Nischarin在脊髓损伤修复中的作用及机制研究

发布时间：2018-07-10 13:42

本文选题：Nischarin + 脊髓损伤　；参考：《浙江大学》2015年硕士论文

【摘要】：目的：探讨Nischarin在脊髓损伤(Spinal cord injury, SCI)后轴突再生中所起的作用,并初步探讨其分子机制。方法： 1Allen' s撞击法建立Sprague-Dawley大鼠SCI模型； 2大鼠脊髓内微量注射针对Nischarin的shRNA质粒或siRNA小片段与转染试剂,进行体内转染； 3针对shRNA质粒转染,利用GFP荧光蛋白检测体内转染的效率； 4通过RT-PCR方法评价shRNA质粒或siRNA小片段抑制Nischarin的效果； 5连续6w的旷场试验对大鼠进行BBB行为学评分,反映大鼠SCI后运动功能恢复情况； 6连续6w爬杆试验对大鼠进行后肢脚掌踏地角度FSA、尾根高度指数RHI和爬杆时间等测试,反映大鼠SCI后运动功能恢复情况； 7在SCI后6w,脊髓损伤平面以下注射荧光金示踪剂,逆行示踪法检测红核区的荧光金分布和强度,反映SCI后轴突再生的情况； 8Western blot和免疫组化法检测脊髓损伤区GAP43蛋白的表达,反映轴突再生的情况。结果： 1成功建立大鼠SCI模型； 2质粒注射区局部可见GFP荧光,提示shRNA质粒成功实现体内转染； 3Nis-shRNA质粒和Nis-siRNA小片段均能有效抑制Nischarin的转录水平； 4体重监测结果证明各组间均无显著差异,提示shRNA和siRNA使用安全； 5旷场试验中,SCI+Nis-shRNA组的BBB运动功能评分较SCI+Ctl-shRNA组和SCI组高,差异显著；SCI+Nis-siRNA组的BBB运动功能评分较SCI+Ctl-siRNA组和SCI组高,差异显著； 6爬杆试验中,SCI+Nis-shRNA组和SCI+Nis-siRNA组的FSA均较各自的对照组和SCI组低,差异显著；SCI+Nis-shRNA组和SCI+Nis-siRNA组的RHI均较各自的对照组和SCI组高,差异显著；但爬杆时间在各组间无显著差异； 7逆行示踪实验中,SCI+Nis-shRNA组和SCI+Nis-siRNA组在红核的荧光金阳性神经元个数均多于各自的对照组和SCI组； 8SCI后1w和6w, western blot结果显示,SCI+Nis-shRNA组和SCI+Nis-siRNA组的GAP43表达均较各自的对照组和SCI组多,差异显著；免疫荧光强度显示,SCI+Nis-shRNA组和SCI+Nis-siRNA组的GAP43表达较各自的对照组和SCI组多。结论：在损伤脊髓处抑制Nischarin的表达,有利于促进SCI大鼠的神经元轴突再生和运动功能的康复。
[Abstract]:Aim: to investigate the role of nischarin in axonal regeneration after spinal cord injury (sci) and its molecular mechanism. Methods: (1) Sprague-Dawley rat sci model was established by Allen's impact method; (2) shRNA plasmids or siRNA fragments targeting Nischarin were microinjected into the spinal cord of rats and transfected with transfection reagent in vivo; (3) shRNA plasmids were transfected with shRNA plasmids. GFP fluorescent protein was used to detect transfection efficiency in vivo; 4 shRNA plasmid or siRNA fragment was used to evaluate the inhibitory effect of nischarin by RT-PCR. The results showed that the recovery of motor function in rats after sci was reflected in 6 consecutive weeks of pole climbing test, which reflected the recovery of motor function of rats after sci by measuring the angle of foot and foot angle, the index of tail root height RHI and the time of climbing pole. (7) at 6 weeks after sci, fluorescent gold tracer was injected below the spinal cord injury plane. The distribution and intensity of fluorescence gold in the red nucleus were detected by retrograde tracing method, which reflected the regeneration of axon after sci. The expression of GAP43 protein in spinal cord injury area was detected by Western blot and immunohistochemistry, which reflected the regeneration of axon. Results: 1 rat sci model was successfully established, 2GFP fluorescence was observed locally in plasmid injection area, indicating that shRNA plasmid was successfully transfected in vivo; 3Nis-shRNA plasmids and Nis-siRNA fragments could effectively inhibit the transcription level of nischarin, and the results of body weight monitoring showed that there was no significant difference among the groups, suggesting that shRNA and siRNA were safe to use. (5) the score of BBB motor function in sci Nis-shRNA group was higher than that in sci Ctl-shRNA group and sci group in open field test, and the BBB motor function score of sci Nis-siRNA group was higher than that of sci Ctl-siRNA group and sci group. 6FSA of sci Nis-shRNA group and sci Nis-siRNA group was lower than that of control group and sci group, and the RHI of sci Nis-shRNA group and sci Nis-siRNA group was higher than that of sci Nis-siRNA group and sci group. 7 in retrograde tracer test, the number of fluorescent gold positive neurons in red nucleus of sci Nis-shRNA group and sci Nis-siRNA group was more than that of control group and sci group. 1 week and 6 weeks after sci, western blot results showed that the expression of GAP43 in sci Nis-shRNA group and sci Nis-siRNA group was significantly higher than that in control group and sci group, and immunofluorescence intensity showed that GAP43 expression in sci Nis-shRNA group and sci Nis-siRNA group was higher than that in sci group and sci group. Conclusion: inhibiting the expression of Nischarin in injured spinal cord is beneficial to promote axonal regeneration and motor function rehabilitation in sci rats.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R651.2

【参考文献】