Cx43组成的间隙连接和半通道对骨-肌相互作用的调控机制研究

Both osteoporosis and sarcopenia affect the living quality of old people. Besides mechanical regulation, the biochemical regulation between bone and skeletal muscle involve in the interaction of the two degenerative diseases. Bone secrete regulatory factors to affect the function of skeletal muscle. In bone tissue, gap junctions and hemichannels formed by connexin43 (Cx43) play important roles in the secretion of regulatory factors and signal transmission. Previous studies have shown the alteration in the structure and functiuon of skeletal muscles in osteoblast selective Cx43 deficient mice. We have generated two osteocyte selective Cx43 dominat negative transgenic mice R76W and ∆130-136, both of gap junctions and hemichannels were blocked in ∆130-136, while only gap junction was blocked in R76W. Our previous studies have shown the alteration of bone structure and secretion of regulatory factors in the two transgenic mice. Also the fast muscle of soleus increased in R76W mice, compared with that of wide type mice. These results indicate that gap junctions and hemichannels play different roles in the regulation of bone on skeletal muscle, but the mechanism is not clear..In the study, the effects of blocking gap junctions and hemichannels on the structure and function of skeletal muscles will be investigated in different month-aged mice. And the role of conditioned media of primary osteocytes isolated from transgenic mice on the function of skeletal muscle cells will be detected. Also the role of signal pathway and some important regulatory factors in the process will be investigated. This study will give some clues for the interaction mechanism between bone and skeletal muscle, and osteoporosis and sarcopenia.

肌肉减少症常伴随着骨质疏松，都严重影响老年人生活质量。这两种退行性疾病间的相互作用除了受骨-肌间力学调控，也受到生化调控。骨能通过分泌调控因子影响骨骼肌功能。而连接蛋白43（Cx43）组成的间隙连接和半通道是骨最主要的信号传递方式，介导调控因子分泌。研究表明成骨细胞敲除Cx43影响了小鼠骨骼肌的结构功能。申请者也发现骨细胞Cx43转基因小鼠R76W（抑制间隙连接）和∆130-136（间隙连接和半通道均抑制）的骨调控因子表达分泌和骨骼肌纤维类型改变，提示两种通道在骨对骨骼肌调控中起重要作用，但潜在机制不明。.本项目拟研究：①不同月龄小鼠骨细胞间隙连接和半通道抑制对骨骼肌结构功能的影响；②转基因小鼠原代骨细胞条件培养基对骨骼肌细胞功能的影响；③代谢因子和信号通路在骨对骨骼肌调控中的作用。为揭示骨-肌间、及骨质疏松和肌肉减少症间的相互作用提供科学依据，并为老年性肌肉减少症康复治疗提供新思路。

本项目选用两种骨细胞显性负突变Cx43的转基因小鼠R76W和∆130-136及其同窝野生型（WT）小鼠，其中∆130-136的间隙连接和半通道都被抑制，R76W只抑制间隙连接，而半通道功能被促进。研究了3月龄、10月龄、18月龄小鼠骨与骨骼肌的结构功能，及骨对骨骼肌的调控机制。.体内实验结果显示，1）骨细胞间隙连接和半通道调控3月龄小鼠快肌肌肉量和功能：间隙连接抑制促使快肌向慢肌纤维转变，抑制半通道影响蛋白合成降解的平衡负调控肌肉量；2）骨细胞半通道功能影响老年小鼠骨丢失：半通道增强促进 ATP 分泌增加了骨重建；而半通道抑制降低骨细胞 PGE2 分泌，促进骨细胞凋亡。3）骨细胞间隙连接和半通道调控老年小鼠骨骼肌结构功能：半通道抑制降低 PGE2 水平促进骨骼肌中胶原蛋白沉积，间隙连接抑制引起骨骼肌活性氧水平增加，都引起骨骼肌收缩力下降。.体外实验提取了小鼠长骨的原代骨细胞，对细胞上清进行了非靶向代谢组学分析，发现半通道功能改变所产生的差异代谢物参与成肌细胞能量代谢调控，其条件培养基影响成肌细胞肌管生成。. 这些研究提示骨细胞Cx43间隙连接和半通道参与调控小鼠骨骼肌的结构和功能，为骨-肌生化交流提供了重要的理论依据，同时也为老年骨质疏松和肌肉减少症的防治提供了潜在分子靶点。