【作者】 王宁；

【导师】 邢丽华；

【作者基本信息】 郑州大学 ， 内科学， 2011， 硕士

【摘要】 背景：肺癌是目前严重威害人类健康和生命的恶性肿瘤之一,根据流行病学相关研究结果,近年来,肺癌的发病率和死亡率呈上升趋势,据统计,占所有恶性肿瘤发病率的12%。在欧美某些国家和我国大中城市,其发病率已居于首位,且发病年龄趋于年轻化,与吸烟、环境污染物密切相关,其治疗难度大、迅速转移、死亡率高为特点。肿瘤的浸润与转移己成为当今导致治疗失败和患者死亡的主要原因,因此寻找关键靶位点抑制肺癌血管生成,为肺癌的临床治疗开辟一条新的途径。核因子-kB (nuclear factor-kappa B, NF-kB)是一种多显性基因转录因子,与多种基因的转录密切相关,在肿瘤细胞中高表达,与多种细胞基因的启动子和增强子发生特异性结合,与血管内皮生长因子(Vascular endothelial growth factor, VEGF)、环氧合酶-2(Cyclooxygenase -2, COX-2)关系密切,参与炎症反应、损伤应激、免疫防御、细胞增殖及凋亡等过程。核因子-кB圈套寡脱氧核苷酸(NF-kB decoy ODN)是目前研究发现进行体内外基因调控的强有力工具,含有转录因子识别序列的寡核苷酸或质粒转入细胞,与核内基因启动子区相应顺势作用元件竞争结合转录因子,从而抑制启动子活化,达到抑制基因转录和翻译的目的,其能明显抑制癌细胞的增殖,并可提高肿瘤细胞对化疗药物(紫杉醇)的敏感性。目的：研究NF-κB圈套寡脱氧核苷酸(NF-κB decoy ODN)联合紫杉醇对肺癌A549细胞增殖、凋亡、血管生成的影响及其作用机制,为临床实践提供理论依据。方法：1、培养人肺癌细胞A549。2、脂质体瞬时转染法介导核因子-кB圈套寡核苷酸(NF-κB decoy ODN)和核因子-кB错义圈套寡核苷酸(NF-κB scramble decoy ODN)处理A549细胞。3、MTT试验观察干预96 h经NF-κB decoy ODN及NF-κB Scramble decoyODN作用后肺癌A549细胞生长曲线。4、设对照组、NF-κB decoy ODN组、紫杉醇组(1000 ng/mL)和NF-κB decoyODN+紫杉醇组,RT-PCR法测环氧合酶-2(COX-2) mRNA水平表达,免疫组织化学法测血管内皮生长因子(VEGF)蛋白表达,流式细胞术检测各组48 h后细胞凋亡率。结果：1、核因子-кB圈套寡脱氧核苷酸(NF-κB decoy ODN)使细胞生长明显受到抑制,时间越长抑制作用越明显。2、核因子-кB圈套寡脱氧核苷酸(NF-κB decoy ODN)、紫杉醇干预后A549细胞的环氧合酶-2(COX-2) mRNA水平表达及血管内皮生长因子(VEGF)蛋白表达均下降,COX-2mRNA水平表达各组浓度分别是：0.71±1.4μg/μL、0.44±1.8μg/μL、0.51±7.2μg/μL、0.34±9.5μg/μL (F=7.8, P<0.05), VEGF蛋白表达各组细胞阳性率分别是：85±0.50%,63±0.26%,57±1.5%,21±0.34%(F=21,P<0.05)。3、核因子-кB圈套寡脱氧核苷酸(NF-κB decoy ODN)联合紫杉醇可促使A549细胞凋亡增加,二者联用呈协同或相加作用,各组细胞的总凋亡率分别是：(16.8±0.01)%、(5.6±0.02)%、(32.6±0.04)%,均高于对照组(6.0±0.02)%(F=14,P<0.05)。结论：1.核因子-кB圈套寡脱氧核苷酸(NF-kB decoy ODN)能明显抑制肺癌A549细胞的生长。2.核因子-кB圈套寡脱氧核苷酸(NF-kB decoy ODN)联合紫杉醇可明显抑制肺癌A549细胞的生长,该作用可能与环氧合酶-2(COX-2)、血管内皮生长因子(VEGF)的表达相关。3.核因子-кB圈套寡脱氧核苷酸(NF-kB decoy ODN)联合紫杉醇可增强对肺癌A549细胞增殖的抑制及凋亡的诱导,二者具有协同抗肿瘤作用。更多还原

