人凝血因子IX基因在小鼠脂肪干细胞中的稳定表达

Stable expression of human coagulation factor IX in mouse adipose-derived stem cells

目的将携带人凝血因子IX(h FIX)基因(F9)的重组腺病毒转染C57BL/6小鼠脂肪间充质干细胞(adipose-derived stem cell,ADSC),经过传代培养后,观察F9是否在ADSC中稳定表达,探索ADSC是否可作为血友病基因治疗的载体细胞。方法取C57BL/6小鼠腹股沟区脂肪,依据组织块悬浮法分离培养小鼠ADSC并进行传代培养,应用携带h FIX基因并含GFP荧光标记的重组腺病毒转染第3代ADSC,转染后再次进行传代培养至第4、5代细胞。荧光显微镜下观察细胞携带荧光数量,RT-PCR检测F9基因表达,ELISA法检测细胞上清液及Western Blot检测细胞内携带目的基因表达蛋白情况。结果 (1)重组腺病毒转染后荧光显微镜下可见荧光表达,细胞传代后仍具有荧光。(2) RT-PCR结果显示:四组ADSC均可表达内参GAPDH基因片段,A、B、C组可检测到目的基因F9的表达,D组未检测到F9的表达。(3) ELISA法检测凝血因子IX抗原(h FIX:Ag)结果显示:A组(81.62±8.82) ng/m L、B组(52.50±3.25) ng/m L、C组(47.41±4.00) ng/m L明显高于D组检测值(0.76±0.44) ng/m L,差异具有显著性(P<0.05)。(4) Western Blot法检测四组细胞内h FIX蛋白表达情况,结果显示A组蛋白表达灰度值(0.68±0.10)、B组(0.49±0.15)、C组(0.18±0.05)明显高于D组蛋白表达灰度值(0.02±0.01),差异具有显著性(P <0.05)。结论重组腺病毒转染ADSC后,经过传代后培养,仍可表达较高的h FIX活性,可以作为血友病基因治疗的载体细胞。

Objective To observe whether h FIX is stably expressed in adipose-derived stem cells( ADSCs) and to explore whether ADSCs can be used as a vector cell line for hemophilia gene therapy following transformation by recombinant adenoviruses carrying human coagulation factor IX( h FIX) gene,F9. Methods ADSCs were isolated from C57 BL/6 mice and cultured by tissue mass suspension. The third generation of ADSCs was transfected by recombinant adenovirus carrying F9 and GFP fluorescence marker. After transfection,the cells were subcultured again to the 4 th and 5 th passages. The level of fluorescence expressed by the cells was observed by fluorescence microscopy,the expression of F9 was detected by RT-PCR,and the protein expression of h FIX was detected by ELISA and western blotting. Results Fluorescence was observed after recombinant adenoviral transfer,and was still identified after cell passaging. RT-PCR revealed that all four groups of ADSCs could express internal reference GAPDH,but there was no expression of the target gene F9 in group A,and the expression of F9 could be detected in group D. Detection of h FIX: Ag by ELISA showed that the detection values of groups A( 81. 62 ± 8. 82) ng/m L,B( 52. 50 ± 3. 25) ng/m L,and C( 47. 41 ± 4. 00) ng/m L were significantly higher than that of group D( 0. 76 ± 0. 44) ng/m L,and there was a significant difference between the two groups( P< 0. 05). h FIX expression in the four groups of ADSCs was detected by western blotting. The result showed that the gray value of histone expression in groups A( 0. 68 ± 0. 10),B( 0. 49 ± 0. 15) and C was significantly higher than that in group D( 0. 02 ± 0. 01),and the difference was statistically significant( P< 0. 05),while the gray value of histone expression in group A was significantly higher than that in group D( P< 0. 05). Conclusions The recombinant adenoviruses were transformed into ADSCs and cultured after passaging,and could still express high h FIX activity,thus demonstrating their potential as vector cells for hemophilia gene therapy.