腺病毒介导人凝血因子IX基因在小鼠脂肪干细胞中的表达

Expression of Adenovirus-mediated Human Clotting Factor IX Gene in Mouse Adipose-derived Stem Cells

目的:探讨腺病毒介导的人凝血因子IX(hFIX)基因在小鼠脂肪干细胞(adipose-derived stem cell,ADSC)中的表达。方法:体外分离培养小鼠脂肪干细胞,观察其细胞形态,CCK-8法测定其生长活力,流式细胞术鉴定其细胞表型CD29、CD90、CD45,成脂成骨诱导鉴定其分化能力。将携带hIX基因的腺病毒转染小鼠脂肪干细胞,对比观察其与未转染时细胞形态及生长曲线有无变化。RT-PCR检测hFIX基因在细胞中的表达,Western blot检测细胞内及培养细胞上清液中hFIX蛋白的表达。ELISA法检测细胞上清液中的hFIX蛋白量(The antigen content of hFIX,FIX:Ag),一期法检测培养细胞上清液中hFIX蛋白活性(The clotting factor activity of hFIX,FIX:C)。结果:小鼠ADSC离体培养最初几小时呈体积较小的圆球形,具有很强的折光性,贴壁生长时间为种瓶后4-6 h,种瓶后72 h细胞变为长梭形纤维状,呈漩涡状排列;第3代ADSC体外培养1-2 d生长速度较缓慢,3-5 d增殖速度增快,呈指数倍扩增,6-7 d生长速度逐渐缓慢,总体生长趋势呈S型;油红O对诱导后细胞进行染色,用倒置显微镜观察,可见红色脂滴形成。茜素红对诱导后细胞进行染色,用倒置相差显微镜观察,可见橙红色钙化骨节。第3代ADSC高表达CD29(99.91%),CD90(99.02%),几乎不表达CD45(0.94%)。RT-PCR结果显示,hFIX基因可以在小鼠脂肪干细胞内表达。Western blot结果显示,小鼠脂肪干细胞内及培养细胞上清液中均可表达hFIX蛋白。ELISA测定转染ADSC培养上清FIX:Ag:第1 d为21.33±3.93 ng/(10^6 cells·24 h),第3 d为12.63±0.86 ng/(10^6 cells·24 h),第9 d为12.63±2.36 ng/(10^6 cells·24 h)。一期法检测培养细胞上清液中FIX:C为8.5%。结论:携带hFIX基因的腺病毒能有效转染ADSC。hFIX基因修饰的ADSC可以分泌具有凝血活性的hFIX蛋白。

Objective:To investigate the adenovirus-mediated expression of human clotting factor IX(hFIX)gene in mouse adipose-derived stem cells(ADSC).Methods:The mouse ADSC were isolated and cultured in vitro,the morphology of cells was observed and its growth viability was detected by using CCK-8.Cell surface markers CD29,CD90,CD45 were identified by flow cytometry,and its diferentiation ability was identified by adipogenic and osteogenic induction.Morphological changes was observed and the growth curve should be drawn after transfecting ADSC with adenovirus containing hFIX gene.The expression of hFIX gene was detected by RT-PCR.The expression of hFIX protein in ADSC or in culture supernatant was detected by Western blot.hFIX protein in the supernatant was measured by ELISA,and the clotting factor activity of hFIX in culture supernatant was measured by one-stage method.Results:The in vitro cultured mouse ADSC displayed microspherical shape and strong refractive property.Anchoring growth was lasted for 4-6 hours after planting into culture flask.After cultured for 72 hours,the cells showed long spindle-shaped fibrous and swirling arrangement.The overall growth trend of the third generation ADSC cultured in vitro was S-shaped.The formation of lipid droplets could be observed in the induced cells with Oil red O staining by inverted microscope.After alizarin red staining,the orange-red calcified bone nodes were observed in the induced cells under inverted phase contrast microscope.CD29(99.91%)and CD90(99.02%)highly expressed in the third generation of ADSC,but CD45(0.94%)almost not expressed.RT-PCR showed the hFIX gene could expressed in mouse ADSC.Western blot showed that hFIX protein expressed in both ADSC and culture supernatant.FIX:Ag in cell supernatant was 21.33±3.93 ng/(10^6 cells.24 h)on the first day,12.63±0.86 ng/(10^6 cells.24 h)on the third day and 12.63±2.36 ng/(10^6 cells.24 h)on the ninth day.FIX:C in culture supernatant was 8.5%.Conclusion:Adenovirus-carried hFIX gene can effectively transfect ADSC.ADSC modified by hFIX gene can secrete hFIX protein with coagulation activity.