中文摘要：

目的：观察shRNA干扰己糖激酶（hexokinase,HK）Ⅱ基因表达对人淋巴瘤Raji和SU-DHL-4细胞代谢、增殖、凋亡及耐药的影响,初步评价HKⅡ基因靶向治疗在淋巴瘤中的潜在应用前景。方法：构建慢病毒介导的shRNA靶向干扰HKⅡ基因表达的淋巴瘤细胞株Raji和SU-DHL-4（HKⅡshRNA转染组）,同时设置阴性shRNA转染对照组。采用实时荧光定量PCR法和蛋白质印迹法分别检测细胞中HKⅡmRNA和蛋白的表达水平。锥虫蓝拒染法检测细胞存活数并绘制生长曲线;CCK-8法检测多柔比星（doxorubicin,DOX）对淋巴瘤细胞的半数抑制浓度（half maximal inhibitory concentration,IC50）。FCM法分析细胞周期分布及细胞凋亡。蛋白质印迹法检测细胞中凋亡相关蛋白caspase-3及其剪切体、Bcl-2和Bcl-6的表达;分别应用乳酸和葡萄糖检测试剂盒检测细胞培养上清液中乳酸和葡萄糖的浓度。结果：与空质粒转染对照组细胞相比,HKⅡshRNA转染组细胞中HKⅡmRNA及蛋白的表达水平均明显降低（P值均〈0.05）。shRNA干扰HKⅡ基因表达能抑制2种淋巴瘤细胞的增殖活性（P值均〈0.01）,并使细胞周期阻滞在G0/G1期（P值均〈0.05）,同时促进细胞凋亡（P值均〈0.01）。与对照组细胞相比,HKⅡ基因沉默可降低DOX对2种淋巴瘤细胞的IC50值（P值均〈0.05）,抑制细胞中葡萄糖消耗（P值均〈0.05）,减少乳酸生成（P值均〈0.01）。与对照组细胞相比,HKⅡshRNA转染后2种淋巴瘤细胞中Bcl-2蛋白的表达水平明显降低（P值均〈0.05）,而caspase-3剪切体表达水平明显升高（P值均〈0.05）;加入DOX作用后,2种淋巴瘤细胞的Bcl-2表达水平无明显差异（P值均〉0.05）,但HKⅡshRNA转染组细胞中caspase-3前体蛋白表达水平明显降低（P值均〈0.01）,而caspase-3剪切体表达水平升高更加明显（P值均〈0.05）。结论：shRNA干扰HKⅡ基因表达可抑制淋巴瘤细胞增殖,促进细胞凋亡,纠正?

英文摘要：

Objective： To investigate the effects of hexokinaseII （HKII） gene-silencing by shRNA interference on cell metabolism, proliferation, apoptosis and drug-resistance of human lymphoma cell lines Raji and SU-DHL-4, therefore to evaluate the potential value of HKII gene-targeted therapy for the treatment of lymphoma.Methods： The lymphoma cell lines Raji and SU-DHL-4 transfected with lentiviral vectors carring shRNAs targeting HKII gene （HKII shRNA） were established, while the control vectors carring negative control shRNAs were used as the control group. The expression levels of HKII mRNA and protein were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. Then the cell viability was assessed using trypan blue staining, and the cell growth curve was drawn. The half maximal inhibitory concentration （ICso） of doxorubicin （DOX） for lymphoma cells was examined apoptosis were analyzed by flow cytom proteins caspase-3, cleaved caspase-3 by Western blotting. The concentrations cell culture were analyzed using Lactic respectively. by CCK-8 method. The cell cycle distribution and etry. The expression levels of apoptosis-related （c-caspase-3）, Bcl-2 and Bcl-6 were detected of lactic acid and glucose in the supernatant of Acid Detection Kit and Glucose （HK） Assay Kit,Results： Compared with the control group, the levels of HKII mRNA and protein were significantly decreased in HKII shRNA transfected Raji and SU-DHL-4 cells （all P 〈 0.05）. After HKII gene-silencing by HKII shRNA interference, the proliferation of lymphoma Raji and SU-DHL-4 cells was significantly inhibited （both P 〈 0.01）, the cell cycle was arrested in Go/G1 phase （both P 〈 0.05）, and the apoptosis was induced （both P 〈 0.01）. Compared with the control group, HKII gene-silencing reduced ICs0 value of DOX in lymphoma Raji and SU-DHL-4 cells （both P 〈 0.05）, inhibited glucose consumption （both P 〈 0.05）, and decreased lactic acid generation （both P 〈 0.01）.