增强型绿色荧光蛋白标记兔脂肪干细胞体外复合膀胱黏膜下层的生长特性

In vitro growth characteristics of green fluorescent protein labeled rabbit adipose derived stem cells on bladder acellullar matrix grafts

背景:绿色荧光蛋白作为细胞示踪标记已得到广泛应用,作者所查尚未见到以增强型绿色荧光蛋白作为标记方法用于研究脂肪干细胞与膀胱脱细胞基质复合后体外生长特性的报道。目的:利用绿色荧光蛋白标记兔来源的脂肪干细胞,观察其作为种子细胞复合膀胱脱细胞基质体外培养的生长特性,为复合物回植体内时间提供参考。设计、时间及地点:细胞标记示踪实验,于2009-01/05在上海组织工程研究与开发中心及上海第六人民医院实验动物房完成。材料:取2只新西兰大白兔,制备膀胱脱细胞基质。取2只新西兰大白兔脂肪分离脂肪干细胞。方法:体外培养增殖后脂肪干细胞转染增强型的绿色荧光蛋白,转染前后根据增殖速度,绘制生长曲线。然后将增强型绿色荧光蛋白转染的第3代脂肪干细胞以30×106的浓度接种于1cm×1cm大小的膀胱脱细胞基质支架材料上培养。主要观察指标:观察细胞转染后的生长特性,检测复合后的细胞总数,荧光显微镜观察复合后细胞形态。结果:脂肪干细胞经转染后生长增殖速度未受明显影响。细胞DNA检测示复合培养7d细胞增殖状态最佳。荧光显微镜观察显示培养7d细胞增殖良好,10d后呈现老化形态。结论:增强型的绿色荧光蛋白转染不影响脂肪干细胞传代、增殖。脂肪干细胞-膀胱脱细胞基质复合物体外培养7d左右回植较合适。

BACKGROUND： Green fluorescent protein has been used widely for cell trace labeling, but according to the author＇s investigation, there is still few reports on the enhanced green ,fluorescent protein （EGFP） using for labeling in the study on the in vitro growth characteristics of adipose derived stem cells on bladder acellullar matrix graft （ADSCs-BAMG）. OBJECTIVE： To observe, with EGFP labelling skill, the in vitro growth characteristics of rabbit ADSCs that were used as seed cells in its co-culture with BAMG and to get the proof of the proper in vivo replantation time of the ADSCs-BAMG compounds. DESIGN, TIME AND SETTING： A cell trace labeling experiment was performed at the Shanghai Tissue Engineering Research and Development Center and the Experimental Animal Department of the Sixth People＇s Hospital of Shanghai between January and May in 2009. MATERIALS： Two New 7ealand rabbits were used to prepare BAMG and the other two were used to isolate ADSCs. METHODS： The in vitro cultured and proliferated ADSCs were labeled by EGFP transfection. Curves were drawn in terms of the proliferation rate of ADSCs before and after tansfection. After that, the EGFP labeled ADSCs of passage 3 were celtured at the concentration of 30×10^6 on the 1 cmxl cm BAMGs scaffol. MAIN OUTCOME MEASURES： The growth characteristics of EGFP labeled ADSCs were observed, the total cell number after combination were detected and the morphology of cells after combination was observed with fluorescent microscope. RSULTS： There was no difference between the grow curves of the ADSCs before and after labeled by EGFP transfectlon. The cell＇s DNA detection showed that ADSCs exhibited the best proliferation state on BAMG at day 7 after combination. The fluorescent microscope showed that the cells grew well on BAMG at day 7 and aged at day 10 after combination. CONCLUSION： The EGFP has no effect in ADSCs passage and proliferation. The ADSCs-BAMG compound is proper to be replanted at about day 7 after co-culture.