Objective To investigate the effect of lentivirus-mediated three transcription factors ( Ascl1-Brn2-Ngn2, ABN) transfer for the treatment of Parkinson's disease (PD).Methods The PD model was established by injecting 6-OHDA unilaterally into the rat striatum, and was divided into high-dose treatment group (n＝11), low-dose treatment group ( n ＝11), and no-load virus PD model group ( no-load virus group, n ＝8).Another PBS negative control group ( n ＝6 ) was established.After stereotactic injection of viral preparations or PBS using micropipettes into the rat striatum , the effect of LV-ABN was evaluated with rotation behavioral test , Western blot was performed to detect the expression of the TH protein in the substance nigra ; The levels of DA and DOPAC in the rats striatal were analyzed by High Performance Liquid Chromatography .Results PBS negative control group rats not apparent rotation behavior .All PD model rats since about 7 -10 d after partial lateral PD symptoms , animal spontaneous rotate to the right , spin to the right after intramuscular injection of APO (10 -17 times/min).Gene therapy before no-load virus group rats compared with the two treatment groups , the average spin laps differences had no statistical significance (all P＞0.05); Light group rats in no-load virus and virus apparent rotation behavior has been observed before and after injection (P＞0.05); And two ABN gene treatment group after treatment for 3 weeks with the rotational behavior significantly improved ( all P＜0.05 ), of which the high dose treatment group after treatment for 2 weeks on average slewing ring number decreases , trend lower dose treatment groups .PBS operation side TH within the substantia nigra express the highest negative control group , no-load virus group rats significantly lower TH protein expression .The ABN gene therapy of two groups of rats after TH protein expression significantly higher than the no-load virus group, but still lower than that of PBS negative control group .Twelve weeks after transplantation,the level of DA and DOPAC no-load virus group were significantly lower than that of PBS negative control group (all P＜0.05).And ABN gene DA and DOPAC content in the high and low dose group were signific- antly higher than the no-load virus group ( all P＜0.05), but still lower than that of PBS negative control group . Conclusion Our results suggest that LV-ABN delivery can ameliorate abnormal rotational behavior in a PD rat model, and the therapeutic effect might be due to promote dopaminergic neuron regeneration or increase expression of dopamine in striatal.%目的 探讨慢病毒介导的三种神经元谱系相关转录因子(Ascl1-Brn2-Ngn2,ABN)脑内转移治疗帕金森病(PD)的疗效.方法 采用SD大鼠单侧纹状体注射6-羟基多巴胺(6-OHDA)法建立PD模型并分为高剂量治疗组(n＝11)、低剂量治疗组(n＝11)和空载病毒PD模型组(空载病毒组, n＝8),另设PBS阴性对照组(n＝6).应用立体定向仪将携带ABN基因的慢病毒(LV)注射至PD模型大鼠纹状体,观察行为学变化并用Western blot方法检测黑质内酪氨酸羟化酶(TH)表达;利用高效液相色谱法检测多巴胺(DA)及二羟苯乙酸(DOPAC)含量.结果 PBS阴性对照组大鼠未出现明显旋转行为;所有PD模型大鼠自术后第7～10 d左右开始出现偏侧PD症状,动物自发向右侧旋转,肌内注射APO后出现快速向右侧旋转(10 ～17 次/min).基因治疗前空载病毒组大鼠与两治疗组相比,平均旋转圈数差异无统计学意义(均P＞0.05);空载病毒组大鼠在空载慢病毒注射前后未见有明显旋转行为改善( P＞0.05);而两ABN基因治疗组在治疗后3周有显著的旋转行为改善(均P＜0.05),其中高剂量治疗组在治疗后2周出现平均旋转圈数减少,趋势较低剂量治疗组更加明显. PBS阴性对照组术侧黑质内TH表达最高,空载病毒组大鼠TH蛋白表达明显降低;经ABN基因治疗后的两组大鼠TH蛋白表达明显高于空载病毒组,但仍低于PBS阴性对照组.移植后12周,空载病毒组DA及DOPAC水平均显著低于PBS阴性对照组(均P＜0.05);而ABN基因高低剂量组中DA及DOPAC含量均明显高于空载病毒组(均P＜0.05),但仍低于PBS阴性对照组.结论 ABN基因脑内转移可显著改善PD大鼠的旋转行为,其作用机制可能与促进多巴胺能神经元再生、增加纹状体多巴胺表达相关.
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