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目的探讨转染精脒/精胺N1乙酰基转移酶2(spermidine/spermine N1-acetyltransferase 2,SSAT2)在低氧条件下对肾癌Caki-1细胞增殖、凋亡和阿霉素敏感性的影响及机制。方法经X-treme GENE HP介导将真核表达质粒pcDNA3.1(D)/V5-His-SSAT2转入肾癌Caki-1细胞,细胞在1%氧浓度下培养,Western blot法检测SSAT2蛋白和低氧诱导因子-1α(hypoxia-inducible factor-1 alpha,HIF-1α)蛋白表达;流式细胞仪检测细胞的凋亡率、死亡率;MTT法检测细胞的增殖,以及不同浓度阿霉素对细胞的抑制率。结果 SSAT2成功转入肾癌Caki-1细胞,与空白细胞组及空载体组比较,转染SSAT2组HIF-1α蛋白表达明显下调(P<0.01),细胞增殖受到明显抑制(P<0.01),细胞凋亡率和死亡率均增加(P<0.05)。阿霉素浓度为2μg/ml时,转染组细胞抑制率可达(67.5±3.7)%,对空白细胞和空载体细胞的抑制率为(54.7±8.4)%、(56.8±7.7)%,差异有统计学意义(P<0.01)。结论转染SSAT2可以通过下调HIF-1α蛋白表达,减少肾癌细胞的增殖,增加肾癌Caki-1细胞的凋亡率、坏死率和对阿霉素的敏感性,针对SSAT2的基因治疗可能成为逆转肾癌耐药的方法之一。
Objective To investigate the effects of transfection of spermidine / spermine N1-acetyltransferase 2 (SSAT2) on the proliferation, apoptosis and doxorubicin sensitivity in renal cell carcinoma Caki-1 cells under hypoxic conditions And mechanism. Methods The eukaryotic expression plasmid pcDNA3.1 (D) / V5-His-SSAT2 was transfected into Caki-1 cells by X-treme GENE HP. The cells were cultured at 1% oxygen concentration. Western blot was used to detect the expression of SSAT2 protein And hypoxia-inducible factor-1α (HIF-1α) protein were detected by flow cytometry. The apoptosis rate and death rate were detected by flow cytometry. The proliferation of cells and the proliferation of adriamycin The rate of inhibition of cells. Results SSAT2 was successfully transfected into Caki-1 cells. The expression of HIF-1α in SSAT2 cells was significantly downregulated (P <0.01) and the cell proliferation was significantly inhibited (P <0.01) compared with the blank cells and empty vector groups. Both apoptosis rate and mortality increased (P <0.05). When the doxorubicin concentration was 2μg / ml, the inhibitory rate of transfected cells was (67.5 ± 3.7)%, that of transfected cells was (54.7 ± 8.4)%, (56.8 ± 7.7)%, The difference was statistically significant (P <0.01). Conclusion Transfection of SSAT2 can reduce the HIF-1α protein expression, reduce the proliferation of renal cell carcinoma and increase the apoptosis rate, necrosis rate and sensitivity to doxorubicin in Caki-1 cells. The gene therapy targeting SSAT2 may become One of the ways to reverse the resistance of kidney cancer.