CD、Flt-1基因共表达重组质粒的构建及对BT325细胞增殖的抑制作用

Construction of recombinant plasmid coexpressing CD and Flt-1 genes and the inhibition effects on BT325 cell proliferation

目的构建CD、Flt-1基因共表达重组质粒,并检测其对BT325细胞增殖抑制的影响。方法从大肠杆菌及人脐静脉内皮细胞中提取总RNA,利用逆转录聚合酶链式反应(RT-RCR)扩增CD基因和FLT-1基因片段,应用基因重组技术将CD基因和Flt-1基因片段克隆到真核表达载体pEGFP-C1中,经酶切鉴定及DNA序列分析证实所构建的载体。转染BT325细胞株,通过MTT法和流式细胞术观察其对细胞的增殖抑制作用。结果 RT-RCR方法获得CD基因和Flt-1基因片段,酶切分析和DNA测序鉴定表明重组质粒pEGFPC1-CD-Flt-1构建成功。转染72h后实验组细胞生长速度是control组的(33.2%±5.4%),流式细胞术发现实验组细胞的G1期细胞比例明显高于control组,细胞出现凋亡,实验组细胞的凋亡率为(19.7%±5.45%)。结论成功构建真核表达重组质粒pEGFP-C1-CD-Flt-1,其体外可抑制BT325增殖并诱导其凋亡。

Objective To construct an recombinant plasmid coexpressing CD and Flt--1 gene and to investigate its effect on proliferation inhibition of BT325 cells. Methods Total RNA was extracted from E. coli and human umbilical vein endothelial cells as the template and the CD gene and Flt--1 gene were amplified by reverse transcription --polymerase chain reaction（RT-- PCR）. By using gene recombination technique, CD and Flt-1 cDNA were inserted into eukaryotic expression vector pEGFP--C1. The recombinant plasmid was identified by restriction enzyme di- gestion and sequence analysis. It was infected into BT325 cells. Proliferation abilities were meas- ured by MTT and FCM. Results The CD gene and Flt--1 gene could be obtained by RT--PCR. Restriction enzyme digestion and PCR suggested that the recombinant plasmids were constructed successfully. 72 hours after transfection, the proliferation rate of cells was inhibited according to MTT, about 33.2 %±6.4 % of that in control group. Cells numbers of G1 phrase were significant- ly higher than those in control group. Cell apoptosis was found and the apoptotic rate was 19. 70%±5.45%. Conclusion The recombinant plasmid pEGFP-C1-CD- Flt-1 was constructed suc- cessfully,which could inhibit BT325 cells proliferation and induce their apoptosis in vitro.