慢病毒转染大鼠破骨样细胞研究

Abstract

目的探讨慢病毒转染大鼠破骨样细胞的可行性。方法诱导大鼠骨髓间充质干细胞(BMSCs)培养成破骨样细胞(OLC)后,将其用绿色荧光蛋白(GFP)标记的对照慢病毒进行转染,荧光显微镜下观察不同感染复数(MOI)时慢病毒转染OLC的荧光率,并用TRAP染色鉴定OLC。选择最适宜的MOI并加入不同干预进行对照,分为OLC对照组、NFAT2抑制剂(VIVIT)组、NFAT2iRNA慢病毒组、对照病毒组,用RT-PCR进行基因验证。结果对照病毒转染后,OLC随着MOI值的升高,荧光转染率增加;MOI=15时荧光数最多(41.00±9.19)个/视野。不同干预后,NFAT2抑制剂组和NFAT2iRNA慢病毒组的NFAT2的基因表达明显低于OLC对照组和对照病毒组(P<0.05)。结论慢病毒可转染大鼠破骨样细胞,并在一定范围内随MOI升高转染率增高。

Objective To explore the feasibility of lentivirus transfect osteoclast-like cells. Methods After the bone mesenchymal stem cells( BMSC) of rat were induced osteoclast-like cells( OLC),the contrast lentivirus that marked Green fluorescent protein( GFP) was used to transfect the OLC. The fluorescent ratio of different multiplicity of infection( MOI) was observed under fluorescent microscope,and then Tartrate resistant acid phosphatase( TRAP) staining was used to identify the osteoclast-like cells. The most appropriate MOI value was selected and then divided into OLC,NFAT inhibitor( VIVIT),NFAT2 iRNA lentivirus,control lentivirus groups,respectively. Results The number of fluorescent cells was increased with elevating of MOI value after contrast lentivirus transfect osteoclast-like cells. The maximum fluorescent number was( 41. 00 ± 9. 19) / view when MOI = 15. After different intervention,the mRNA expression of NFAT2 in NFAT inhibitor group and NFAT2 iRNA lentivirus group were more decreased than those of OLC and control lentivirus groups( P < 0. 05). Conclusions Lentivirus could transfect osteoclast-like cells of rat. Furthermore,fluorescent transfect rate is increased with the rise of MOI value in a certain range.