免疫分子B7-H3对骨关节炎成纤维样滑膜细胞生物学功能的影响

The biological effect of B7-H3 on fibroblast-like synoviocytes in osteoarthritis

目的探讨免疫分子B7-H3对OA成纤维样滑膜细胞(FLS)生物学功能的影响。方法获取OA膝关节滑膜组织5例和正常膝关节滑膜组织4例,并分离、培养原代细胞株。以OA滑膜组织和原代OA-FLS为研究对象,利用免疫组织化学染色、realtime-PCR、流式细胞术研究OA滑膜组织B7-H3表达情况。根据B7-H3996及1041位点设计相应siRNA沉默并下调OA-FLS中B7-H3的表达,通过蛋白质印迹法、流式细胞术测定靶细胞B7-H3蛋白抑制情况,划痕实验、Transwell实验分析其迁移、侵袭能力,CCK-8法检测细胞增殖能力,CBA法检测细胞培养上清细胞因子及趋化因子表达。使用GraphPad Prism 8.0软件分析实验数据,正态分布数据用±s表示,数据间两两比较采用t检验,以P<0.05为差异有统计学意义。结果B7-H3分子在OA膝关节滑膜组织中FLS异常高表达。相比于siNC,si996、si1041可以抑制OA-FLS中B7-H3的表达。在Transwell迁移实验中,OA-FLS迁移能力下降[siNC组和si996组(100.3±3.7)个/视野和(48.7±1.2)个/视野,t=13.24,P<0.001;siNC组和si1041组(100.3±3.7)个/视野和(59.7±1.9)个/视野,t=9.80,P<0.001]。在Transwell侵袭实验中,表明侵袭能力下降[siNC组和si996组(127.3±5.6)个/视野和(39.7±3.3)个/视野,t=13.49,P<0.001;siNC组和si1041组(127.3±5.6)个/视野和(57.3±1.9)个/视野,t=11.85,P<0.001]。沉默B7-H3基因可以抑制OA-FLS细胞因子IL-6[siNC组和si996组(248±21)pg/ml和(111±12)pg/ml,t=24.08,P=0.002;siNC组和si1041组(248±21)pg/ml和(46±5)pg/ml,t=13.21,P=0.006]、IL-8[siNC组和si996组(118.1±15.6)pg/ml和(47.1±5.4)pg/ml,t=6.68,P=0.022;siNC组和si1041组(118.1±15.6)pg/ml和(10.0±1.3)pg/ml,t=13.08,P=0.006]和趋化因子CXCL8[siNC组和si996组(178.8±6.4)ng/ml和(83.2±2.7)ng/ml,t=13.77,P=0.005;siNC组和si1041组(178.8±6.4)ng/ml和(93.5±2.8)ng/ml,t=12.23,P=0.007],CCL2[siNC组和si996组(184.1±5.1)ng/ml和(109.4±5.9)ng/ml,t=9.57,P=0.011;siNC组和si1041组(184.1±5.1)ng/ml和(97.1±1.5)ng/ml,t=16.39,P=0.004]的分泌。结论B7-H3可能对OA-FLS的迁移、侵袭及细胞因子分泌等生物学功能有一定的调控作用,为进一步研究B7-H3参与OA发病机制提供了线索。

Objective:To investigate the effect of B7-H3 gene on the biological function of fibr-oblastlike synoviocytes (FLS) in osteoarthritis (OA).Methods:Synovial tissue of five cases of OA and synovial tissue of 4 normal knee were obtained, and the primary cell lines were isolated and cultured. The expression of B7-H3 in OA synovial tissue and primary OA-FLS were studied by immunohi-stochemistry, real time-poly merase chain reaction (PCR) and FACS. According to sites 996 and 1041 of B7-H3, corresponding siRNA was designed and the expression of B7-H3 in FLS was silenced and down-regulated. The inhibition of B7-H3 and its protein in target cells was determined by Western blot and FACS. The migration and invasion ability of B7-H3 in target cells were analyzed by scratch assay and Transwell assay. CCK8 assay was used to detect cell proliferation ability, and CBA assay was used to detect cytokines and chemokines in cell culture supernatant. GraphPad Prism 8.0 software was used to analyze the experimental data. The normal distribution data was expressed as mean±standard deviation ( Mean± SD). The comparison between data was performed by T test, and P<0.05 was considered statistically significant. Results:The abnormally high expression of B7-H3 in fibro-blast-like synoviocytes of OA was detected. Compared with siNC, si996 and si1041 inhibited the expression of B7-H3 in OA-FLS. In the Transwell migration experiment, the mean cells number of random view in the siNC group, the si996 group, and the si1041 group indicating decreased migration ability of OA-FLS [siNC vs si996 (100.3±3.7) /view vs (48.7±1.2) /view, t=13.24, P<0.001;siNC vs si1041 (100.3±3.7) /view vs (59.7±1.9) /view, t=9.80, P<0.001). In the Transwell invasion experiment, the mean cells number of random view in the siNC group, in the si996 group, and in the si1041 group indicating decreased invasion ability of OA-FLS [siNC vs si996 (127.3±5.6) /view vs (39.7±3.3) /view, t=13.49, P<0.001;siNC vs si1041 (127.3±5.6) /view vs (57.3±1.9) /view, t=11.85, P<0.001]. The secretion of IL-6 [siNC vs si996 (248±21) pg/ml vs (111±12) pg/ml, t=24.08, P=0.002;siNC vs si1041 (248±21) pg/ml vs (46±5) pg/ml, t=13.21, P=0.006], IL-8 [siNC vs si996 (118.1±15.6) pg/ml vs (47.1±5.4) pg/ml, t=6.68, P=0.022;siNC vs si1041 (118.1±15.6) pg/ml vs (10.0±1.3) pg/ml, t=13.08, P=0.006], CXCL8 [siNC vs si996 (178.8±6.4) ng/ml vs (83.2±2.7) ng/ml, t=13.77, P=0.005;siNC vs si1041 (178.8±6.4) ng/ml vs (93.5±2.8) ng/ml, t=12.23, P=0.007] and CCL2 [siNC vs si996 [(184.1±5.1) ng/ml vs (109.4±5.9) ng/ml, t=9.57, P=0.011;siNC vs si1041 (184.1±5.1) ng/ml vs (97.1±1.5) ng/ml, t=16.39, P=0.004] was decreased . Conclusion:B7-H3 may regulate the migration, invasion, cytokine secretion and other biological functions of OA-FLS, providing clues for further study of B7-H3's involvement in the pathogenesis of OA.