siRNA干扰slug基因表达对IEC-6细胞放射线敏感性影响

EFFECTS OF siRNA INTERFERENCE ON SLUG GENE IN IEC-6 CELLS ON RADIATION SENSITIVITY

目的探讨siRNA干扰slug基因表达对体外肠隐窝上皮细胞IEC-6接受X线照射的敏感性影响及其机制。方法以体外培养大鼠小肠隐窝上皮细胞IEC-6为研究对象,将转染siRNA-slug(实验组)、转染siRNA(阴性对照组)和未转染siRNA(空白对照组)的细胞进行X射线照射处理,24h后,用Western blot法检测各组细胞的Slug和细胞P53正向凋亡上调因子(PUMA)蛋白的表达,CCK8法检测细胞在不同剂量X射线照射下的生长抑制情况,流式细胞仪检测细胞凋亡情况。结果实验组Slug蛋白表达与阴性对照组和空白对照组比较明显降低,PUMA蛋白表达明显高于阴性对照组和空白对照组,差异有显著性(F=1 760、421,P<0.01)。与阴性对照组和空白对照组比较,实验组细胞的凋亡率增加,差异有统计学意义(F=33.02,P<0.01)。在0~6Gy剂量范围内,随着X线照射剂量的增加,各组细胞的增殖活性均降低(F=6.595~31.470,P<0.05);在6Gy剂量X线照射下,实验组细胞生长抑制率、早期+晚期凋亡率较阴性对照组和空白对照组明显增高,差异有统计学意义(F=47.700、33.020,P<0.01)。结论 siRNA干扰slug基因的表达能够使促凋亡的PUMA蛋白表达增加,从而增强IEC-6细胞对X射线的敏感性。

Objective To investigate the effects of siRNA interference on sluggene expression on radiosensitivity of intestinal crypt cells（IEC-6）. Methods Rat intestinal crypt epithelial cells（IEC-6）cultured in vitro were used as research object.Cells that transfected with siRNA-slug（experimental group）,that transfected with siRNA（negative control group）and that nontransfected with siRNA（blank control group）were treated with X-ray irradiation.Twenty-four hours later,Western Blot was used to detect the protein expression of Slug and PUMA in each group,CCK8 assay was used to observe the growth inhibition of cells under different doses of X-ray,and flow cytometry was used to detect cell apoptosis. Results Compared with the negative control and blank control groups,the protein expression of Slug in the experimental group was lower,and the expression of PUMA protein was higher（F=1 760,421;P〈0.01）.The apoptosis rate in the experimental group was higher than that in the negative and blank control groups（F=33.02,P〈0.01）.In the dose range of 0-6Gy,cell proliferation activity decreased with the increase of X-ray dose（F=6.595-31.470,P〈0.05）.Under the 6Gy dose of X-ray irradiation,the growth inhibition rate,early and late apoptosis rate in the experimental group were obviously higher than that in the negative control and blank control groups,the differences were statistically（F=47.700,P〈0.01）. Conclusion sluggene targeting siRNA can increase the protein expression of proapoptotic gene PUMA,and thus enhance the sensitivity of IEC-cells to X-rays.