摘要
目的:探讨细胞巨自噬与Runx2诱导C2C12细胞成骨分化的关系。方法:在强力霉素(doxycyc-line,Dox)诱导Runx2表达的细胞系C2C12/Runx2Dox中进行研究。Dox(10 mg/L)处理0 d、1 d、3 d及6 d后,real-time qPCR检测LC3b、Beclin-1、p62和LAMP-2表达情况,Western blotting分析LC3-I/LC3-Ⅱ比值。设置不同的3-甲基腺嘌呤(3-methyladenine,3-MA)或雷帕霉素(rapamycin,Rap)浓度,Dox处理14 d后分析碱性磷酸酶(alkalinephosphatase,ALP)活性。用3-MA(5 mmol/L)或Rap(10μmol/L)与Dox共同处理1 d、3 d及6 d后检测ALP及骨钙素(osteocalcin,OC)表达情况。结果:(1)C2C12细胞向成骨分化时,LC3b与Beclin-1显著下调,p62与LAMP-2无明显变化;(2)LC3-I向LC3-Ⅱ转换的过程被抑制;(3)3-MA(5 mmol/L)可增强ALP活性,而Rap(10μmol/L)则抑制其活性;(4)3-MA可上调ALP及OC表达,Rap则下调二者表达。结论:Runx2通过下调LC3和Beclin-1、抑制LC3-I向LC3-Ⅱ转换的方式阻碍自噬体形成,以诱导C2C12细胞分化为成骨细胞。
AIM： To explore the relationship between macroautophagy and Runx2-induced osteogenic differen- tiation of C2C12 cells. METHODS： The cells of C2C12/Runx2Dox subline were used in the study, which expresses Runx2 in response to doxycycline （Dox）. The cells were treated with Dox （10 rag/L） for 0 d, 1 d, 3 d, and 6 d, and the expres- sion levels of LC3b, Beclin-1, p62, and LAMP-2 were detected at each time point using real-time qPCR. The LC3-I/LC3- II ratio was measured by Western blotting. The cells were treated with different concentrations of 3-methyladenine （3-MA） or rapamycin （Rap） for 14 days in the presence of Dox, and the alkaline phosphatase （ALP） activity was measured. The cells were treated with 3-MA （5 mmol/L） or Rap （10 μmol/L） for 1 d, 3 d, and6 d in the presence of Dox, and the ex- pression levels of ALP and osteocalcin （OC） were detected. RESULTS： The expression levels of LC3b and Beclin-1 were dramatically down-regulated during Runx2-induced osteogenic differentiation of C2C12 cells, and the levels of p62 and LAMP-2 were not changed during this process. The conversion of LC3-I into LC3-II was inhibited. The ALP activity was increased in 3-MA （5 mmol/L）-treated cells, but decreased in Rap （10 μmol/L）-treated cells. The expression levels of ALP and OC were up-regulated by 3-MA treatment and were down-regulated by Rap treatment. CONCLUSION： Runx2 induces osteogenic differentiation of C2C12 cells by inhibiting the formation of autophagosome through down-regulating LC3 and Beclin-1 as well as inhibiting the conversion of LC3-I to LC3-II.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2013年第3期481-487,共7页
Chinese Journal of Pathophysiology
基金
国家自然科学基金重大研究计划培育项目(No.90919050)
