GFP+Luc双报告基因标记脂肪干细胞裸鼠体内示踪研究

In vivo tracking of adipose-derived stem cells labeled with green fluorescent protein and luciferase dual reporter gene

目的探索慢病毒载体GFP+Luc双报告基因标记和生物发光成像技术活体内示踪脂肪干细胞的可行性。方法利用慢病毒载体将绿色荧光蛋白(GFP)和荧光素酶(Luc)双基因标记脂肪干细胞,利用荧光显微镜观察人脂肪干细胞(ADSCs)体外转染率,裸鼠皮下注射脂肪干细胞后1 h,1、2、4、6和8周利用生物发光成像系统连续监测ADSCs在裸鼠活体内的发光强度。离体组织DAPI染色鉴定移植细胞的存在及其存在的组织层次。结果荧光显微镜下观察慢病毒感染后的人ADSCs的GFP表达率高,流式细胞术测定转染率为87.3%,传至25代的人ADSCs仍稳定表达GFP。慢病毒载体对人ADSCs的生长和增殖没有影响。裸鼠注射ADSCs后活体内生物发光时间可持续8周,ADSCs主要分布在移植部位。离体组织切片鉴定标记的脂肪干细胞存在于移植部位皮下。结论慢病毒介导GFP+Luc双报告基因标记和生物发光技术可用于监测脂肪干细胞在动物体内的生物行为。

Objective To explore the feasibility of in vivo tracking of adipose-derived stem cells （ADSCs） by using lentiviral vectors containing green fluorescent protein （GFP） and luciferase （Luc） dual reporter gene transfection and bioluminescent imaging techniques. Methods The ADSCs were isolated from human adipose tissue samples of cosmetic subdermal liposuction. The ADSCs were transfected by lentiviral vectors containing GFP and Luc. The labeling efficiency was evaluated by fluorescence microscope and flow cytometry/n vitro. The transfected ADSCs were subcutaneously injected into BALB/C mice, and then a non-invasive bioluminescence imaging procedure was performed 1 h, 1, 2, 4, 6 and 8 weeks after transplantation. The DAPI staining of skin dssues was performed to identify the survival and the location of transplanted ADSCs. Results High GFP expression was observed in transfected ADSGs by lendviral vector under fluorescence microscope. The transfecdon efficiency of lendviral vectors containing GFP and Luc was 87. 30/0. Transfected ADSCs can express GFP and Luc after 25 successive generations, and the transduction of ADSCs with lentiviral bioluminescence reporter vectors had no effect on cell proliferation. The bioluminescence of ADSCs in vivo can be detectable as long as 8 weeks after transplandon, and the ADSCs mainly survive in the inoculating site of the thigh. Histological analysis of sections showed that the labeled ADSCs located in the subcutaneous layer of the implantation site. Conclusion Using lentiviral vectors containing GFP and Luc dual reporter gene transfecfion and bioluminescent imaging techniques can monitor ADSCs hz vivo.