人CRIF1基因调控骨髓间充质干细胞细胞周期分布初步研究

Primary study on the role of human CRIF1 gene in cell cycle regulation of bone marrow mesenchymal stem cells

目的探讨人CRIF1基因对骨髓间充质干细胞(hBMSCs)细胞周期调控机制。方法通过构建慢病毒载体改变CRIF1表达水平,合成发夹shRNA模板寡核苷酸链,构建pGreenPuro-sh-CRIF1干扰载体;PCR扩增CRIF1基因CDS全长,链接pMD18-T载体经测序鉴定后,构建pLentis-CMV-CRIF1-SFFV-GTP过表达载体。载体成功构建后包装慢病毒,并转染hBMSCs,以pLentis-CMV-SFFV-GTP空载体病毒作为对照,定量PCR及Western blot(WB)检测CRIF1基因和蛋白表达,流式细胞仪检测hBMSCs细胞周期分布,并通过免疫荧光观察CRIF1与CDK2细胞共定位。结果成功构建CRIF1 shRNA及过表达慢病毒载体,测定后病毒滴度达到108TU/mL。以MOI=10转染hBMSCs,并以0.8μg/mL嘌呤霉素筛选1周后,感染效率可达到>95%,WB及定量PCR证实,CRIF1基因和蛋白的表达水平,在pGreenPuro-sh-CRIF1载体转染后hBMSCs细胞受到抑制:与对照组比较,mRNA相对表达水平0.42±0.03 vs1.00±0.03(P<0.01),蛋白相对表达水平0.13±0.024 vs 1.00±0.04(P<0.01),而pLentis-CMV-CRIF1-SFFV-GTP载体转染后得到增强:与对照组比较,mRNA相对表达水平5.54±0.53 vs 1.00±0.03(P<0.01),蛋白相对表达水平4.21±0.22 vs 1.00±0.04(P<0.01)。同时,CRIF1基因过表达诱导hBMSCs G0/G1期阻滞(与对照组比较,84.99±2.66 vs 77.28±0.86,P<0.01),抑制CRIF1基因表达促进其进入细胞周期循环:G0/G1期比例与对照组比较,61.60±3.02 vs 77.28±0.86(P<0.01)。激光共聚焦显示,CRIF1与CDK2共定位于胞浆与胞核。结论 CRIF1可能通过与CDK2结合,负性调控hBMSCs细胞周期分布。

Objective To study the function of human CRIF1 gene in cell cycle regulation of hBMSCs. Methods Hairpin siRNA template oligonucleotides and CRIF1 CDS were inserted into lentiviral vector pLentis-CMV-SFFV-GTP, and Plasmid DNA were verified by restriction enzyme analysis and DNA sequencing. Lentiviral vectors were eo-transfected to 293FT cells with pMD2. G and pSPAX2. Viral particles were harvested and then used for hBMSCs transfection. Expression of CRIF1 mRNA and protein were detected by qPCR and WB. Cell cycle of hBMSCs was analyzed by flow cytometry. Co-localization of CRIF1 and CDK2 was Observed by immunofluorescence. Results Lentiviral vectors were successfully constructed and named pGreenPuro-sh-CRIF1 and pLentis-CMV-CRIF1-SFFV-GTP respectively. The virus titer was 10s TU/mL. Recombinant lentivirus were transfected into hBMSCs （MOI = 10 ） and the efficiency of infection was above 95% after puromycin （0.8 μg/ mL） selection, qPCR and WB showed that expression of CRIF1 mRNA and protein were significantly higher （5.54 ± 0. 53 vs 1. 00±0. 03,4. 21±0. 22 vs 1.00 ± 0. 04 at the mRNA and protein level respectively, P 〈 0. 01 ） in pGreenPuro-sh-CRIF1 transfected h BMSCs and lower（0. 42 ±0. 03 vs 1.00± 0. 03,P 〈 0.01 ;0. 13 ±0. 024 vs 1. 00 ±0. 04 at the mRNA and protein tevel respectively, P 〈 0. 01 ） in pLentis-CMV-CRIF1-SFFV-GTP transfected hBMSCs when compared with control group. Also,increased expression of CRIF1 can make hBMSCs arrested in ＂ G0/G1＂ phase compared with control group,84. 99 ±2. 66 vs 77. 28 ±0. 86, （P 〈0. 01 ） and impress the expression of CRIF1 to facilitate cell cycle GO/GI phase ratio 61.60 ± 3.02 vs 77. 28± 0. 86, （P 〈 0. 01 ）. Confocal microscopy images showed a colocalization of CRIF1 and CDK2 in both cytoplasm and nucleus. Conclusion CRIF1 can negatively regulate hBMSCs proliferation by combining with CDK2.