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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 a plasmid vector for bcl-2-specific RNAi eukaryotic cells and study its effect on Raji cells of B cell lymphoma. Methods According to the bcl-2 cDNA sequence provided by GenBank, two pairs of short hairpin cDNAs were designed and synthesized. After annealed to form a complementary double-stranded DNA, the recombinant plasmid was cloned into vector pGenensil-1 to construct a recombinant plasmid. Liposome-mediated recombination plasmids were transfected into Raji cells. The effect of the recombinant plasmids on the expression of bcl-2 mRNA was observed by RT-PCR and the apoptosis was detected by flow cytometry. Results The results of restriction enzyme digestion and sequencing showed that plasmids pGenesil-1-bcl-2-544 and pGenesil-1-bcl-2-1009 were successfully constructed. The expression of bcl-2mRNA in Raji cells was down-regulated by (31.95 ± 3.02)% and (47.57 ± 2.88)%, respectively, 48h after transfection with Raji cells. Compared with the blank control group, the difference was statistically significant (P <0.05). The results of flow cytometry showed that the apoptotic rates of Raji cells were (14.25 ± 0.84)% and (11.96 ± 0.79)%, respectively, which were significantly higher than those of the control group (P <0.05). Conclusion The bcl-2 specific RNAi eukaryotic plasmid expression vector was constructed successfully. It can effectively silence the expression of bcl-2 gene in B cell lymphoma and promote the apoptosis of B cell lymphoma Raji cells.