iPSC-MSCs体外抑制ADAMTS保护骨关节炎患者软骨基质  

Inhibition of ADAMTS by iPSC-MSCs in vitro protects cartilage matrix in patients with osteoarthritis

作　　者：张峰 程刚 吴玉娇 储柱平 王旭蕾 王惠敏 王康 魏伟[1] 严尚学[1] Zhang Feng;Cheng Gang;Wu Yujiao;Chu Zhuping;Wang Xulei;Wang Huimin;Wang Kang;Wei Wei;Yan Shangxue(Institute of Clinical Pharmacology,Anhui Medical University,Key Laboratory of Anti-Inflammatory and Immune Medicine,Ministry of Education,Anhui Collaborative Innovation Centre for Anti-inflammatory and Immune Drugs,Hefei 230032)

机构地区：[1]安徽医科大学临床药理研究所,抗炎免疫药物教育部重点实验室,抗炎免疫药物安徽省协同创新中心,合肥230032

出　　处：《安徽医科大学学报》2023年第9期1522-1528,共7页Acta Universitatis Medicinalis Anhui

基　　金：安徽高校自然科学研究重大项目(编号:KJ2020ZD15)。

摘　　要：目的研究诱导多能干细胞来源的间充质干细胞(iPSC-MSCs)体外对膝骨关节炎(KOA)患者软骨基质的保护作用及其部分机制。方法收集KOA患者关节置换手术切除的软骨组织,分别进行组织和细胞实验。软骨组织剪成小块,随机分为对照组、白介素-1β(IL-1β)诱导组和iPSC-MSCs组。除对照组外,各组软骨组织予以IL-1β(10 ng/ml)刺激96 h,再与不同数量iPSC-MSCs(1×10^(4),1×10^(5),1×10^(6))细胞共培养72 h后取出软骨组织进行石蜡包埋切片,免疫组化法检测组织中带有血小板凝血酶敏感蛋白结构域的解聚蛋白样金属蛋白酶家族(ADAMTS-4、ADAMTS-5)及Ⅱ型胶原(COL2)表达,ELISA法检测共培养上清中基质金属蛋白酶13(MMP13)、白介素-6(IL-6)、白介素-10(IL-10)水平,苏木精伊红(HE)染色检测离体软骨组织的病理改变。分离KOA患者软骨组织中软骨细胞,经IL-1β(10 ng/ml)刺激48 h后,将软骨细胞与不同数量iPSC-MSCs(1×10^(4),1×10^(5),1×10^(6))共培养72 h。免疫荧光和蛋白免疫印迹法检测软骨细胞中矮小相关转录因子2(RUNX2)、ADAMTS-4、ADAMTS-5的表达。结果与对照组比较,IL-1β可诱导软骨组织RUNX2、ADAMTS-4、ADAMTS-5水平升高、COL2水平降低,培养上清中MMP-13、IL-6水平升高(P<0.05),IL-10水平减少(P<0.05);与IL-1β诱导组比较,不同数量iPSC-MSCs共培养可降低上清中MMP-13、IL-6水平,降低RUNX2、ADAMTS-4、ADAMTS-5的表达,促进COL2表达,并升高IL-10水平。结论iPSC-MSCs体外可抑制ADAMTS-4、ADAMTS-5表达,减少软骨细胞外基质降解,起到关节软骨保护作用。Objective To study the protective effect and mechanism of iPSC-MSCs on cartilage matrix in knee osteoarthritis(KOA)patients in vitro.Methods Cartilage tissues removed from KOA patients with joint replacement surgery were collected and subjected to tissue and cellular experiments,respectively.Cartilage tissue was cut into small pieces and randomly divided into a control group,an IL-1β(10 ng/ml)induction group,and iPSC-MSCs groups.Except for the control group,cartilage tissues from each group were stimulated with IL-1β(10 ng/ml)for 96 h and then co-cultured with different amounts of iPSC-MSCs(1×10^(4),1×10^(5),1×10^(6))cells for 72 h.For in tissues,the pathological changes of isolated cartilage tissues were examined by HE staining.The levels of ADAMTS-4,ADAMTS-5,and type II collagen expression were analyzed by immunohistochemistry,while the levels of MMP13,IL-6,and IL-10 in culture supernatants were detected by ELISA kits.The 2 to 5 generations of chondrocytes,which were extracted from cartilage tissue of KOA patients,were stimulated with IL-1β(10 ng/ml)for 48 h and then co-cultured with different concentrations of iPSC-MSCs(1×10^(4),1×10^(5),1×10^(6))cells for 72 h.Immunofluorescence and Western blot detected the expression of RUNX2,ADAMTS-4,and ADAMTS-5 in chondrocytes.Results Comparison with the control group,in the IL-1β-induced group,the levels of RUNX2,ADAMTS-4,and ADAMTS-5 increased,the level of type II collagen decreased,the levels of MMP-13 and IL-6 in the culture supernatant increased(P<0.05),and the level of IL-10 decreased(P<0.05);Compared with the IL-1β-induced group,co-culture of different numbers of iPSC-MSCs reduced the levels of MMP-13 and IL-6 in the supernatant,decreased the expression of RUNX2,ADAMTS-4,and ADAMTS-5,promoted type II collagen expression and elevated IL-10 levels.Conclusion iPSC-MSCs inhibited ADAMTS-4 and ADAMTS-5 expression in vitro,reduced cartilage extracellular matrix degradation,and played a role in articular cartilage protection.

关 键 词：骨关节炎 诱导多能干细胞 关节软骨 带有血小板凝血酶敏感蛋白结构域的解聚蛋白样金属蛋白酶家族 

分 类 号：R684.3[医药卫生—骨科学]