慢病毒介导的shRNA干扰PTEN表达促皮层神经元轴突再生及脊髓损伤修复

摘要

目的 观察慢病毒介导特异性短发夹RNA (short hairpin RNA,shRNA)干扰第10号染色体同源丢失性磷酸酶张力蛋白基因(phosphatase and tensin homology deleted on chromosome ten,PTEN)表达对皮层神经元轴突再生及脊髓损伤修复的影响.方法 体内实验和体外实验两部分各分四组:阴性对照组(DMEM)、空载慢病毒组(Lenti-control)、空白载体组(Lenti-scramble)、慢病毒介导shRNA组(Lenti-shRNA).体外神经元转染72 h后,Western blot检测各组PTEN表达情况,免疫荧光检测神经元轴突再生能力;体内载体注射1周后,Western blot检测各组PTEN表达情况;6周后荧光显微镜观察皮质脊髓束穿越脊髓损伤局部周围增强绿色荧光蛋白荧光强度及突触素表达情况.采用大鼠脊髓损伤评分评价大鼠后肢运动恢复情况.结果 慢病毒介导shRNA组体外转染神经元后PTEN表达水平比阴性对照组下降83.75％±2.85％,与其他组比较具有统计学意义(F=4277,P＜ 0.05);轴突长度(249.70±10.70)μm,大于阴性对照组(95.71±20.24) μm、空载慢病毒组(97.00±22.82)μm及空白载体组(87.57±19.34)μm,差异具有统计学意义(F=84.74,P＜ 0.05);每个神经元一级突起数量(5.800±0.359)个,大于阴性对照组(2.800±0.678)个、空载慢病毒组(2.900±0.389)个及空白载体组(3.000±0.877)个,其差异具有统计学意义(F=16.47,P＜ 0.05);神经元突起穿越硫酸软骨素蛋白多糖基质的百分比20.60％± 1.80％,大于阴性对照组6.70％±1.45％、空载慢病毒组5.50％±1.69％、空白载体组5.60％±1.77％,其差异具有统计学意义(F=94.90,P＜0.05).慢病毒介导shRNA注射大脑皮层运动区后,第6周大鼠脊髓损伤评分达(13.29±0.42)分,高于阴性对照组(7.00±1.48)分,空载慢病毒组(6.43±1.43)分,空白载体组(6.29±1.22)分,其差异具有统计学意义(F=44.85,P＜ 0.05).皮层组织PTEN表达水平下降84.57％±1.87％,损伤中心尾端见绿色荧光,突触素染色阳性面积明显增大.结论 慢病毒介导shRNA下调PTEN基因表达后可明显提高脊髓损伤后轴突再生能力,促进神经功能修复.%Objective To observe the effects of PTEN silencing on axon regeneration and functional recovery after spinal cord injury (SCI).Methods Four groups were designed:DMEM、lenti-control、lenti-scramble、lenti-shRNA.Cortical neurons were seeded on or adjacent to chondroitin sulfate proteoglycans (CSPGs).Three days after lentivirus infection,the expression of PTEN were assessed by Western blot,and immunocytochemistry method were applied to examine the length,number and crossing behavior of neurites;Lentivirus was locally injected into the conical motor area of the rats with spinal cord injury.1 week later,the expression of PTEN was assessed by Western blot.6 weeks later,motor function of hind limbs was evaluated by Basso Beattie Bresnahan (BBB) score,while the expressions of green fluorescence in injured spinal cord were observed by fluorescence microscope,and immunohistochemical method was used to observe the expression of synaptophysin changes in spinal tissues.Results Less expression of PTEN was detected in lentivirus-shRNA,and neurites with PTEN silencing exhibited significant enhancements in elongation,initiation and crossing ability when they encountered CSPGs in vitro.Better hindlimb functional recovery,less expression of PTEN and more synaptophysin positive response area were detected in rats injected with lentivirus-shRNA compared with other groups.Moreover,green fluorescence was found in the caudal region of injured spinal cord of rats injected with lentivirusshRNA.Conclusion Lentiviral-mediated PTEN-shRNA can significantly improve the axon regeneration and neurological functional recovery after spinal cord injury.