背景:目前尚未见脂肪间质干细胞体外诱导分化成多巴胺能神经元的报道,且有关脂肪间质干细胞维持多巴胺能神经元存活的机制也缺乏实验证据。目的:观察腺病毒介导胶质细胞系源性神经营养因子基因修饰的脂肪间质干细胞对共培养条件下多巴胺能神经元存活的影响。设计、时间及地点:细胞学体外对比观察,于2007-03/12在吉林省耳鼻咽喉研究所和教育部吉林大学人兽共患病重点实验室完成。材料:3周龄Wistar大鼠、孕14 d Wistar大鼠由吉林大学白求恩医学院实验动物中心提供。方法:采用pAdTrackCV和pAdEasy-1系统构建重组胶质细胞系源性神经营养因子腺病毒。取孕14d大鼠,采用酶消化法培养中脑多巴胺能神经元。取Wistar大鼠腹股沟处脂肪,酶消化法分离培养脂肪间质干细胞,体外培养至第3代当细胞生长至60%融合时,以病毒滴度为1×109vp/mL的胶质细胞系源性神经营养因子重组腺病毒作用细胞1h后,转移到生长培养基继续培养,通过ELISA法检测培养上清胶质细胞系源性神经营养因子水平。设立3组:Ad-GDNF转染共培养组、Ad-GFP转染共培养组分别在脂肪间质干细胞经相应病毒转染24h后加入分离的多巴胺能神经元,继续培养7d;单纯多巴胺能神经元培养组不加入脂肪间质干细胞。主要观察指标:采用免疫荧光技术检测共培养环境对多巴胺能神经元存活的影响,共培养环境对胶质细胞系源性神经营养因子修饰脂肪间质干细胞分化的影响。结果:脂肪间质干细胞在pAd-GDNF转染24h后细胞上清中出现胶质细胞系源性神经营养因子蛋白,72h达高峰,pAd-GDNF对脂肪间质干细胞的转染效率约为80%。酪氨酸羟化酶免疫荧光染色结果发现,Ad-GDNF转染共培养组多巴胺能神经元存活率明显高于单纯多巴胺能神经元培养组、Ad-GFP转染共培养组(55%,15%,25%,P<0.01)。对共培养7d的细胞进行酪氨酸羟化酶免疫荧光染色,分别以波长为488nm和563nm进行单通道扫描,未发现同时表达绿色荧光蛋白和酪氨酸羟化酶的细胞,表明此共培养环境可能不具备诱导脂肪间质干细胞分化为多巴胺能神经元的条件。结论:胶质细胞系源性神经营养因子基因修饰的脂肪间质干细胞与胚胎中脑分离出的多巴胺能神经元共培养,能够维持和促进多巴胺能神经元的存活,但可能不具备诱导脂肪间质干细胞分化为多巴胺能神经元的作用。 BACKGROUND: At present, no evidence has been reported that adipose-derived mesenchymal stem cells differentiate into dopaminergic neurons in vitro and there is no experimental evidence that the mechanism of adipose-derived mesenchymal stem cells maintaining the survival of dopaminergic neurons. OBJECTIVE: To observe the effect of adenovirus-mediated adipose-derived mesenchymal stem cells modified by glial cell line-derived neurotrophic factor gene on the survival of dopaminergic neurons under co-culture conditions. DESIGN, TIME AND SETTING: Comparative cytology in vitro was performed at the Department of Otorhinolaryngology, Jilin Provincial Key Laboratory of Zoonosis, Jilin Institute of Otolaryngology, Ministry of Education. MATERIALS: Wistar rats of 3 weeks old and 14th Wistar rats of pregnancy were provided by Experimental Animal Center of Bethune Medical College of Jilin University. Methods: Recombinant glial cell line-derived neurotrophic factor adenovirus was constructed by using pAdTrackCV and pAdEasy-1 system. Pregnant 14-day-old rats, the use of enzyme digestion of midbrain dopaminergic neurons. Adipose-derived mesenchymal stem cells were isolated and cultured in the inguinal region of Wistar rats, and cultured in the third passage. When the cells grew to 60% confluence, the glial cell line derived from the glioma cell line with virus titer of 1 × 109 vp / mL The neurotrophic factor recombinant adenovirus cells after 1h, transferred to the growth medium for further culture, the culture supernatant glial cell line-derived neurotrophic factor levels were detected by ELISA. Three groups were established: Ad-GDNF transfected co-culture group, Ad-GFP transfected co-culture group were added to the isolated dopaminergic neurons after transfection of ADSCs for 24 hours respectively, Meta-culture group did not add adipose-derived mesenchymal stem cells. MAIN OUTCOME MEASURES: The effects of co-culture environment on the survival of dopaminergic neurons and the effects of co-culture environment on the differentiation of glial cell line-derived neurotrophic factor-derived adipose-derived mesenchymal stem cells were detected by immunofluorescence technique. RESULTS: Adipose-derived mesenchymal stem cells showed glial cell line-derived neurotrophic factor in the supernatant of the cells transfected with pAd-GDNF for 24 hours and reached the peak at 72 hours. The transfection efficiency of adipose-derived mesenchymal stem cells by pAd-GDNF was about 80% . Tyrosine hydroxylase immunofluorescence staining showed that the survival rate of dopaminergic neurons in Ad-GDNF-transfected co-culture group was significantly higher than that in simple dopaminergic neuron culture group and Ad-GFP transfected co-culture group (55%, 15 %, 25%, P <0.01). Tyrosine hydroxylase immunofluorescent staining was performed on the 7th day co-cultured cells, single-channel scanning was performed at wavelengths of 488nm and 563nm respectively, and no cells expressing both green fluorescent protein and tyrosine hydroxylase were found, indicating that this co-culture The environment may not have the conditions that induce adipose-derived mesenchymal stem cells to differentiate into dopaminergic neurons. CONCLUSION: Adipose-derived mesenchymal stem cells modified with glial cell line-derived neurotrophic factor can coexist with dopaminergic neurons isolated from the midbrain of embryos, which can maintain and promote the survival of dopaminergic neurons but may not have the effects of inducing adipose tissue Differentiation of mesenchymal stem cells into dopaminergic neurons.