特异性针对大鼠甲状旁腺激素受体1基因SiRNA表达载体的构建、鉴定及对高糖状态下INS-1细胞周期的影响

Construction and identification of siRNA recombinant expression vector targeting parathyroid hormone 1 receptor gene and its effect on the cell cycle of INS-1 cells

目的构建并筛选携带针对大鼠甲状旁腺激素受体1基因(PTH1R)的pSUPERretro-GFP/Neo干扰逆转录病毒载体以及鉴定及观测高糖下对INS-1细胞周期的影响。方法根据Genbank筛选PTH1R三个阳性克隆及一个阴性克隆序列,定向克隆入逆转录病毒载体pSUPERretro-GFP/Neo,并用RT-PCR和测序鉴定,将其转染至INS-1细胞中,Westernblotting观察PTH1R表达,鉴定其转染效率,筛选出最佳抑制基因后,应用流式细胞仪检测25mmol/LD-葡萄糖处理INS-1细胞周期。结果菌落RT-PCR及基因测序鉴定结果表明序列正确。并能成功转染到INS-1细胞株中,Westernblot筛选最佳抑制基因。细胞周期检测提示PTH1R沉默后抑制INS-1细胞从G0/G1期进入S期。结论成功构建并筛选出特异性沉默大鼠甲状旁腺激素受体1基因的pSUPERretro-GFP/Neo干扰逆转录病毒载体,高糖状态下PTH1R表达可能为INS-1细胞自我保护的一种作用。

Objective To constructthe expressionvectorof siRNA targeting parathyroid hormone 1 receptor（PTH1R） gene and evaluate its effectonthe cell cycle of INS-1 cells.Methods The sequences of PTH1Rgene was retrievedfromGenbank,and4 pairs of oligonucleotides were synthesizedandinsertedinto pSUPERretro RNAi,whichwas identifiedby RT-PCRandsequence analysis.The vectors were thentransfectedinto INS-1 cells,inwhichthe expressionof PTH1Rwas observedby Westernblotting to evaluate the transfection efficiency.The cell cycle of INS-1 cells in high glucose medium was detected by flow cytometry.Results RT-PCR and sequence analysis confirmed the correct construction of the siRNA recombinant expression vector targeting PTH1R gene.The vectors were successfully transfected into INS-1 cells,and the most effective vectorwas selected by Westernblotting.Transfectionwiththe siRNA forPTH1Rgene silencing resultedinthe inhibitionof INS-1 formentering the S phase.Conclusion The successful constructionof the recombinantPTH1R-siRNA vectors establishes abasis forfurtherstudy of protective role of the PTH1Rgene inINS-1 cells inhighglucose medium.