手法调控雪旺氏细胞表达及其诱导失神经支配骨骼肌卫星细胞成肌分化的动态研究

It was very difficult to repair the denervated skeletal muscle .It was important to protect the activity of muscle satellite cells, promote the differentiation of muscle satellite cells, prevent its irreversibility atrophy.these were the key of denervated skeletal muscle regeneration.The study found that the Schwann cells could promote the proliferation and differentiation of muscle satellite cells, bying a lot of nutritional factors to a certain extent on the continuous supply of muscle satellite cells.Our previous studies have shown that regeneration and repairing techniques of denervated skeletal muscle could be directed into the muscle differentiation by promoting muscle satellite cells to be normal muscle. We speculated that the way may be by promoting the expression of Schwann cells in improving denervated muscle microenvironment to promote muscle satellite cells into muscle differentiation, but the specific mechanism of action was still unclear.So intends of this project was to establish denervated rabbit skeletal muscle model firstly,and vitro cell culture experiment,then to study the way that the Schwann cells activity, secretion capacity and expression of skeletal muscle, to observe the repair process of skeletal muscle regeneration in Schwann cells into muscle regulation factor expression,these results could illustrate muscle satellite cells regeneration pathway after denervation muscle.Tt could provide new theoretical basis and research directions for the clinical treatment of skeletal muscle atrophy.

失神经支配后骨骼肌的再生修复异常困难，保护肌卫星细胞的活性，促进肌卫星细胞的成肌分化，保持失神经后骨骼肌的再生活性，防止其不可逆性萎缩，是解决失神经骨骼肌重获神经再支配后有效功能恢复的关键。研究发现，雪旺氏细胞通过持续供给肌卫星细胞大量的营养因子，在一定程度上促进肌卫星细胞的增殖和分化。我们前期研究显示：手法可以通过促使肌卫星细胞成肌分化来促进失神经支配骨骼肌再生修复，我们推测手法可能是通过改善失神经支配骨骼肌的微环境，促进雪旺氏细胞的表达，来促进肌卫星细胞成肌分化的，但具体作用机理仍不甚明确。本项目拟通过建立失神经支配骨骼肌家兔模型，结合体外细胞培养实验，研究手法对失神经支配骨骼肌雪旺氏细胞增殖表达作用，观察骨骼肌再生修复过程中雪旺氏细胞活性、分泌能力及成肌相关因子的表达规律，来阐释推拿手法促进失神经支配骨骼肌卫星细胞成肌分化的作用途径，为临床骨骼肌萎缩的治疗提供新的理论依据和研究方向。

目的：通过观察失神经支配家兔骨骼肌肌湿重，组织病理结构，损伤神经超微结构，成肌因子及神经生长因子表达水平的变化，探讨推拿手法对失神经支配骨骼肌成肌分化的影响。.方法：新西兰家兔，随机分为四大组：正常组30只、模型观察组30只、NGF治疗组30只、手法治疗组30只，手法干预应用按揉法，分别于手法干预2周、3周、1月、2月、4月后，每组各取6只家兔，测量肌湿重比值；术侧腓肠肌行光镜下HE染色观察；损伤神经远端透射电镜检查；用荧光定量PCR法测定MHC mRNA、Myf-5 mRNA、Myogenin mRNA、NGF mRNA表达水平。.结果：①腓肠肌肌湿重比：手法治疗组均显著升高（P<0.05），2月、4月好于NGF治疗组（P<0.05）。②腓肠肌病理结构：手法治疗组肌细胞萎缩程度、结缔组织及脂肪组织增生程度均明显轻于模型观察组与NGF治疗组。③损伤神经超微结构：手法治疗组后期髓鞘结构、雪旺细胞内细胞器的丰富程度均明显优于模型观察组，与NGF治疗组疗效相当。④骨骼肌MHC mRNA、Myf-5 mRNA、Myogenin mRNA的表达：手法治疗组可以使Myogenin含量表达显著升高，与模型对照组及NGF治疗组相比较，有显著性差异（P＜0.05）。⑤损伤神经NGF mRNA表达：模型观察组、NGF治疗组、手法治疗组的Myogenin mRNA相对表达量早期时高于正常组，后期则低于正常组。NGF治疗组在2周、3周、1月，手法治疗组在1月、2月、4月高于模型观察组，手法治疗组在1月、2月、4月均高于NGF治疗组，差异均有统计学意义（P＜0.05）。⑥免疫组化法检测雪旺氏细胞S-100蛋白的表达：NGF治疗组、手法治疗组均高于模型观察组，手法治疗组在1月、2月均高于NGF治疗组，差异有统计学意义（P＜0.05）。.结论：①推拿手法可以促进成肌相关因子的表达，疗效好于NGF治疗。②在生肌调节因子表达方面，推拿手法好于NGF治疗。③推拿手法可以延缓周围神经损伤后骨骼肌萎缩、变性，促进损伤神经中雪旺细胞增殖及髓鞘的新生、成熟。④推拿手法治疗可以促进损伤神经NGF蛋白与mRNA的表达，保持残存神经的活性，促进肌卫星细胞的成肌分化。