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目的:观察抑制孕激素膜受体1(PGRMC1)对子宫内膜癌细胞化疗敏感性的影响。方法:设计合成以PGRMC1为靶标的shRNA1、2质粒,对照质粒shRNA3,转染入瘤细胞。转染1周后,检测细胞绿色荧光蛋白(GFP)阳性率,RT-PCR检测转染shRNA后PGRMC1基因mRNA水平变化,蛋白质印迹法检测蛋白表达变化。CCK8法检测转染后第2代的瘤细胞对化疗药物ADM、5-FU及DDP的敏感性,流式细胞仪检测AnnexinⅤ标记的转染组与对照组细胞加化疗后细胞凋亡率的差异;双氢二氯荧光染色阳性率判定细胞内活性氧簇(ROS)水平的差异。结果:shRNA可在细胞稳定表达>1周,并可传代。shRNA1、2可显著抑制PGRMC1基因mRNA及蛋白表达,提高子宫内膜癌细胞化疗敏感性。相同化疗压力下,PGRMC1抑制的子宫内膜细胞凋亡率及细胞内ROS水平明显高于对照组。结论:抑制PGRMC1基因表达能够增加子宫内膜癌细胞对化疗的敏感性。
Objective: To observe the effect of inhibiting the expression of progesterone receptor 1 (PGRMC1) on the chemosensitivity of endometrial carcinoma cells. Methods: shRNA1,2 plasmid targeting PGRMC1 was designed and synthesized, and the control plasmid shRNA3 was transfected into tumor cells. One week after transfection, the positive rate of green fluorescent protein (GFP) was detected. The mRNA level of PGRMC1 was detected by RT-PCR and the protein expression was detected by Western blotting. The sensitivity of chemotherapeutic drugs ADM, 5-FU and DDP was detected by CCK8 assay. The difference of apoptosis rate between AnnexinⅤ-labeled transfected group and control group after chemotherapy plus chemotherapy was detected by flow cytometry ; The difference of intracellular reactive oxygen species (ROS) levels was determined by the positive rate of DHBF staining. Results: shRNAs can be stably expressed in cells for> 1 week and can be passaged. shRNA1,2 can significantly inhibit the mRNA and protein expression of PGRMC1 and improve the chemosensitivity of endometrial cancer cells. The same chemotherapy pressure, PGRMC1 inhibition of endometrial apoptosis and intracellular ROS levels were significantly higher than the control group. Conclusion: Inhibition of PGRMC1 gene expression can increase the sensitivity of endometrial cancer cells to chemotherapy.