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目的观察人脐带Wharton胶细胞外基质(hWJECM)对兔软骨细胞增殖的影响。方法用差速离心法制作hWJECM,制成0.5%浓度浆料分别铺于六孔细胞培养板和96孔细胞培养板上,形成1 mm厚的薄膜,包被有此膜的培养板作为试验组,未铺膜单纯培养液组作为空白对照组,在培养板上培养兔关节软骨细胞。通过倒置显微镜观察,甲苯胺蓝染色观察两个组的兔软骨细胞5,10,15天的生长形态和增殖情况,用CCK8细胞增殖实验比较两组的1、3、5、7 d的增殖情况,来评估软骨种子细胞的增殖和表型维持情况。结果倒置显微镜观察5、10、15 d的细胞增殖情况好于单纯培养液组,5、15 d的甲苯胺蓝染色的结果也证明这个观点,CCK8细胞增殖试验1,3天时试验组和对照组的增殖没有明显的统计学差异(P=0.7142;P=0.0657),第5,7天显示hWJECM组在细胞增殖方面明显地优于单纯培养液的对照组(P=0.0001;P=0.0006)。结论 hWJECM免疫原性低,无细胞毒性作用,能够很好地促进软骨细胞的增殖。
Objective To observe the effects of human umbilical cord Wharton glue extracellular matrix (hWJECM) on the proliferation of rabbit chondrocytes. Methods hWJECM was prepared by differential centrifugation, and 0.5% concentration slurry was made on six-hole cell culture plate and 96-well cell culture plate to form a 1 mm thick film. The culture plate coated with this film was used as a test group , Untreated pure culture medium group as a blank control group, cultured rabbit articular chondrocytes. The growth morphology and proliferation of rabbit chondrocytes in 5, 10 and 15 days were observed by inverted microscope and toluidine blue staining. The proliferation of 1, 3, 5 and 7 days in CCK8 cells was compared , To assess the proliferation and phenotypic maintenance of cartilage seed cells. Results The results of inverted microscope showed that the proliferation of cells at 5, 10 and 15 days was better than that of the culture medium alone. The results of toluidine blue staining on day 5 and day 15 also proved that CCK8 cells proliferated at 1,3 days (P = 0.7142; P = 0.0657). On the 5th and 7th day, hWJECM group was obviously better than the control group (P = 0.0001; P = 0.0006). Conclusion hWJECM has low immunogenicity and no cytotoxic effect, which can well promote the proliferation of chondrocytes.