Mcl-1论文：靶向Mcl-1基因沉默对胰腺癌PANC-1细胞增殖和凋亡影响的实验研究.docVIP

Mcl-1论文：靶向Mcl-1基因沉默对胰腺癌PANC-1细胞增殖和凋亡影响的实验研究 【中文摘要】通过siRNA介导的RNAi技术对人类胰腺癌细胞株PANC-1中的Mcl-1基因进行特异性靶向沉默,然后检测分析下调Mcl-1蛋白表达对PANC-1细胞的增殖和凋亡的影响,为胰腺癌的基因治疗提供一定的实验和理论基础。方法①将Lipofectamine?2000与FAM标记的空白链(FAM-NC-siRNA)按照几个不同比例混合后分组转染PANC-1细胞。流式细胞仪检测不同混合比转染相应的转染效率,然后选出最佳转染条件。②通过反转录PCR (RT-PCR)和Westernblot技术检测siRNA(Mcl-1)对PANC-1细胞的Mcl-1表达的抑制情况。③通过MTT法检测转染后不同时间点各实验组细胞的活力,然后绘制各实验组的生长曲线。运用流式细胞仪检测各实验组细胞于转染后48h的凋亡情况,然后进行比较分析。结果①经过实验摸索,本次实验最佳转染效率为87.5%。②siRNA(Mcl-1)组PANC-1细胞的Mcl-1的mRNA和蛋白均明显下调(p0.05),沉默效果于转染后48h时达到高峰,随后沉默效果有所下降。③与空白对照组、脂质体组和空白链组(NC-siRNA组)相比, siRNA(Mcl-1)组PANC-1细胞的增殖速度明显放缓;转染后48h流式细胞仪检测各实验组细胞凋亡率结果显示, siRNA(Mcl-1)组、空白对照组、脂质体组和NC-siRNA组的凋亡率依次为14.6±0.36%,4.1±0.35,4.2±0.41,4.4±0.26,siRNA(Mcl-1)组凋亡率明显高于另外三组,差别有统计学意义(p0.05)。结论脂质体介导的siRNA(Mcl-1)能够明显下调PANC-1细胞Mcl-1的表达。下调该基因表达后,PANC-1细胞增殖活力明显下降,凋亡显著增加。可见靶向Mcl-1的RNAi技术在胰腺癌的基因治疗上具有潜在价值。 【英文摘要】By RNAi technology Mcl-1 in Human pancreatic cancer cell line PANC-1 was specifically knocked down with siRNA, then detect and analyze the impact on proliferation and apoptosis of PANC-1 cells in vitro. From this research we hope to provide the experimental foundation for gene treatment of the pancreatic cancer.Methods 1 The lipofectamine ? 2000 and FAM-NC-siRNA were mixed in different proportions then transfected into PANC-1 cells. Transfection efficiency was detected by flow cytometry, and then select the best transfection conditions. 2 The inhibition of Mcl-1 expression was measured by reverse transcription PCR (RT-PCR) and Westernblot after transfecting siRNA(Mcl-1) . 3 Cell viability in different time after transfection was assayed By MTT and then drew the growth curve of each experimental group. The apoptosis of PANC-1 cells each experimental group was measured by flow cytometry in 48h after transfection.Results 1 After experimental exploration, the best transfection efficiency was 87.5%. 2 The Mcl-1 mRNA and protein of siRNA (Mcl-1) group were significantly downragulated (p 0.05). This effect reached a peak at 48h after transfection the