SPARC基因过表达对SKM-1细胞凋亡的影响

Effects of SPARC Gene Overexpression on Proliferation and Apoptosis of SKM-1 Cells

构建SPARC基因过表达载体,转染人骨髓增生异常综合征(myelodysplastic syndrome,MDS)细胞系SKM-1细胞,探讨SPARC基因过表达对人MDS细胞系SKM-1细胞凋亡的影响。以pcDNA-SPARC为引物,PCR扩增SPARC基因;将靶基因克隆入慢病毒载体pGC-GV,构建含有SPARC基因的重组慢病毒载体pGC-GV-SPARC,测序检测正确性;将构建载体pGC-GV-SPARC转染人MDS细胞系SKM-1,流式细胞术检测转染效率,RT-PCR检测SKM-1细胞中SPARC mRNA表达,Western blot检测SPARC蛋白表达,MTS法测定小剂量阿糖胞苷(30 ng/mL)对实验组增殖抑制的影响,Annexin V检测SPARC基因转染后对人SKM-1细胞凋亡的影响。结果显示,构建含有SPARC基因的重组慢病毒载体pGC-GV-SPARC转染效率为(64.25±1.42)%;转染后,SPARC mRNA及蛋白表达在靶细胞中较对照组增多。小剂量阿糖胞苷对转染组的增殖抑制率明显高于其他组。SPARC基因转染后人SKM-1细胞凋亡率较未转染组明显增高,加入阿糖胞苷后人SKM-1细胞凋亡率较对照组明显增高。由此说明,作者成功构建了携带人SPARC基因的慢病毒载体,转染人SKM-1细胞系后稳定表达SPARC基因,SPARC过表达可抑制细胞增殖,且联合小剂量阿糖胞苷(30 ng/mL)更有效地抑制SKM-1细胞的增殖,并诱导其凋亡。

To construct a recombinant lentiviral vector carrying SPARC gene, we investigated the altera- tion on the proliferation of MDS cell line SKM-I cells. The SPARC was obtained using pcDNA-SPARC by PCR. The SPARC gene was cloned into a lentiviral vector pGC-GV to construct a recombinant lentiviral vector carrying SPARC gene named pGC-GV-SPARC, which was confirmed to be correct by DNA sequencing, pGC-GV-SPARC was used to transfect human MDS cell line SKM-1. The transfection efficiency, SPARC mRNA and protein ex- pression were detected by flow cytometry, RT-PCR and Western blot, respectively. The MTS method was used to determine inhibition effects of low doses of Ara-C in different groups. Results showed that the recombinant len- tiviral vector pGC-GV-SPARC was confirmed to be correct by DNA sequencing. The transfection efficiency was （64.25±1.42）%, and stably expressed. PT-PCR and Western blot showed that the expression of SPARC was increased. MTS results showed that the proliferation effect of low doses of Ara-C on the inhibition rate of transfection group was significantly higher than those of negative group and SKM-1 group. The results showed that lentiviral vector carrying SP4RC was constructed successfully, and the expression of SPARC could effectively increased by pGC-GV- SPARC, which could inhibit the proliferation of SKM- 1 cell and promote its apoptosis by combining with low doses of Ara-C.