【Abstract】 Background:Lung cancer is a serious threat to harm human health and life of the malignant tumor, according to the results of epidemiological studies in recent years, incidence and mortality lung cancer is rising, according to statistics, the incidence of all malignant tumors accounted for 12%. Some countries in Europe, America and our cities, the incidence rate has primacy, and the younger age of onset tends to, and smoking, environmental pollutants are closely related, the treatment is difficult, rapid transfer, high mortality characterized. Tumor invasion and metastasis has become the lead to treatment failure and the leading cause of death in patients, so look for the key target sites of angiogenesis inhibition of lung cancer, for lung cancer treatment to open up a new way.Nuclear factor (nuclear factor- kappa B, NF-kB) is a multi-dominant gene transcription factor, and transcription of multiple genes closely related to high expression in tumor cells, with a variety of cellular gene promoter and enhancer specific binding occurs, and cyclooxygenase 2 (Cyclooxygenase -2,COX2), vascular endothelial growth factor (Vascular endothelial growth factor,VEGF) is closely related, is involved in inflammation, injury stress, immune defense, cell proliferation and apoptosis process. NF-kB decoy oligodeoxynucleotide (NF-kB decoy ODN) is carried out in vivo study found a powerful tool for gene regulation, transcription factor recognition sequence containing oligonucleotides or plasmids into cells, and nuclear genes the role of the promoter region of the corresponding advantage of the opportunity component competitive binding transcription factor, thereby inhibiting the activation of the promoter, to inhibit gene transcription and translation purposes, its value can inhibit cancer cells, and enhance the tumor cells to chemotherapeutic drugs (paclitaxel) sensitivityObjective:Research NF-kB decoy oligodeoxynucleotide (NF-kB decoy ODN) combined with paclitaxel on lung cancer A549 cell proliferation, apoptosis, angiogenesis and its mechanism of action, provide the theoretical basis for clinical practice.Methods:1. Cultivate human lung cancer cell A549.2. Using liposome-mediated transient transfection method full phosphorothioate NF-kB decoy oligonucleotides (decoy ODN), and all missense phosphorothioate decoy oligonucleotides (scramble decoy ODN) treatment A549 cells.3. MTT test interventions observed 96 h after NF-kB decoy ODN and NF-kB Scramble decoy ODN role in lung cancer A549 cells after growth curve.4. Divided into the control group, NF-kB decoy ODN group, paclitaxel group (1000ng/ml) and NF-KBdecoy ODN + paclitaxel group, RT-PCR method to measure the level of Cyclooxygenase -2(COX-2)mRNA expression measured by immunohistochemistry the expression of Vascular endothelial growth factor (VEGF) protein in the flow Type cells 48 h after surgery to detect the apoptotic rate. Results:1. NF-κB decoy oligodeoxynucleotide (NF-κB decoy ODN) significantly inhibited the cell growth, the more obvious the longer inhibited.2. NF-κB decoy oligodeoxynucleotide (NF-κB decoy ODN), paclitaxel A549 cells after the intervention levels of Cyclooxygenase -2(COX-2)mRNA expression and VEGF protein expression decreased, COX-2 mRNA expression levels of concentration in each group were:0.71±1.4μg/μL,0.44±1.8μg/μL,0.51±7.2μg/μL,0.34±9.5μg/μL (F=7.8, P<0.05), Vascular endothelial growth factor(VEGF) protein expression positive rate of cells in each group were:85±0.50%,63±0.26%,57±1.5%,21±0.34%(F= 21, P＜0.05).3. NF-κB decoy oligodeoxynucleotide (NF-κB decoy ODN) combined with paclitaxel can promote apoptosis of A549 cells, the two together were synergistic or additive effect with each group the apoptosis of cells is:(16.8±0.01)%, (5.6±0.02)%, (32.6±0.04)%, were higher (6.0±0.02)%(F=14, P<0.05).Conclusion:1. Nuclear factor-κB decoy oligodeoxynucleotide (NF-κB decoy ODN) significantly inhibited the growth of lung cancer A549 cells.2. The nuclear factor-κB snare widowed deaeration nucleotide (NF-κB decoy ODN) unites the taxus mellowly to be possible to suppress the lung cancer A549 cell’s growth obviously, this function is possible and epoxy synthase-2 (COX-2), blood vessel bast growth factor (VEGF) expression related.3. The nuclear factor -κB snare widowed deaeration nucleotide (NF-κB decoy ODN) unites the taxus mellowly to be possible to strengthen the induction which and perishes weakly to lung cancer A549 cell multiplication’s suppression, the two have coordinate the anti-tumor function更多还原