转染CDK2-shRNA对黑色素瘤B16-F1细胞达巴卡嗪敏感性的影响及其机制

发布时间：2018-05-04 21:40

本文选题：黑色素瘤 + 周期素依赖性蛋白激酶　；参考：《山东医药》2017年23期

【摘要】：目的探讨转染周期素依赖性蛋白激酶2短发夹RNA(CDK2-shRNA)对黑色素瘤B16-F1细胞达巴卡嗪敏感性的影响及其机制。方法将黑色素瘤B16-F1细胞分为观察组、空质粒组和对照组,观察组转染重组慢病毒pUL-CDK2-shRNA质粒,空质粒组转染重组慢病毒pUL-NC-shRNA质粒,对照组不转染。转染12 h后用250μmol/L的达巴卡嗪孵育三组细胞72 h。采用MTT法检测三组细胞吸光度值,采用集落形成实验检测三组细胞集落形成率(CFR),采用Annexin V-FITC/PI双染法检测三组细胞凋亡率,采用Western blotting法检测三组细胞Bcl-2、Bax、Caspase-3蛋白相对表达量,计算Bax/Bcl-2。结果观察组、空质粒组和对照组细胞吸光度值分别为0.221±0.019、0.684±0.049、0.699±0.051。观察组细胞吸光度值与空质粒组和对照组相比,P均0.05。观察组、空质粒组和对照组细胞CFR分别为33.00%±1.80%、67.50%±7.26%、69.83%±5.25%。观察组细胞CFR与空质粒组和对照组相比,P均0.05。观察组、空质粒组和对照组细胞凋亡率分别为43.6%±4.1%、17.6%±3.8%、15.1%±4.0%。观察组细胞凋亡率与空质粒组和对照组相比,P均0.05。观察组、空质粒组和对照组细胞Bax/Bcl-2分别为2.56±0.28、0.84±0.04、0.86±0.11,Caspase-3相对表达量分别为0.92±0.07、0.69±0.08、0.57±0.05。观察组细胞Bax/Bcl-2、Caspase-3相对表达量与空质粒组和对照组相比,P均0.05。结论转染CDK2-shRNA可以提高黑色素瘤B16-F1细胞对达巴卡嗪的敏感性。其机制是转染CDK2-shRNA可以提高达巴卡嗪共培养的黑色素瘤B16-F1细胞Bax/Bcl-2和Caspase-3表达水平,促进其凋亡。
[Abstract]:Objective to investigate the effect of CDK2-shRNA-transfected cyclin dependent protein kinase 2 short hairpin on the sensitivity of melanoma B16-F1 cells to Vaca and its mechanism. Methods melanoma B16-F1 cells were divided into observation group, blank plasmid group and control group. The observation group was transfected with recombinant lentivirus pUL-CDK2-shRNA plasmid, the blank plasmid group was transfected with recombinant lentivirus pUL-NC-shRNA plasmid, and the control group was not transfected. After transfection for 12 h, the cells were incubated with 250 渭 mol/L of Dazarizine for 72 h. The absorbance of three groups of cells was detected by MTT assay, the colony forming rate was detected by colony forming assay, the apoptosis rate of three groups was detected by Annexin V-FITC/PI double staining, the relative expression of Bcl-2BaxCaspase-3 protein was detected by Western blotting method, and the expression of Bax-2Bcl-2 protein was calculated. Results the cell absorbance values of observation group, empty plasmid group and control group were 0.221 卤0.019 卤0.684 卤0.049 卤0.699 卤0.051, respectively. The cell absorbance in the observation group was 0.05 compared with that in the blank plasmid group and the control group. The CFR of the observation group, the empty plasmid group and the control group were 33.00% 卤1.80% and 67.50% 卤7.26% respectively. Compared with blank plasmid group and control group, the CFR of observation group was 0.05 (P < 0.05). The apoptotic rate of the observation group, the empty plasmid group and the control group were 43.6% 卤4.1% and 17.6% 卤3.8% respectively. The rate of apoptosis in the observation group was 0.05 compared with that in the blank plasmid group and the control group. The relative expression of Caspase-3 in the observation group, the empty plasmid group and the control group was 2.56 卤0.28 卤0.84 卤0.04 卤0.86 卤0.11 respectively, and the relative expression of Caspase-3 was 0.92 卤0.07 卤0.08 卤0.57 卤0.05, respectively. Compared with blank plasmid group and control group, the relative expression of Bax-Bcl-2Caspase-3 in observation group was 0.05 (P < 0.05). Conclusion transfection of CDK2-shRNA can enhance the sensitivity of melanoma B16-F1 cells to Dharizine. The mechanism is that transfection of CDK2-shRNA can increase the expression of Bax/Bcl-2 and Caspase-3 in B16-F1 cells and promote the apoptosis of B16-F1 cells.
【作者单位】：鹤壁职业技术学院医学院;湖北省食品药品监督检验研究院;郑州大学第一附属医院;
【基金】：河南省科技计划项目(122300410193) 河南省高等学校青年骨干教师资助计划项目(2011GGJS-262)
【分类号】：R739.5

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