慢病毒介导artemin基因修饰的骨髓间充质干细胞对帕金森病大鼠的治疗作用  

Therapeutic effects of transplantation of lentivirus-mediated artemin gene modified bone marrow mesenchymal stem cells on the rat model of Parkinson＇s disease

作　　者：刘凯[1] 阴晓峰[1] 相恒伟[1] 邓文帅[1] 孙鹏[2] 

机构地区：[1]青岛大学医学院临床系,266071 [2]青岛大学医学院附属医院黄岛分院

出　　处：《中华神经科杂志》2015年第8期697-703,共7页Chinese Journal of Neurology

基　　金：国家自然科学基金项目（81171208）;山东省科技发展计划项目（2011gsf11831）;山东省自然科学基金项目（Z2008C06）

摘　　要：目的 探讨慢病毒介导的artemin（ARTN）基因修饰的骨髓间充质干细胞（MSCs）对帕金森病模型大鼠潜在性治疗作用及对脑内相关蛋白表达的影响.方法 分离培养大鼠MSCs,将携带ARTN的慢病毒载体转染MSCs;6-羟多巴胺（6-OHDA）制备帕金森病大鼠模型,按随机数字表法随机分为假手术（Sham）组、帕金森病组、移植MSCs（MSCs）组、移植空病毒转染的MSCs（LV-MSCs）组、移植慢病毒介导的ARTN基因修饰的MSCs （LV-ARTN-MSCs）组,对帕金森病大鼠左侧纹状体进行细胞移植;术后2、4、6、8周,腹腔注射阿扑吗啡,进行行为学检测;采用Western印迹及免疫荧光法检测各组大鼠黑质酪氨酸羟化酶（TH）表达变化,移植ARTN修饰的MSCs在大鼠脑组织内表达;高效液相色谱仪检测各组大鼠纹状体内多巴胺、二羟基苯乙酸、高香草酸含量.结果 LV-ARTN-MSCs组在阿扑吗啡诱发下30 min内旋转圈数（179.33±18.74）明显低于帕金森病组、MSCs组和LV-MSCs组（235.83±18.95、203.67±11.50和206.33±11.86;q=8.828,P＜0.01;q=3.802,P＜0.05;q=4.219,P＜0.05）.LV-ARTN-MSCs组大鼠黑质TH阳性细胞百分比（64.05％±5.49％）显著高于帕金森病组（34.18％±3.35％）、MSCs组（52.59％±4.48％）和LV-MSCs组（50.57％±4.41％;q=13.280、5.135、6.028,均P＜0.01）,黑质TH蛋白表达也明显提高,同时慢病毒介导ARTN修饰的MSCs可以在帕金森病大鼠脑内存活至少6周,主要集中在移植侧的纹状体区.细胞移植8周后,MSCs组（2.34±0.54）、LV-MSCs组（2.28±0.45）、LV-ARTN-MSCs组（6.54±0.51）与帕金森病组（0.87±0.07）大鼠相比,纹状体多巴胺表达明显提高（q =5.233,P＜0.05;q=5.020,P＜0.01;q=20.190,P＜0.01）,以LV-ARTN-MSCs组最明显.结论 纹状体移植慢病毒介导ARTN基因修饰的MSCs对帕金森病模型大鼠有一定的治疗作用.Objective To investigate the potential therapeutic efficacy of lentivirus-mediated artemin （ARTN） gene modified bone marrow mesenchymal stem cells （MSCs） transplantation on the rat model of Parkinson＇ s disease （PD） and the effects on expression of brain-related proteins.Methods MSCs were isolated and cultured in vitro,transfected by recombinant lentiviral vectors carrying ARTN gene.The PD rat model established by 6-hydroxydopamine （6-OHDA） was randomly divided into 5 groups：Sham group,PD group,MSCs group,MSCs transfected with empty lentiviral vectors transplanted （LV-MSCs）group and MSCs transfected with recombinant lentiviral vectors carrying ARTN gene transplanted （LVARTN-MSCs） group.The MSCs,LV-MSCs and LV-ARTN-MSCs groups were transplanted into the left striatum of each rat model of PD and ethology tests in every group were made with intraperitoneal injection of apomorphine （APO） 2,4,6,8 weeks after transplantation.The expression of tyrosine hydroxylase （TH） protein in substantia nigra （SN） was measured by Western blotting and immunohistochemistry,and immunofluorescence showed ARTN gene modified MSCs expression in rat brain tissue.The levels of dopamine （DA）,dihydroxy-phenylacetic acid and homovanillic acid in striatum of each group were detected by high performance liquid chromatography.Results After injection of APO,rotation frequency decreased in LV-ARTN-MSCs group,i.e.（179.33 ± 10.74） circles/30 min vs （235.83 ± 18.95）,（203.67 ±11.50） and （206.33 ± 11.86） circles/30 min in PD,MSCs and LV-MSCs groups （q =8.828,P 〈 0.01;q =3.802,P 〈 0.05;q =4.219,P 〈 0.05）.The percentage of TH-positive cells in SN after cell transplantation was increased significantly in LV-ARTN-MSCs group （64.05% ± 5.49%） when compared with PD group （34.18% ±3.35%）,MSCs group （52.59% ±4.48%） and LV-MSCs group （50.57% ± 4.41%）,respectively （q =13.280,5.135,6.028,all P 〈0.01）.At the same time,TH protein in SN after cell transplantation was als

关 键 词：帕金森病 间质干细胞 神经组织蛋白质类 慢病毒属 转染 疾病模型 动物 

分 类 号：R742.5[医药卫生—神经病学与精神病学]