人羊膜上皮细胞对大鼠软骨损伤的修复作用

Investigation of the repair effect of human amniotic epithelial cells on rat cartilage damage

目的 探讨人羊膜上皮细胞(hAECs)对大鼠软骨损伤的修复作用及其调控机制。方法 采用酶解法消化分离hAECs,使用改良Hulth法建立大鼠骨性关节炎模型,将已造模成功的大鼠随机分为实验组和阴性对照组,另设空白对照组(健康大鼠)。实验组:注射100μlDMEM-F12(含1×10^(6)个hAECs细胞);阴性对照组:注射100μl的DMEM-F12;空白对照组不做处理。治疗1个月后取膝关节软骨镜下观察,采用免疫组化染色检测Ⅱ型胶原蛋白(COL2)、金属基质蛋白酶抑制因子(TIMP)和金属基质蛋白酶(MMP)表达;采用Masson染色、番红固绿染色、ELISA检测肿瘤坏死因子(TNF)-α、白介素(IL)-1β、γ-干扰素(γ-IFN)、IL-6及IL-7含量;采用Western-bolt检测JAK2、p-JAK2、STAT3和p-STAT3蛋白表达。结果 阴性对照组相比于空白对照组潮线模糊,软骨排列紊乱,软骨表面磨损严重;而实验组出现了明显的软骨增生,染色几乎完整着染,表层光整。阴性对照组血清及滑膜中TNF-α、IL-1β、γ-IFN、IL-6和IL-7含量均高于空白对照组(均P <0.05),实验组血清及滑膜中TNF-α、IL-1β、γ-IFN、IL-6和IL-7含量均低于阴性对照组(均P <0.05)。阴性对照组COL2、TIMP表达强度弱于空白对照组,MMP表达强于空白对照组;实验组p-JAK2、p-STAT3、COL2、TIMP表达强度高于阴性对照组,而MMP表达弱于阴性对照组(均P <0.05)。结论 hAECs可通过JAK2/STAT3信号通路来调控炎症因子、COL2、TIMP和MMP的分泌,从而促进骨性关节炎软骨损伤修复。

Objective To investigate the repair effect and mechanism of human amniotic epithelial cells on cartilage injury in rats.Methods Human amniotic epithelial cells were digested and isolated by enzymatic hydrolysis.The modified Hulth method was used to establish the rat model of osteoarthritis.After four weeks of modeling,successfully modeled rats were randomly divided into experimental group and negative control group,and blank control group(healthy rats)was setted up.100μl DMEM-F12(containing 1×10^(6) cells)was injected in experimental group.100μl DMEM-F12 was injected in negative control group.The blank control group did not accept any treatment.After one month,immunohistochemical staining was used to detect the expressions of collagen typeⅡ(COL2),matrix metalloproteinase(MMP)and tissue inhibitor of metalloproteinases(TIMP).MASSON staining and saffranine solid green staining were used to detect the contents of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),γ-interferon(γ-IFN),interleukin-6(IL-6)and interleukin-7(IL-7)by ELISA.The protein expressions of JAK2,p-JAK2,S TAT3 and p-STAT3 were detected by Westernbolt.Results Compared with the blank control group,the negative control group had blurred tidal lines,disordered cartilage arrangement,and severe cartilage surface wear,while the experimental group had significant cartilage hyperplasia,with almost complete staining and smooth surface.The contents of TNF-α、IL-1β,γ-IFN、IL-6 and IL-7 in serum and synovium of the negative control group was significantly higher than those of the blank control group(all P 0.05).The levels of TNF-α、IL-1β、γ-IFN、IL-6 and IL-7 in serum and synovium of the experimental group were significantly lower than those of the negative control group(all P 0.05).The expressions intensity of COL2 and TIMP in the negative control group were significantly weaker than those in the blank control group,while the expression of MMP was increased(all P 0.05).The expressions intensity of p-JAK2,p-STAT3,COL2,and TIMP in the experimental group were significantly higher than those in the negative control group,while the expression of MMP was decreased(all P0.05).Conclusions Human amniotic epithelial cells can regulate the secretion of inflammatory factors,COL2,MMP and TIMP through JAK2/P-STAT3 signaling pathway to promote the repair of cartilage injury in osteoarthritis.