GFP标记干细胞修复椎间盘退变中的示踪研究

目的探讨绿色荧光蛋白(GFP)示踪时效及GFP标记的骨髓间充质干细胞(MSCs)在移植后参与退变修复过程中的细胞增殖情况。方法使用针吸髓核法建立兔的椎间盘退变模型,体外培养MSCs,并用含有绿色荧光蛋白标记的腺病毒载体(Adeno-XTM-GFP)转染,成为GFP标记的MSCs(GFP-MSCs),于造模2周后植入造模的椎间盘内。在移植后第2、4、6、8周用激光共聚焦显微成像技术,观察MSCs的增殖情况,对比GFP本身荧光和免疫荧光的示踪时间。结果非免疫荧光组GFP本身荧光强度能够持续到4周,后较微弱甚至难以辨别;免疫荧光组用FITC荧光二抗标记的GFP-MSCs在8周内都能够持续稳定的显现荧光,4个时间点GFP-MSCs的阳性率的可信区间分别是:2周(14.64±2.05)%;4周(21.85±2.45)%;6周(31.03±4.03)%;8周(36.37±4.42)%。结论GFP-MSCs在椎间盘微环境中能够稳定增殖。若示踪研究需要持续超过4周,则应对GFP行免疫荧光染色,可以使有效示踪时间持续至少8周。

Tracing of GFP-labeled stem cells transplanted to discs for treating degenerative discs

Objective To evaluate the tracing efficacy of the green fluorescent protein(GFP) and the proliferation of the GFP-labeled bone marrow mesenchymal stem cells(MSCs) which was transplanted to discs to treat the degenerative intervertebral discs.Methods An in vivo model was established by aspiration of the nucleus pulposus.MSCs were cultured in vitro,and transfected by recombinant Adeno-XTM-GFP expression vector.Then they were transplanted to the model.By the 2nd,4th,6th,and 8 th week,confocal laser scanning microscope was used to evaluate the proliferation of transplanted GFP-MSCs and contrast the endurance of the GFP-self-fluorescence with that of immunofluorescence.Result The GFP-self-fluorescence sustained until the 4th week.The immunofluorescence could sustain until at 8th week.By the 2nd,4th,6th,and 8th week,95% confidence interval for the mean rate of GFP-positive cells were(14.64±2.05)% at the 2nd week,(21.85±2.45)% at the 4th week,(31.03±4.03)% at the 6th week,(36.37±4.42)% at the 8th week.Conclusion GFP-MSCs can proliferate in the degenerative discs and GFP can be used to trace the transplanted MSCs until the 4th week.By immunofluorescence staining,GFP can be traced until the 8th week at least.