bcl-2 RNAi表达载体的构建、鉴定及其对Raji细胞的干扰效果

Construction, Identification of Bcl-2 RNAi Expression Plasmid and Interference Effects on Raji Cells

目的构建bcl-2特异性RNAi真核细胞质粒表达载体,并研究其对B细胞淋巴瘤Raji细胞的干扰效果。方法根据GenBank提供的bcl-2cDNA序列,设计并合成两对短发夹结构的互补DNA序列,经退火形成互补双链,克隆至载体pGenensil-1构建重组质粒,予以酶切和DNA测序鉴定。脂质体介导重组质粒转染Raji细胞,采用RT-PCR法观察重组质粒对bcl-2mRNA表达的影响,流式细胞仪测定细胞凋亡。结果酶切及测序鉴定表明,成功构建bcl-2shRNA的质粒表达载体pGenesil-1-bcl-2-544和pGenesil-1-bcl-2-1009。转染Raji细胞48h后,RT-PCR结果显示Raji细胞中bcl-2mRNA表达分别下调了(31.95±3.02)%和(47.57±2.88)%,与空白对照组相比,差异有统计学意义(P<0.05);流式细胞仪检测结果显示Raji细胞凋亡率分别为(14.25±0.84)%和(11.96±0.79)%,明显高于对照组,差异有统计学意义(P<0.05)。结论成功构建bcl-2特异性RNAi真核细胞质粒表达载体,它能有效沉默bcl-2基因在B细胞淋巴瘤中的表达并促进B细胞淋巴瘤Raji细胞凋亡。

Objective To construct short hairpin RNA （shRNA） interfering eukaryotic expression plasmid vector specific to bcl-2 gene and detect the interfering effects on B-lymphoma Raji cell. Methods Two pairs of complementary shDNA sequence were designed and synthesized according to bcl-2 eDNA from GenBank. After annealing, complementary double strands were formed and cloned into pGenesil-I vector to construct recombinant plasmids, which were identified by restriction enzyme digestion and DNA sequence analysis. Recombinant plasmids were transfected into Raji cells mediated by lipofectamineTM 2000, then bcl-2 mRNA expression and cell apoptotic rate was detected by RT-PCR and flow eytometry respectively. Results The results of restriction enzyme digestion and sequence analysis showed that pGenesil-l-bcl-2-544 and pGenesil-1-bc]-271009 plasmid vectors specific to bcl-2 shRNA were successfully constructed. At the forty-eighth hour post-transfection, bcl-2 mRNAs expression of Raji cells were reduced to （31.95±3.02）% and （47.57±2.88）% in pGenesil-1-bcl-2-544 group and pGenesil-1-bc1-2-1009 group respectively. Compared with the control group, statistically significant difference was observed （P〈0.05）. The ratio of apoptosis cells were （14.25±0.84） % and （11. 96±0.79）% in pGenesil-1-bel-2-544 group and pGenesil-1-bel-2-1009 group respectively, which was significantly higher than that in the control group（P〈0.05）. Conclusion The eukaryotic shRNA plasmid expression vectors specific to bcl-2 gene were successfully constructed, which can silence bcl-2 gene and induce apoptosis effectively on B cell lymphoma.