硫氧还蛋白1对胃癌细胞系BCG823生长的影响

Effects of Human Thioredoxin-1 on the Growth of Gastric Cancer Cell BCG823

目的探讨硫氧还蛋白1(thioredoxin-1,Trx1)对胃癌细胞系细胞周期及增殖速度的影响。方法对BCG823、AGS、MNK45、NUGC3、PHM82五种胃癌细胞系的质粒转染效率进行测定,选择出转染效率高的细胞系;并用实时PCR(RT-PCR)测定胃癌细胞系中Trx1的表达水平,选择出表达较低的细胞系。将所构建的Trx1真核重组表达载体pcDNA3.1myc-His-Trx1转染入上述实验筛选出的胃癌细胞系,使细胞中Trx1过表达,用流式细胞仪检测过表达Trx1的胃癌细胞进入S期的比例。结果 BCG823、AGS两种细胞系的转染效率高,满足实验需要。BCG823的Trx1表达较AGS低[BCG823细胞系的ΔCT(Trx-GAPDH)=1.43,AGS细胞系的ΔCT(Trx-GAPDH)=1.10,ΔCT值与表达量成反比],选择BCG823作为实验对象。转染pcDNA3.1myc-His-Trx1的细胞Trx1表达升高(与内参蛋白的比值为0.45±0.02和0.26±0.03,n=3,t=9.127,P=0.000),进入S期的细胞比例升高(转染带有Trx1质粒的细胞进入S期的比例为28.54%,而转染了空载体者进入S期的比例为22.24%,χ2=104.759,P=0.000)。结论 Trx1表达水平上调可以促进胃癌细胞进入S期。

Objective To study the effects of human thioredoxin-1（Trx1） on the cell cycle and proliferation of gastric cancer cells.Methods Transfection efficiency of five gastric cancer cell line,BCG823,AGS,MNK45,NUGC3,and PHM82 was analyzed to choose the one with highest efficiency for our experiment.At the same time,the expression of Trx1 in the gastric cancer cell lines was measured by real-time PCR,so that to select the cell lines with lowest expression rate,which could avoid the interference from the endogenous Trx1 to the transfected Trx1,and to ensure the obvious effects of transfection.Then,the pcDNA3.1myc-His-Trx1 recombinant vector was transfected into the cell line selected to make the Trx1 enhanced.The percentage of the cells in S phase was detected by flow cytometry.Results The transfection efficiency of BCG823 and AGS was high enough.The Trx1 expression in BCG823 was lower than AGS [ΔCT（Trx-GAPDH） of BCG823 =1.43,ΔCT（Trx-GAPDH） of AGS= 1.10,and ΔCT is reverse to the level of Trx expression],so BCG823 was selected.The expression of Trx1 was up-regulated by transfecting with pcDNA3.1myc-His-Trx1（the ratios to the referent protein are relatively 0.45±0.02 and 0.26±0.03,n=3,t=9.127,P=0.000）.The percentage of the cells in S phase was significantly increased（after interfecting by the plasmid with Trx1,the rate of cells coming into S phase was 28.54%,compared with the rate of the cells interfected by the plasmid without Trx1,which was 22.24%,χ2=104.759,P=0.000）.Conclusions The up-regulation of Trx1 can promote the entry of BCG823 cells into S phase.