TH基因修饰的永生化神经干细胞移植对帕金森病大鼠行为和多巴胺代谢的影响

 目的 评价脑内移植酪氨酸羟化酶(tyrosine hydroxylase, TH)基因修饰的温度敏感型永生化神经干细胞(immortalized neural stem cells, iNSCs)的生物学稳定性和安全性,探讨TH基因表达对帕金森病(Parkinson's disease, PD)大鼠旋转行为和多巴胺代谢的影响. 方法 6-羟基多巴胺(6-OHDA)制备PD模型大鼠73只,随机分为3组.治疗组(36只,TG)脑内纹状体移植体外转染稳定表达TH基因的iNSCs株(RMN-TH),对照组(27只,CG)同部位植入未转染TH基因的RMN细胞株,余10只为模型组(MG).分别比较细胞移植后不同时间点实验大鼠肿瘤发生、排斥反应和阿扑吗啡(APO)诱导旋转行为、免疫组织化学染色观察TH表达;高效液相色谱电化学方法检测多巴胺(dopamine, DA)及其代谢产物3、4-二羟苯乙酸(DOPAC)和高香草酸(HVA)水平. 结果移植细胞在受体大鼠纹状体内存活均超过6个月,并广泛迁移、稳定表达TH,而未诱发肿瘤和排斥反应.RMN-TH TG组的APO诱导旋转行为得到明显改善,移植后第8周与CG组、MG组比较,旋转圈数分别下降64.17%和61.54%(P＜0.01);TG组大鼠移植侧纹状体内TH表达显著高于健侧(P=0.0029)、CG组移植侧和MG组(P=0.0031).TG组纹状体DA及其代谢产物水平明显高于CG组和MG组(P=0.0027).与MG组比较,CG组大鼠旋转行为、TH表达,以及DA,DOPAC和HVA水平均无统计学差异.结论 TH基因修饰的iNSCs细胞株RMN-TH可以在PD大鼠纹状体内长时间安全存活并稳定表达TH,显著增加内源性DA及其代谢产物水平,有效改善模型大鼠PD症状.

The effect of intracerebral transplanted conditional immortalized neural stem cells modified to express tyrosine hydroxylase gene on rotational behavior and dopamine metabolism in rodents with Parkin's disease

Objective To evaluate the safety and biological stability of intracerebral transplanted conditional immortalized neural stem cells(iNSCs) modified to express tyrosine hydroxylase(TH) gene,and to explore the therapeutic effect of expression of TH gene on rotational behavior and dopamine metabolism in hemiparkinsonian rodents.Methods Seventy - three Sprague—Dawley rats were randomly divided into three groups after a PD model was developed using 6 - hydroxydopamine.Thirty - six of them enrolled as therapy group(TG) were striatally transplanted with a iNSCs line,RMN -TH,which had been characterized in vitro before being transduced with a retrovirus encoding TH gene to obtain TH - expressing cells.Another twenty - seven of them were isotopically transplanted RMN cell line without transduction with TH gene as control group(CG),and the 10 remaining rats that received no intervention served as model group(MG).Tumorigenesis,rejective events,and ratational behavior induced by apomorphine were monitored posttransplantation in the three groups.Immunohistochemistry and high performance liquid chromatography coupled with electrochemistry were used to detect the expression of TH,and levels of dopamine,dihydroxy - phenyl acetic acid(DOPAC ) and homovanillic acid (HVA),separately.Results Transplanted cells,RMN -TH,survived in recipients’ striatum for a minimum of 6 months,migrating extensively,expressing TH persistently but without tumorigenesis or cellular rejection.Rotational behavior induced by APO was ameliorated in TG.Compared with CG or MG,the circling number of TG eight weeks after transplantation was reduced by 64.17%and 61.54%,respectively(P<0.01).The expression of TH on the transplant side of striatum in TG was higher than on the uninjured side,the transplant side of striatum in CG,or either side of MG(P = 0.0031).Levels of DA,DOPAC and HVA in TG were markedly higher than those in CG and MG(P = 0.0027 ).However,there was no significant difference in the rotational behavior,expression of TH, level of DA or its metabolic product between CG and MG.Conclusion Transplanted iNSCs line modified by TH gene,RMN - TH, can safely survive in striatum and persistently express TH.Cellular transplantation with RMN - TH effectively ameliorates symptoms in rats suffering from PD,up—regulates the level of endogenous DA and its metabolic product,which can shed light on the underlying mechanism.