厚涂型防火涂料个人简介 (40)
摘要
目的:扫把头
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；建立增强型绿荧光蛋白(enhanced Green Fluorescent Protein，eGFP)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eGFP-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eGFP标记阳性率；eGFP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
观察SPIO 标记结果。采用Alcian Blue、碱性磷酸酶、油红―O‖染检测双标记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eGFP/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。番茄加速
2、eGFP成功标记SMSCs后，荧光显微镜下可见细胞内出现绿荧光，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eGFP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MOI>100时，细胞形态出现改变。
3、以50μg/ml 的SPIO标记经筛选后的eGFP细胞，经普鲁士蓝染，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。
聚酰亚胺板材4、双标记SMSCs经成软骨细胞定向诱导21天后，诱导细胞Alcian Blue染结果显示：细胞外基质明
显染成蓝；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‖染结果显示：细胞内出现明显的红颗粒状脂滴。
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（P>0.05），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。棱镜片
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、eGFP/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。
关键词：滑膜间充质干细胞；组织块培养法；eGFP；超顺磁性氧化铁纳米颗粒
The Study on Isolation, Culture and the Cell Labeling of Rabbit Synovial Mesenchymal Stem Cells in vitro
Abstract
Objective：
To investigate the feasibility of isolation, purification, expansion rabbit synovial mesenchymal stem cells (SMSCs) with the method of explant culture, and study the multi-lineage potential in vitro; establish the enhanced Green Fluorescent Protein(eGFP) and the superparamagnetic iron oxide nanoparticles(SPIO) double labeling technique to provide an efficient tracer methods for imaging SMSCs repair articular cartilage in the intra-articular.
Methods：
SMSCs were isolated from rabbit knee fat pad synovial tissue through the method of explant culture, and purified by the limiting dilution method in sterile, then proliferation in vitro; Morphology and growth characteristics was examined by electron microscopy; Best Multiplicity Of Infection（MOI) of eGFP- lentiviral infected the cells in logarithmic growth phase, the infection efficiency were analyzed by both fluorescent microscope and flow cytometry; eGFP labeled cells fully screened by puromycin, then labeled with superparamagnetic iron oxide nanoparticles(SPIO), and the mark result were observed by Prussian blue staining and transmission electron microscope. Double- labeled SMSCs
differentiated into chondrocytes, osteoblast and dipocytes. And the differentiated cells were identified by alcian blue staining, alkaline phosphatase staining, oil red O staining, respectively. Lastly, the cell activity and proliferation ability of the dual-labeled SMSCs were evaluated by cck-8.
Results：
1. Rabbit primitive synovial cells began to appear around the synovial tissue with the method of explant culture 2-3 days, and after 10-12 days later large scale we will harvest, cell morphology became more homogenized by the limiting dilution method in vitro.
2.Visible green fluorescence could see after SMSCs GFP labeled under the fluorescent microscope, and fluorescence expression rate gradually strengthen with the
increases MOI; Over (38.4±2.7)% SMSCs were eGFP-positive at 3days after transfection by lentivirus at MOI of 100, and could get above 95% eGFP-positive by screening with puromycin;After 30d, fluorescent labeled cells still remained stable expression.
3. The Prussian blue staining showed that the eGFP-positive purified SMSCs got above （98.4±1.2）% marker efficiency after SPIO labeled by electron microscope, and considerable high
electron density particles could be observed in the cytoplasm and Pinopodes through the transmission electron microscope, but the cells of the control group were negative.
4. After chondrocytes induction 21 days later, alcian blue staining showed that extracellular matrix stained blue; Alkaline phosphatase staining showed that alkaline phosphatase was positive after osteogenetic induction; Oil red O staining showed that red lipid droplet existed in cells after dipocytes induction.
5．CCK-8 test result：eGFP/SPIO double-labeled SMSCs still remained good activity and proliferative state, showed no significant difference with unlabeled group (P> 0.05), cell macker did not produce significant cytotoxicity.
Conclusions：
1. the explant culture method can efficiently obtain primary SMSCs with simple operation and little probability of cell contamination.
2. SMSCs which we separated and purified in this study have good potentiality of proliferation.
3. eGFP / SPIO double labeling technique will provide an high efficient and feasible tracer method fo
r imaging SMSCs repair articular cartilage in the intra-articular.And the labeled cells still has potential to induce differentiation, did not appear significant cytotoxicity,
Keywords: Synovial mesenchymal stem cells;Explant culture;eGFP; Superparamagnetic iron oxide蝶形螺栓
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