摘要
目的:观察对中脑腹侧多巴胺能神经元分化、成熟及存活起重要作用的Nurr1基因修饰大鼠骨髓间充质干细胞脑内移植后,对帕金森病大鼠旋转行为及纹状体多巴胺、二羟苯乙酸及高香草酸含量比值的影响。方法:实验于2003-08/2004-12在中山大学附属第一医院神经病学国家重点实验室进行。取SD雄性大鼠,右侧中脑黑质致密区注入6-羟基多巴(2g/L)3μL制备帕金森病模型,4周后将69只成功帕金森病模型随机分为3组:①对照组24只:右侧纹状体尾状核头部注入生理盐水4.5μL。②骨髓间充质干细胞组24只:相同部位注入培养纯化的骨髓间充质干细胞4.5μL。③转Nurr1基因骨髓间充质干细胞组21只:用脂质体转染法转染PcDNA3.1(+)-Nurr1入大鼠骨髓间充质干细胞并稳定表达,然后移植入模型大鼠脑内,部位和剂量与其他两组相同。观察各组大鼠旋转行为学变化,并用免疫组化、反转录-聚合酶链反应等方法检测移植细胞的Nurr1,DAT和酪氨酸羟化酶表达;利用高效液相色谱检测大鼠脑内多巴胺、二羟苯乙酸和高香草酸含量。结果:59只大鼠进入结果分析。①旋转行为学变化:与对照组相比,移植前1周无明显差异(P>0.05),移植后2～8周,另2组大鼠旋转行为明显改善(P<0.05);转Nurr1基因骨髓间充质干细胞组在移植后2～4周较骨髓间充质干细胞组轻(P<0.05),但至8周时两组无差异(P>0.05)。②Nurr1及DAT和酪氨酸羟化酶阳性表达:免疫组化结果显示转Nurr1基因骨髓间充质干细胞组移植区能够稳定表达Nurr1且少量细胞表达DAT,但未发现酪氨酸羟化酶阳性细胞,其他两组移植区未发现阳性细胞。③Nurr1及DAT和酪氨酸羟化酶mRNA表达:移植后2～8周,聚合酶链反应产物经电泳、3组大鼠术侧纹状体染色后均可见1030bp片段(DAT),241bp片段(Nurr1),438bp片段(β-actin),但未发现酪氨酸羟化酶条带(647bp)。④脑内纹状体多巴胺、二羟苯乙酸、高香草酸术侧与健侧比值:移植后2,8周,两治疗组均较对照组增高(P<0.05),但8周时两治疗组比较无差异(P>0.05)。结论:转Nurr1基因大鼠骨髓间充质干细胞在帕金森病大鼠模型纹状体内可以存活(8周以上),在该环境中Nurr1基因的过表达可以促进大鼠骨髓间充质干细胞的DAT基因表达,能显著改善帕金森病大鼠的旋转行为,并在一定时期内维持纹状体内相对较高的多巴胺、二羟苯乙酸、高香草酸水平。
s AIM: To observe the changes of rotational behavior and the content ratio of corpus striatum dopamine, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA)in rats with Parkinson disease following the transplantation of marrow mesenchymal stem cells transfected by Nurr 1 gene which plays an important role in the differentiation,maturity and survival of mesencephalic ventral dopaminergic neuron. METHODS: The experiment was conducted in the State Key Laboratory for Neurology,the First Affiliated Hospital of Sun Yat-sen University from August 2003 to December 2004.SD male rats were chosen.3 μL 6-hydroxydopamine(2 g/L) was injected into substantia nigra dense area in the right-side midbrain to make Parkinson disease model.Four weeks later, 69 Parkinson disease models were randomly divided into 3 groups:①Control group(n=24):4.5 μL normal saline was injected into head of caudate nucleus of right-side corpus striatum.②BMMCs group(n=24):4.5 μL purified BMMCs was injected into the same part.③BMMCs transfected by Nurr1 group(n=21): the transfected PcDNA3.1(+)-Nurr1 with liposomes transfection method were transplanted into the brain in rats following transplanted into BMMCs with stable expression and the injected part and injected dosage were the same as that in the above two groups. The change of rotational praxiology was observed in the rats. The expression of Nurr1,DAT and tyroxine hydroxylase of transplanted cells were detected with immunohistochemical staining and reverse transcription-polymerase chain reaction methods;The content of corpus striatum dopamine,DOPAC and HVA were measured with high performance liquid chromatography. RESULTS: Fifty-nine rats entered the result analysis.①The change of rotational praxiology: Compared with the control group, there was no difference in the first week after transplantation(P >0.05),the rotational behavior of the rats improved significantly in the other 2 groups 2-8 weeks after transplantation(P< 0.05);that in the BMMCs modified by Nurr1-gene group was less obvious than that in the BMMCs group(P< 0.05)and no difference existed at week 8(P >0.05).②the positive expression of Nurr1,DAT and tyroxine hydroxylase:Immunohistochemical result showed that transplanted area in the BMMCS modified by Nurrl gene group can stably express the Nurrsssand few cells expressed DAT, but the positive cells of tyroxine hydroxylase were not found. There were not positive cells in the other 2 groups. ③The expression of Nurr1, DAT and tyroxine hydroxylase mRNA:Electrophoresis was used on the product of polymerase chain reaction. After corpus striatum was stained on the operation side of the rats, 1 030 bp segment (DAT),241 bp segment(Nurr1),438bp segment(β-actin)were found , but tyroxine hydroxylase band (647 bp)was not found. ④Comparison of the ratio of striatum dopamine, DOPAC and HVA between the operation side and health side: those in the two treatment groups were both higher than those in the control group 2 and 8 weeks after transplantation (P< 0.05),but there was no difference between the two groups at week 8(P >0.05). CONCLUSION: BMMSCs transfected by Nurr1-gene can survive in the striatum of rat model at least 8 weeks after administration.The over-expression of Nurr1-gene can promote the DAT gene expression of BMMCs,improve significantly the rotational behavior of the rats with Parkinson's disease and maintain the relatively high level of Nurr1,DAT and tyroxine hydroxylasem in corpus striatum in certain period.
出处
《中国临床康复》
CSCD
北大核心
2005年第22期56-59,i002,共5页
Chinese Journal of Clinical Rehabilitation
基金
国家自然科学基金资助项目(30370509)
教育部留学回国人员基金(2003-406)~~
