外泌体对大鼠骨关节炎软骨细胞凋亡的影响

Effect of exosomes on chondrocyte apoptosis in osteoarthritis rats

目的探讨人脐带间充质干细胞来源外泌体(huc-MSCs-exo)对骨关节炎(OA)大鼠软骨凋亡的影响。方法取健康、足月剖宫产新生儿脐带,分离并提取huc-MSCs-exo,选取Wistar大鼠30只,随机分为假手术组、模型组及治疗组(各10只),模型组及治疗组制备OA模型大鼠,治疗组成模后关节腔内注射huc-MSCs-exo 100μg,治疗8周后;比较各组大鼠步态障碍评分、软骨组织形态学变化、软骨细胞凋亡情况及一氧化氮(NO)表达;Real-time PCR检测软骨组织肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β、IL-4、B细胞淋巴瘤基因-2(Bcl-2)、Bcl-2相关X蛋白(Bax)mRNA表达。另取12只大鼠,6只正常饲养、6只进行膝OA造模,模型成功后分离、培养OA软骨细胞并染色鉴定;正常软骨细胞为对照组,OA软骨细胞培养后分为OA组和实验组,实验组软骨细胞加入1 g/L huc-MSCs-exo 0.2 ml培养,对照组及OA组软骨细胞给予同体积磷酸盐缓冲液培养,3组细胞均培养24 h,LSD t检验比较各组细胞增殖抑制率、凋亡情况及NO表达。结果治疗组与模型组比较,治疗组大鼠步态障碍评分降低(t=5.49,P<0.001),NO含量降低(t=15.69,P<0.001),凋亡指数(AI)值降低(t=7.42,P<0.001),TNF-α表达量降低(t=5.74,P<0.001),IL-1β表达量降低(t=17.22,P<0.001)、IL-4表达量降低(t=11.33,P<0.001),Bax mRNA表达量降低(t=19.51,P<0.001),Bcl-2 mRNA表达量升高(t=10.12,P<0.001),差异均具有统计学意义。结论Huc-MSCs-exo可有效改善膝骨OA大鼠软骨细胞凋亡情况,其机制可能与改善炎症反应有关。

Objective To investigate the effect of human umbilical cord mesenchymal stem cell derived exosomes(huc-MSCs-exo)on cartilage apoptosis in osteoarthritis(OA)rats.Methods Huc-MSCs-exo was isolated and extracted from the umbilical cord of healthy and full-term cesarean section newborns.Thirty Wistar rats were randomly divided into sham operation group,model group and treatment group(10 rats in each group).OA model rats were prepared in the model group and treatment group.Huc-MSCs-exo 100μg was injected into the articular cavity after treatment.After eight weeks of treatment,the gait disorder score,cartilage histomorphological changes,chondrocyte apoptosis and nitric oxide(NO)expression were compared.Detection of tumor necrosis factor-α(TNF-α)in cartilage by real time PCR、interleukin(IL)-1β、IL-4,B-cell lymphoma gene-2(Bcl-2)and Bcl-2 related X protein(Bax)mRNA expression.Another 12 rats were chosen,among which sixratswere normalfed and the other six rats were used for knee OA modeling.After the models were succeeded,OA chondrocytes were isolated,cultured and stained.The normal chondrocytes were used as the control group,and the OA chondrocytes were also cultured and divided into the OA group and the experimental group.The experimental group were cultured with 1g/L huc-MSCs-exo 0.2 ml,while the control group and the OA group were cultured with phosphate buffer.The three groups of cells were cultured for 24 h.The inhibition rate of cell proliferation,apoptosis and NO expression were compared by LSD t test.Results Compared with the model group,in the treatment group,the gait disorder score decreased(t=5.49,P<0.001),the content of no decreased(t=15.69,P<0.001),the value of apoptosis index(AI)decreased(t=7.42,P<0.001).The expression of TNF-αand IL-1βdecreased(t=5.74,P<0.001)(t=17.22,P<0.001),the expression of IL-4 decreased(t=11.33,P<0.001),the expression of Bax mRNA decreased(t=19.51,P<0.001),while the expression of bcl-2 mRNA increased(t=10.12,P<0.001).Conclusion Huc-MSCs-exocan effectively improve chondrocyte apoptosis in knee OA rats,and its mechanism may be related to improving inflammatory response.