formation and osteogenesis, respectively. CCK8 reagent was used to detect the proliferation ability and cell activity of SMSCs labeled with e GFP/SPIO. Results: 1. The synovial tissue of rabbit fat pad was inoculated with tissue mass for 3 days. A large number of synovial cells could be obtained after 10 ~ 12 days of primary synovial cell growth around the tissue block. After purification and amplification with a limited dilution method, the cells were more homogeneous. 2e GFP was successfully labeled with SMSCs, and green fluorescence could be seen in the cells under fluorescence microscope. The fluorescence expression rate increased with the increase of MOI value, when lentivirus with MOI value of 100 was infected with SMSCs, the transfection rate of e GFP was 38.4 卤2.7%, and the cell remained in good condition, and the fluorescence expression rate was over 95% after purine mycin screening. After 30 days of labeling, the fluorescence of the labeled cells was still stable. When MOI100 was observed, the morphology of the cells was changed. The e GFP cells were labeled with 50 渭 g/ml SPIO and stained with Prussian blue. Under microscope, the cell labeling rate was 98.4 卤1.20.The results of transmission electron microscopy showed that a large number of high electron density particles could be seen in the cytoplasm of SMSCs and in the vesicles of swallowing and drinking. However, in the control group, no high electron density granules were found in the cytoplasm of the control group and in the vesicles of swallowing drink. The double labeled SMSCs was induced by chondroblastoblast for 21 days. The results of Alcian Blue staining showed that the extracellular matrix was blue obviously. After 21 days of osteoblast induction with double labeled SMSCs, alkaline phosphatase expression in induced cells was positive, while double labeled SMSCs was induced by adipoblast for 14 days. The results showed that the cells were in a good proliferative state after double labeling. The results showed that there were obvious red granular lipid droplets. 5 CCK-8 assay showed that the cells were still in a good proliferative state after double labeling. There was no significant difference between the unlabeled group and the labeled group (P 0.05) and no obvious cytotoxicity. Conclusion: 1: 1, the primary SMSCs can be obtained efficiently by the tissue mass culture method, and the operation is simple, the cell contamination rate is small, and the cell is isolated. The purified SMSCs cells have good proliferative ability, and have obvious morphological characteristics of mesenchymal stem cells. 3e GFP/SPIO double labeled SMSCs has high efficiency. After double labeling, the cells still have multidirectional differentiation potential and no obvious cytotoxicity. It has certain feasibility, which lays an experimental foundation for the tracer study of intraarticular cartilage repair by SMSCs.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R684

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