Basic study on detecting strategy for drug/gene doping for athlete

为了研究建立一种检测力量型运动员基因兴奋剂的方法的可行性，我们设计了一个实验模型系统。肌生长抑制素是骨骼肌发育和生长的有效负调节因子，并且肌生长抑制素基因敲除小鼠表现出双肌表型。为了实现敲除，我们构建了表达针对肌生长抑制素的短发夹干扰RNA（shRNA）的质粒。将这些shRNA转染到C2 C12培养细胞中或注射到成年小鼠的胫骨前肌（TA）中。通过进行体外和体内实验，我们发现一些shRNAs有效地降低了myostatin的表达，TA肌肉显示出高达27.9%的肥大。然后，使用真实的- time PCR，我们尝试检测注射了shRNA质粒的小鼠的血清或肌肉中的shRNA质粒。虽然我们无法在血清样品中检测到质粒，但在诱导后至少四周，在处理的肌肉中可以检测到质粒。我们还能够在TA附近的肌肉中检测到质粒。该基因兴奋剂模型系统将有助于进一步的研究，旨在兴奋剂控制。

To investigate the feasibility of developing a method for detecion of gene doping in power-athletes, we devised an experimental model system. Myostatin is a potent negative regulator of skeletal muscle development and growth, and myostatinknockout mice exhibit a double-muscle phenotype. To achieve knockdown, we constructed plasmids expressing short hairpin interfering RNAs (shRNAs) against myostatin. These shRNAs were transfected into C2C12 cultured cells or injected into the tibialis anterior (TA) muscle of adult mice. By performing in vitro and in vivo experiments, we found that some shRNAs effectively reduced the expression of myostatin, and that the TA muscle showed hypertrophy of up to 27.9%. Then, using real- time PCR, we tried to detect the shRNA plasmid in the serum or muscles of mice into which it had been injected. Although we were unable to detect the plasmid in serum samples, it was detectable in the treated muscle at least four weeks after induction. We were also able to detect the plasmid in muscle in the vicinity of the TA. This gene doping model system will be useful for further studies aimed at doping control.