iPSC-MSCs体外对骨关节炎患者关节软骨组织的基质保护作用

Matrix protective effect of iPSC-MSCs on articular cartilage tissue of patients with osteoarthritis in vitro

目的研究多能诱导干细胞来源的间充质干细胞(iPSC-MSCs)体外对膝骨关节炎(KOA)患者关节软骨组织的基质保护作用及部分机制。方法经知情同意,收集KOA患者关节软骨,剪碎处理后加入IL-1β(10 ng/ml)刺激96 h,再分别加入不同数量的iPSC-MSCs(1×10^(4)、1×10^(5)、1×10^(6))细胞,在37℃、5%CO培养箱内培养3 d。另设IL-1β(10 ng/ml)诱导组和培养液对照组。硫酸软骨素含量测定法检测软骨组织中糖胺聚糖含量,免疫组化法检测组织中Ⅰ型胶原、Ⅱ型胶原表达,ELISA法检测共培养上清液中MMP13、IL-6、IL-10水平,HE染色检测离体软骨组织的病理改变。结果与对照组比较,IL-1β可诱导KOA软骨组织中软骨细胞肿胀死亡,炎性细胞比例升高,Ⅰ型胶原水平升高、Ⅱ型胶原水平降低,培养上清液中MMP-13、IL-6水平升高(P<0.05),IL-10和糖胺聚糖含量减少(P<0.05);与IL-1β诱导组比较,iPSC-MSCs不同细胞数共培养可降低上清液中MMP-13、IL-6水平和Ⅰ型胶原水平,促进Ⅱ型胶原表达,升高软骨组织中糖胺聚糖含量和IL-10水平。结论iPSC-MSCs体外可调节炎症因子水平,抑制软骨细胞基质降解,对关节软骨组织具有保护作用。

Objective To study the protective effect of mesenchymal stem cells(iPSC-MSCs)derived from pluripotent induced stem cells on the articular cartilage tissue of patients with knee osteoarthritis(KOA)in vitro and its partial mechanism.Methods With informed consent,the articular cartilage of KOA patients was collected,shredded and treated with IL-1β(10 ng/ml)to stimulate 96 h,and then different numbers of iPSC-MSCs(1×10^(4),1×10^(5),1×10^(6))cells were added and cultured for 3 d in a 37℃,5%COincubator.In addition,IL-1β(10 ng/ml)induction group and culture medium control group were set up.Chondroitin sulfate assay method was used to detect glycosaminoglycan content in cartilage tissue,immunohistochemical method was used to detect the expression of typeⅠcollagen and typeⅡcollagen in the tissue,and ELISA method was used to detect the level of MMP13,IL-6 and IL-10 in the co-culture supernatant,HE staining was used to detect the pathological changes of cartilage tissuein vitro.Results Compared with the control group,IL-1βcould induce swelling and death of chondrocytes in the cartilage tissue of KOA,increase the proportion of inflammatory cells,increase the level of type I collagen,decrease the level of typeⅡcollagen,and the level of MMP-13 and IL-6 in the culture supernatant significantly increased(P<0.05),and the content of IL-10 and glycosaminoglycan was significantly reduced(P<0.05);compared with the IL-1βinduction group,the co-culture of iPSC-MSCs with different cell numbers could reduce the MMP-13,IL-6 level and type I collagen level in the supernatant,promote the expression of typeⅡcollagen,increase glycosaminoglycan content and IL-10 level in cartilage tissue.Conclusion iPSC-MSCs can regulate the level of inflammatory factorsin vitro,inhibit the degradation of chondrocyte matrix,and have a protective effect on articular cartilage tissue.