中文摘要：

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进行研究。Dox（10mg／L）处理0d、1d、3d及6d后，real-timeqPCR检测LC3b、Beclin-1、p62和LAMP-2表达情况，Westernblowing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14d后分析碱性磷酸酶（alkalinephosphatase，ALP）活性。用3-MA（5mmol／L）或Rap（10μmol／L）与Dox共同处理1d、3d及6d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（5mmol／L）可增强ALP活性，而Rap（10μmog/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。

英文摘要：

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.