人脐带Wharton胶细胞外基质对软骨种子细胞的作用

Effects of Wharton's jelly extracellular matrix from human umbilical cord on rabbit chondrocytes

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 proliferation of the rabbit chondrocytes on the human Wharton＇s jelly extracellular matrix in vitro. Methods The extracellular matrix of the human Wharton＇s jelly was prepared using the differential centrifugation. The chondroeytes were isolated from articular cartilage of the New Zealand white rabbits and cultured on WJECM-coated plates. These chondrocytes were cultured on the hWJECM-plated wells as the experiment groups. The cells cultured in simple medium was set as the control group. The cells were analyzed regarding cell growth and morphology using an inverted microscope, the toluidine blue staining, and cell counting Kit-8 assay. Results The cell proliferation of hWJECM group was better than that of the control group under the inverted microscope on the 5th day, the 10th day, and the 15th day. The result of the toluidine blue staining also proved this. There was no statistically significant difference in proliferation of the experiment group and the control group （ P = 0. 7142; P =0. 0657） according to the CCK8 cell proliferation assay on the 1st day and the 3rd day. The cell proliferation of hWJECM group was obviously better than the control group （P = 0. 0001 ;P = 0. 0006） on the 5th day and the 7th day. Conclusion bWJECM has low immunogenicity and no cytotoxic effects. It can promote the proliferation of the chondrocytes. In addition, human umbilical cord is discarded in delivery, so it has advantages of wide variety of sources, simple operation, and no ethical problems. It could be an ideal biological material.