DEPTOR-mTOR信号通路介导的自噬在多发性骨髓瘤中对破骨细胞分化的调控作用  

The role of DEPTOR-mTOR signaling pathway-mediated autophagy on osteoclasts differentiation and maturation in multiple myeloma

作　　者：张浩然[1] 乔旭旭 毕明宏[1] 翟云芝[1] 钱立宇[2] 潘成武[2] 赵论[1] 

机构地区：[1]蚌埠医学院第一附属医院肿瘤内科,安徽蚌埠233004 [2]蚌埠医学院第一附属医院肿瘤外科,安徽蚌埠233004

出　　处：《中国医药导报》2018年第4期18-22,共5页China Medical Herald

基　　金：蚌埠医学院科技发展基金项目(BYKF1403);安徽省高校自然科学研究项目(KJ2016A483)

摘　　要：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 sh RNA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 sh RNA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 m RNA表达水平。结果 DEPTOR sh RNA可明显抑制RPMI-8226细胞中DEPTOR及RANKL蛋白表达(P<0.05),DEPTOR sh RNA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 sh RNA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 sh RNA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。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 eontactless 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-assoeiated 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. Osteoelast-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 GVll5 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 ,ositive multinuclear cells. RPMI-8226 oromoted 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 in- hibition of autophagy will restrain osteoclast maturation.

关 键 词：骨髓瘤骨病 DEPTOR RNA干扰 自噬 THP-1 

分 类 号：R733.3[医药卫生—肿瘤]