胶质细胞系源性神经营养因子修饰脂肪间质干细胞与共培养多巴胺能神经元的存活

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14 d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1×109 vp/mL的胶质细胞系源性神经营养因子重组腺病毒作用细胞1 h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24 h后加入分离的多巴胺能神经元，继续培养7 d；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24 h后细胞上清中出现胶质细胞系源性神经营养因子蛋白，72 h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7 d的细胞进行酪氨酸羟化酶免疫荧光染色，分别以波长为488 nm和563 nm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。

Effect of glial cell-derived neurotrophic factor-modified adipose-derived stem cells on survival of co-cultured dopaminergic neurons

BACKGROUND: Reports regarding adipose-derived stem cells (ADSCs) differentiation into dopaminergic (DN) neurons are few; in addition, there is not experimental evidence of the effect of ADSCs on maintaining the survival of DN neurons. OBJECTIVE: To investigate the effect of glial cell-derived neurotrophic factor (GDNF)-modified adipose-derived stem cells on survival of DN neurons under co-cultured condition. DESIGN, TIME AND SETTING: The in vitro cytology experiment was conducted at the Institute of Otolaryngology-Head and Neck Surgery and Key Laboratory of Zoonoses of Ministry of Education between March and December 2007. MATERIALS: Wistar rats with 3-weeks-old, or 14 days of pregnancy were provided by Norman Bethune College of Medicine, Jilin University. METHODS: The GDNF recombinant adenovirus was constructed by using pAdTrackCMV and pAdEasy-1 system. DN neurons were obtained from the rostral mesencephalic tegmentum of Wistar rat embryos by using trypsin and collagenase method. ADSCs isolated from rat inguinal fat pads were digested with collagenase II, cultured and passaged in vitro. When the cells reached 60% confluency at the 3rd passage, cells were transfected with 1×109 vp/mL of Ad-GDNF for 1 hour and then transferred into growth medium for another 24 hours, and GDNF level in cell supernatant was detected by ELISA assay. Meanwhile, the co-cultured of ADSCs and DN neurons were carried out for following 7 days. With GFP-modified ADSCs was served as a control group. MAIN OUTCOME MEASURES: The effect of co-cultured condition on the survival of DN neurons, as well as the differentiation of GDNF-modified ADSCs was detected by immunofluorescence staining. RESULTS: GDNF appeared in ADSCs supernatant at 24 hours after Ad-GDNF transfection and reached a peak at 72 hours. There was approximately 80% GFP-positive labeled in ADSCs. The tyrosinase hydroxylase staining results demonstrated that the rate of survival DN neurons were significantly increased than in DA neurons cultured alone, co-cultured group of GFP-modified ADSCs and GDNF-modified ADSCs groups (55%, 15%, 25%, P < 0.01). However, there were no co-expressing TH and GFP positive cells appeared at 7 days of co-culture, which indicated that the co-cultured condition was not available to ADSCs differentiation. CONCLUSION: The co-cultured of GDNF modified ADSCs and DN neurons can promote the survival and growth of cultured DN neurons, however, it can not induce ADSCs differentiate into DN neurons.