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目的体外分离培养猪滑膜源性MSCs(synovium-derived MSCs,SMSCs),探讨其在体外多向分化潜能。方法2月龄长枫杂交猪3头,雌雄不限,体重8～10kg。取猪膝关节滑膜组织分离SMSCs并行原代及传代培养,待第3代SMSCs长满培养皿后,吸去基础培养液,分别加入特定培养液进行成软骨、成骨、成脂诱导分化,作为实验组;以基础培养液培养作为对照组。成软骨诱导21d后行甲苯胺蓝染色、免疫组织化学染色和实时荧光定量PCR检测;成骨诱导10d后行ALP染色检测,21d后行茜素红染色检测;成脂诱导21d后行油红O染色检测。结果SMSCs培养24h后细胞形态细长或呈多角形;48h后细胞数量增多;72h后可见大量纺锤形细胞,其间散在分布少许小圆形细胞。实验组成软骨诱导21d后甲苯胺蓝染色呈阳性,细胞外有蛋白多糖(Aggrecan)形成;免疫组织化学染色示特异软骨基质ColⅡ表达;实时荧光定量PCR检测示ColⅡA1、Aggrecan、SOX9 mRNA表达量与对照组比较,差异有统计学意义(P<0.05)。成骨诱导10d后ALP染色阳性,21d后茜素红染色阳性,有钙结节形成。成脂诱导21d后油红O染色示细胞内有脂滴形成。对照组除ALP染色观察呈弱阳性外,余染色均呈阴性。结论猪膝关节滑膜组织可分离获取SMSCs,其在体外具有向成软骨、成骨、成脂多向分化潜能,有望成为组织工程种子细胞来源。
Objective To isolate and culture synovium-derived MSCs (SMSCs) in vitro and investigate their potential of multidrug differentiation in vitro. Methods 2-month-old Fengfeng crossbred pigs 3, male or female, body weight 8 ~ 10kg. The primary and subcultures of SMSCs were isolated from synovial tissue of swine and knee joints. After the third generation of SMSCs were covered with culture dishes, the basal culture fluid was aspirated, and the specific culture medium was added for cartilage, osteogenic and adipogenic differentiation, As the experimental group; basic culture medium as a control group. Toluidine blue staining, immunohistochemical staining and real-time fluorescent quantitative PCR were used to detect the cartilage formation 21 days after osteogenic induction. ALP staining was performed 10 days after osteogenic induction, and alizarin red staining was performed 21 days after osteogenic induction. Oil red O Staining test. Results After cultured for 24 hours, the cells were slender or polygonal in shape. After 48 hours, the number of cells increased. After 72 hours, a large number of spindle cells were scattered, and scattered small round cells. After 21 days of cartilage induction, toluidine blue staining was positive and extracellular proteoglycan (Aggrecan) was formed. The expression of ColⅡ was detected by immunohistochemical staining. The expression of ColⅡA1, Aggrecan and SOX9 mRNA was detected by real-time fluorescence quantitative PCR The difference was statistically significant (P <0.05). ALP staining was positive 10 days after osteogenic induction. Alizarin red staining was positive 21 days later with calcium nodule formation. Lipid red staining after 21 days of adipogenic induction showed the formation of lipid droplets in the cells. The control group except ALP staining showed weak positive, the residual staining were negative. Conclusions SMSCs can be isolated from the synovial tissue of swine and knee joint and have the potential to differentiate into chondrogenic, osteogenic and adipogenic cells in vitro. It is expected to be the source of tissue engineering seed cells.