移植脑源性神经生长因子修饰骨髓间充质干细胞对帕金森病大鼠模型治疗作用的实验研究

Experimental study on efficacy of bone marrow mesenchymal stem cell modified by plasmid pIRESneo-EGFP-BDNF in treatment of rats with Parkinson’s disease

摘要:

目的：观察移植pIRESneo-EGFP-BDNF修饰骨髓间充质干细胞（mesenchymal stem cells，MSCs）对帕金森病（Parkinson’s disease，PD）大鼠的研究。方法：将pEGFP（N1）-BDNF质粒进行改造与pIRESneo相连结，构建高拷贝质粒pIRESneo-EGFP-BDNF，采用电穿孔法转染骨髓MSCs；制备PD大鼠模型，随机分为假手术组、PD组、MSCs组、脑源性神经生长因子（brain-derived neurotrophic factor，BDNF）组，经侧脑室移植MSCs或pIRESneo-EGFP-BDNF修饰骨髓MSCs术后2、4、8周，腹腔注射阿朴吗啡诱导PD大鼠旋转行为；采用Western blot方法测定中脑黑质BDNF、酪氨酸羟化酶（tyrosine hydroxylase，TH）蛋白的含量。结果：双酶切鉴定pIRESneo-EGFP-BDNF构建成功；移植术后2、4、8周MSCs组和BDNF组大鼠与PD组比较旋转次数明显减少（P<0.05），以BDNF组较MSCs比较改善更为明显（P<0.05）；移植细胞干预PD模型8周后中脑黑质BDNF、TH蛋白的含量，BDNF组较MSCs组、PD组明显增高（P<0.05）。结论：经侧脑室移植pIRESneo-EGFP-BDNF修饰的骨髓MSCs能明显改善PD大鼠的行为能力，显著提高中脑黑质BDNF、TH蛋白的含量。

Abstract:

Objective：To investigate efficacy of bone marrow mesenchymal stem cells（MSCs） modified by plasmid pIRESneo-EGFP-BDNF in treatment of rats with Parkinson’s disease（PD）. Methods：pIRESneo-EGFP-BDNF plasmid was established by reconstructing pEGFP（N1）-BDNF plasmid and bone marrow MSCs was transfected by electroproation. PD rat models were set up by 6-OHDA and then divided into four groups randonly：sham group，PD group，MSCs group and brain-derived neurotrophic factor（BDNF） group. Rotating behaviors of rats were induced by intraperitoneal injection of Apomorphine at 2，4，8 weeks after bone marrow MSCs transplantation or MSCs modification of plasmid pIRESneo-EGFP-BDNF through cerebral lateral ventricle. BDNF protein and tyrosine hydroxylase（TH） protein in mesencephalic nigra were measured by Western blot. Results：Plasmids pIRESneo-EGFP-BDNF were constructed successfully，which was identified by double digestion. Rotation numbers were significantly decreased in MSCs group and BDNF group than in PD group at 2，4，8 weeks after the transplantation（P<0.05），with more evident improvement in BDNF group than in MSCs group（P<0.05）. Levels of BDNF protein and TH protein in nigra at 8 weeks after PD model being intervened by cell transplantation were increased significantly in BDNF group than in MSCs group and PD group. Conclusions：Transplantation of pIRESneo-EGFP-BDNF modified MSCs through cerebral lateral ventricle can improve PD rats’ behavior and increase BDNF and TH protein levels.