Department of Orthopedics, the Affiliated Lianyungang Hospital of Xuzhou Medical University, The First People's Hospital of Lianyungang, Lianyungang, China.
Eur Rev Med Pharmacol Sci. 2017 Nov;21(21):4989-4998.
To investigate the changes in cell autophagy and the molecular mechanism of rapamycin affecting the fracture healing.
Sprague-Dawley (SD) rats were used to establish the right femoral shaft fracture models, and then underwent immunofluorescence assay to detect the autophagy level in bone tissues. After model establishment, SD rats were divided into two groups, the control group and the rapamycin group (1 mg/kg/d). Respectively, at the 2nd, 4th, and 6th week, rats were randomly selected from each group for X-ray and Micro-computed tomography (Micro-CT) examinations to determine callus growth, immunofluorescence assay to detect the protein expression of light chain 3 II (LC3 II), immunohistochemistry to evaluate the autophagy level through detecting the expression of Beclin1 in rats, Western blotting assay to detect cell apoptosis in tissues, hematoxylin and eosin staining (HE staining) to evaluate the osteoblastic activity through count of osteoblast in bone tissue at the end of fracture, and measure the expression of vascular endothelial growth factors (VEGF).
Significant increases were seen in protein expression of cells in bone tissues at the end of fracture. In rapamycin group, callus formation and calcification level in rats were all higher than those in control group; compared with control group, for rats in rapamycin group, cell autophagy was significantly elevated in bone tissues, while cell apoptosis at the end of fracture was reduced with a significant increase in osteoblastic activity. The expression of VEGF in rapamycin group was higher than that in control group.
Rapamycin can facilitate fracture healing through inducing cell apoptosis and suppressing cell apoptosis in bone tissues.
研究细胞自噬的变化及雷帕霉素影响骨折愈合的分子机制。
选用 SD 大鼠建立右侧股骨干骨折模型，采用免疫荧光法检测骨组织中的自噬水平。建模后，SD 大鼠分为对照组和雷帕霉素组（1mg/kg/d），分别于第 2、4、6 周时，每组随机选择部分大鼠进行 X 射线和 Micro-CT 检查以确定骨痂生长情况，采用免疫荧光法检测 LC3 II 蛋白表达，免疫组化法检测 Beclin1 在大鼠中的表达，评价自噬水平，Western blot 法检测组织细胞凋亡，HE 染色法计数骨组织中破骨细胞以评估成骨细胞活性，检测血管内皮生长因子（VEGF）的表达。
骨折端骨组织细胞的蛋白表达明显增加。与对照组相比，雷帕霉素组大鼠的骨痂形成和钙化水平均较高；与对照组相比，雷帕霉素组大鼠骨组织中的细胞自噬明显升高，而骨折端细胞凋亡减少，成骨细胞活性明显增加。雷帕霉素组 VEGF 的表达高于对照组。
雷帕霉素通过诱导骨组织中的细胞凋亡和抑制细胞凋亡促进骨折愈合。
