摘要
目的 探讨Artemin（ARTN）对6-羟基多巴胺（6-OHDA）诱导的多巴胺能神经元损伤的保护作用及对帕金森病（PD）大鼠脑内相关蛋白表达的影响。 方法 分离培养大鼠骨髓间充质干细胞（MSCs）,将携带ARTN基因的慢病毒载体转染MSCs,获得稳定表达ARTN的MSCs细胞株。应用MTT法测定细胞存活率,Hochest33258荧光染色观察凋亡细胞形态学改变,同时检测Lv-ARTN-MSCs细胞上清液对6-OHDA诱导SH-SY5Y细胞凋亡作用的影响。制备PD大鼠模型,按照随机数字表法分为PD组（左侧纹状体输注生理盐水5μL）、MSCs组（左侧纹状体移植MSCs,1.0×10^5个/5 μL）、Lv-MSCs组（左侧纹状体移植Lv-MSCs,1.0×10^5个/5 μL）、Lv-ARTN-MSCs组（左侧纹状体移植Lv-ARTN-MSCs,1.0×10^5个/5 μL）,每组各6只;另取6只大鼠设为假手术组（以等量生理盐水代替6-OHDA,手术步骤及坐标同PD模型大鼠制备）。Western blotting检测各组大鼠纹状体酪氨酸羟化酶（TH）和多巴胺转运体（DAT）表达。 结果 Lv-ARTN-MSCs组可见明显的ARTN蛋白条带。Lv-ARTN-MSCs上清液可明显拮抗6-OHDA引起的细胞存活率降低,与6-OHDA组相比,Lv-ARTN-MSCs组细胞存活率提高13.67％,差异有统计学意义（P＜0.05）。细胞移植8周后,MSCs组、LV-MSCs组、LV-ARTN-MSCs组与PD组大鼠相比,纹状体TH、DAT蛋白表达明显提高,差异有统计学意义（P＜0.05）,以LV-ARTN-MSCs组最明显。 结论 ARTN作为一种分泌蛋白,可以抑制6-OHDA诱导的多巴胺能神经元损伤,推测机制为上调TH、DAT蛋白表达。
Objective To discuss the protective effect of Artemin （ARTN） on 6-hydroxydopamine （6-OHDA）-mediated neurotoxic injury,and the alternations of related proteins in Parkinson＇s disease （PD） model rats.Methods Bone marrow mesenchymal stem cells （MSCs） were isolated and cultured in vitro.After being transfected with recombinant lentiviral vectors carrying A RTN gene,MSCs stably expressed ARTN were chosen （Lv-ARTN-MSCs cells）.The SH-SY5Y cells were treated with supematant of Lv-ARTN-MSCs prior to 6-OHDA treatment,and then,cell survival rate was measured by MTT assay and morphologic changes in cultured SH-SY5Y cells were observed by fluorescence microscope （Hochest33258 staining）.PD rat models were established and randomly divided into four groups （n=6）：PD group,MSCs group,Lv-MSCs group and Lv-ARTN-MSCs group; and sham-operated group （n=6） was also chosen.The PD,MSCs,Lv-MSCs and Lv-ARTN-MSCs groups were transplanted with 5 μL of saline,MSCs （1.0×l0^5 cell/5 μL）,empty virus modified MSCs （1.0×10^5 cell/5 μL） and ARTN gene modified MSCs （1.0×10^5 cell/5 μL）,respectively,into the left striatum; rats in the sham-operated group were injected with saline when rats in the other groups were received 6-OHDA injection （the same surgical procedures and coordinates）.The expressions of tyrosine hydroxylase （TH） and dopamine transporter （DAT） in the striatum were measured by Western blotting.Results Western blotting indicated obvious ARTN protein expressions in the Lv-ARTN-MSCs groups.The supematant of Lv-ARTN-MSCs could effectively reduce the apoptosis rate induced by 6-OHDA; as compared with that of SH-SY5Y cells in the 6-OHDA group,the cell survival rate in the Lv-A R TN-MSCs group increased by 13.67%,with significant difference （P〈0.05）.Eight weeks after transplantation,the levels of TH and DAT protein in the striatum were elevated significantly in MSCs group,Lv-MSCs group and Lv-ARTN-MSCs group as compared with those in the PD group （P〈0.05）,and the L
出处
《中华神经医学杂志》
CAS
CSCD
北大核心
2015年第2期125-130,共6页
Chinese Journal of Neuromedicine
基金
国家自然科学基金（81171208）
关键词
帕金森病
骨髓间充质干细胞
基因转染
Artemin
Parkinson＇s disease
Mesenchymal stem cell
Artemin gene
Gene transfection
