Objective To study the role of DEPTOR knockdown in RPMI-8226 cells on the protein expression of RANKL and differentiation of THP-1 into osteoclast-like cells in a contactless co-culture system and its possible mechanism.Methods Constructed DEPTOR shRNA expression vector GV115-shRNA was transferred into RPMI-8226 cell to produce packaged lentivirus.Western blot was applied to measure the protein levels of DEPTOR and RANKL.The expression of autophagy-associated proteins LC-3 and Atg5 were confirmed by Western blot analysis.Three grouPs were divided:THP-1 group,THP-1 + RPMI-8226 group and THP-1 + DEPTOR shRNA group.Osteoclast-like cells were identified by TRAP.The mRNA levels of calcitonin receptor (CTR) and Cathepsin-K were examined using RTPCR.Results The results showed that protein expression levels of DEPTOR and RANKL were significantly lower in RPMI-8226 cells transfected with GV115 DEPTOR shRNA compared with that in untransfected cells (P ＜ 0.05).The expression levels of autophagy-associated proteins LC-3 and Atg5 in the DEPTOR shRNA group were significantly lower than those in the control shRNA group and the parental group (P ＜ 0.05).In the co-culture system,THP-1 cell could differentiate into TRAP positive multinuclear cells.RPMI-8226 promoted mRNA exoression of CTR and Cathepsin-K (P ＜ 0.05).DEPTOR shRNA suppressed osteoclast-like cells formation and decreased CTR and Cathepsin-K mRNA expression in co-cultures,the differences were statistically significant (P ＜0.05).Conclusion In the coculture system,DEPTOR shRNA inhibits the differentiation of THP-1 cells into TRAP positive multinuclear cells,which may be due to its inhibition on RANKL expression in RPMI-8226 cells,and the inhibition of autophagy will restrain osteoclast maturation.%目的 探讨沉默RPMI-8226 DEPTOR基因表达在非接触式共培养体系下,对破骨细胞分化因子(RANKL)蛋白表达及THP-1细胞向破骨样分化的作用,并研究其可能机制.方法 构建DEPTOR shRNA重组慢病毒载体转染RPMI-8226细胞.通过Western blot技术从蛋白水平检测RPMI-8226细胞中DEPTOR及RANKL的表达.Western blot检测自噬相关蛋白LC-3和Atg5表达.构建共培养体系,分三组:THP-1细胞单培养组、THP-1+RPMI-8226细胞组和THP-1+DEPTOR shRNA组.应用抗酒石酸酸性磷酸酶(TRAP)染色法检测THP-1向破骨样细胞分化过程中抗酒石酸酸性磷酸酶活性改变,应用RT-PCR法检测THP-1细胞降钙素受体(CTR)和组织蛋白酶(Cathepsin)-K mRNA表达水平.结果 DEPTOR shRNA可明显抑制RPMI-8226细胞中DEPTOR及RANKL蛋白表达(P＜0.05),DEPTOR shRNA组自噬相关蛋白LC-3和Atg5的蛋白的表达水平较阴性对照转染组及未转染组下降(P＜0.05).THP-1细胞与RPMI-8226细胞共培养条件下可诱导THP-1细胞破骨样分化,CTR和Cathepsin-K基因表达上调(P＜0.05);DEPTOR shRNA共培养条件下可抑制THP-1细胞向破骨样细胞分化,CTR和Cathepsin-K基因表达减弱,差异有统计学意义(P＜0.05).结论 DEPTOR shRNA能明显抑制共培养体系中THP-1细胞的破骨样分化,该作用可能与DEPTOR下调RPMI-8226细胞RANKL有关,抑制自噬可阻碍破骨细胞的分化成熟.
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