摘要目的 探讨移植酪氨酸羟化酶(TH)和neurturin (NTN)基因修饰的骨髓间充质干细胞(BMSC)对帕金森病(PD)模型大鼠的作用及其脑内相关蛋白表达的影响.方法 6-羟基多巴胺(6-OHDA)单侧两点注射法,制备PD动物模型,成功模型大鼠按随机数字法分为PD组(未移植组)、BMSC组(移植BMSC)和TH-NTN-BMSC组(移植TH-NTN-BMSC),微量进样器将BMSC和TH-NTN-BMSC移植到PD模型大鼠右侧纹状体;移植后各组大鼠腹腔注射阿朴吗啡(APO),进行行为学检测;高效液相-电化学法检测各组大鼠右侧纹状体区多巴胺(DA)及二羟苯乙酸(DOPAC)含量;免疫组化法和Western印迹检测移植后各组大鼠右侧纹状体区TH和NTN的表达,最后采用后包埋免疫金颗粒电镜技术定量分析纹状体区突触后膜上的多巴胺受体密度变化.结果 TH-NTN-BMSC组APO诱发下30 min内旋转圈数(5.7±1.3) 圈/min明显低于PD组和BMSC组的(10.8±2.2)、(9.9±1.2)圈/min(P＜0.05),右侧纹状体区DA、DOPAC含量,(0.421±0.113)、(0.093±0.012) nmol/L,显著高于PD组(0.208±0.043)、(0.043±0.017) nmol/L和BMSC组(0.231 ±0.082)、(0.044±0.023)nmol/L(P＜0.05),TH和NTN蛋白表达也显著增加,同时该组多巴胺D2受体亚型在突触后膜上的密度(623 ±96)个/μm2显著低于PD组和BMSC组的(923±132)、(860±116)个/μm2 (P＜0.05).结论 将TH和NTN结合起来进行基因治疗,既可增加脑内DA的合成,同时又能保护DA能神经元,二者共同作用达到双重治疗效果.

abstractsObjective To explore the effects of tyrosine hydroxylase-neurturin (TH-NTN) gene modified bone marrow mesenchymal stem cell (BMSC) transplantation in Parkinson's disease (PD) model rats and the alternations of correlated proteins.Methods The PD rat model was established by the 2-point injection of 6-hydroxydopamine (6-OHDA) into unilateral (right) striatum.Successful modeling rats were separated into PD,BMSC and TH-NTN-BMSC groups.BMSC and TH-NTN-BMSC groups were transplanted into BMSCs and TH-NTN gene modified BMSC cells separately into right striatum.After transplantation,ethology detection in all groups was made with an intraperitoneal injection of apomorphine (APO).Dopamine (DA) and dopacetic acid (DOPAC) in striatum were detected by high performance liquid electrochemical analysis.TH and NTN proteins in right striatum were also analyzed by immunohistochemistry and Western blot.Finally the density of dopamine receptors in post synaptic density of dopaminergic synapses of corpus striatum were compared between each group by post-embedding immunogold electron microscopy.Results After an injection of APO,rotation frequency decreased in TH-NTN-BMSC group,i.e.(5.7 ± 1.3) circles/min versus (10.8 ± 2.2),(9.9 ± 1.2) circles/min in PD and BMSC groups (P ＜0.05).For proteins in right striatum,DA,(0.421 ±0.113) and DOPAC,(0.093 ±0.012) nmol/Lincreased significantly versus (0.208 ± 0.043),(0.043 ± 0.017) nmol/L in PD and (0.231 ± 0.082),(0.044 ± 0.023) noml/L in BMSC groups (P ＜ 0.05).Also a lower density of D2 receptors at (623 ± 96) /μm2 in TH-NTN-BMSC group versus (923 ± 132)/μm2 in PD and (860 ± 116)/μm2 in BMSC groups was also found.Conclusion The combined therapy of TH and NTN genes increases the synthesis of DA and also protects the dopaminergic neurons to achieve double therapeutic effects.It may provide potential innovations of PD genetic therapy.