中文摘要：

研究表明,葡萄糖-6-磷酸脱氢酶（G6PD）与肿瘤的发生、发展、临床表型及 治疗和预后有关.为阐明G6PD与肿瘤的关系及其机理,针对人G6PD基因设计3条siRNA和一条无关序列,再据每一序列,合成两条互补并含siRNA正反义链的DNA,退火后与含GFP的载体pRNAT-U6.2/Lenti连接,转染人皮肤黑色素瘤A375细胞,Real-timePCR筛选有效的一条siRNA,经病毒颗粒包装和病毒生产并感染A375细胞,G418筛选后,挑取单个阳性克隆放大培养,Western blotting检测siRNA干扰G6PD效率为88.83%,构建成G6PD缺陷型A375稳转细胞（A375-G6PD!）.与野生型A375细胞（A375-WT）比较,A375-G6PD!的G6PD活性仅为21.53%,细胞倍增时间延长,增殖被抑制,克隆形成率降低25%（P〈0.05）,凋亡细胞数增加2.86倍（P〈0.01）,SPF增加33.8%（P〈0.05）,PI增加59.7%（P〈0.01）,G0/G1期下降27.7%（P〈0.01）,凋亡相关蛋白P53下降54.7%（P〈0.01）、Caspase-3增加2.2倍（P〈0.01）和Bcl-2降低21.7%（P〉0.05）,提示,G6PD缺陷可能通过下调P53蛋白表达和上调Caspase-3的表达,抑制G2/M期向G0/G1期转换的进程,促进A375的凋亡,机理有待于进一步探讨.

英文摘要：

Glucose-6-phosphate dehydrogenase （G6PD） derives from the expression of the house-keeping gene G6PD. Recent studies have indicated that G6PD is related to tumor genesis, growth, clinical phenotype, therapy, and prognosis. To elucidate the relationship between G6PD and cancer, three siRNA sequences and one negative control sequence were designed based on the 3＇ noncoding region of the human G6PD gene. Two complementary single-strand DNA （sense and antisense） were designed and synthesized based on siRNA sequences. The DNA fragments were annealed and ligated to the GFP expression vector pRNAT-U6.2/Lenti. One siRNA with higher interference efficiency than the other two was found after siRNA plasmid transfecting human skin A375 melanoma cells. After lentivirus particle packaging and virus production, the A375 cells were infected, and the single cell clone was acquired and cultured to establish the stable cell strain. Western blotting showed that the endogenous G6PD in the stable A375 cell strain was 0.257 ± 0.074, which was 11.17% of G6PD expression （2.301 ± 0.286） in wild type A375 cells. The final siRNA interference efficiency in this stable cell strain was 88.83%. The G6PD activity of A375-G6PDA was 21.53% of A375-WT. Further study showed that A375-G6PDA doubling generation time prolonged, and its proliferation was greatly inhibited and the cloning efficiency lowered 25% （P 〈 0.05）, compared with A375-WT cell. FCM analysis indicated that apoptosis cell in A375-G6PDA was 2.86 times as that ofA375-WT（P 〈 0.01） with 33.8 % increase of SPF（P〈 0.05）, 59.7 % raise of Pl（P〈 0.01）, and 27.7 % decrease of G0/G1 phase（P 〈 0.01）. Furthermore, apoptosis-associated protein check showed that Caspase-3 was 2.86 times as that of A375-WT（P 〈 0.01） with 54.7% descend of P53（P 〈 0.01）. It is proposed that G6PD can maintain the growth and proliferation of A375 cell. G6PD deficiency probably restrains the change proceeding of G2/M phase to G0/G1 phase in A375A cell cycle through