【作者】 祝亮方；

【导师】 胡建达；

【作者基本信息】 福建医科大学 ， 内科学， 2004， 硕士

【摘要】 目的：bcl-2是调控细胞凋亡的重要基因，它编码的蛋白通过阻止细胞凋亡而延长细胞存活。高达90％的肺癌组织和细胞株高表达bcl-2。细胞因子IL-6是一种重要的正性细胞调控因子，它通过与IL-6R结合发挥其生物学作用，目前的研究表明IL-6高表达与肺癌的发生、发展和转移密切相关。反义技术因其高效、特异和简便的特点，正逐渐成为人们研究肿瘤发病机制和基因治疗肿瘤的重要手段。本文应用bcl-2硫代磷酸寡脱氧核糖核苷酸(简写为bcl-2 AS-PS-ODN，下类同)、IL-6 AS-PS-ODN、IL-6R AS-PS-ODN三种反义寡核苷酸，研究它们分别单独作用以及bcl-2 AS-PS-ODN与IL-6 AS-PS-ODN联合作用对小细胞肺癌细胞株NCI-H446增殖和凋亡的影响，检测bcl-2、IL-6、IL-6R、Mcl-1等基因mRNA表达水平的变化，探讨三种AS-PS-ODN对NCI-H446细胞生长的影响及其调控机制，了解反义药物联合作用的效果。 方法：通过MTT实验、细胞克隆形成实验观察各组反义药物对细胞增殖的影响，应用细胞凋亡线粒体流式检测和DNA倍体分析观察各组药物对细胞凋亡的影响，建立半定量RT-PCR，检测各组药物作用后的NCI-H446细胞株bcl-2、IL-6、IL-6R、Mcl-1等相关基因mRNA表达水平的变化。 结果：1、各组反义寡核苷酸均能抑制NCI-H446细胞增殖和细胞克隆的形成，5μmol／L AS-PS-ODN作用48小时后，细胞增殖抑制率分别为：bcl-2 AS-PS-ODN 15.46％，IL-6 AS-PS-ODN 22.79％，IL-6R AS-PS-ODN 19.85％，bcl-2 AS-PS-ODN与IL-6 AS-PS-ODN联合作用组41.91％，与空白对照组及正义或无义对照组比较均有显著性差别(P＜0.05)。AS-PS-ODN作用7天后，细胞克隆形成率分别为：10μmol／L bcl-2 AS-PS-ODN 13.25％，5μmol／L IL-6 AS-PS-ODN 7.00％，10μmol／L bcl-2 AS-PS-ODN与5μmol／L IL-6 AS-PS-ODN联合作用组1.63％，10μmol／L IL-6RAS一PS一ODN 11 .88%，与空白对照组及正义或无义对照组比较均有显著性差别(尸<0.01)。各组反义寡核普酸均能诱导细胞凋亡，细胞凋亡线粒体流式检测结果显示:单药作用的凋亡率均在32.0%以下，而bel一2 AS一PS一ODN与IL一6 AS一PS一ODN联合作用时则达到64.7%，与空白对照组及正义或无义对照组比较均有显著性差别(尸<0.01)。DNA倍体分析显示:单药作用的凋亡率均在23.5%以下，而bel一2AS一PS一ODN与lL一6 AS一PS一ODN联合作用时则达到36.4% 2、半定量Rl’- PCR检测发现bel一2、IL一6、IL一6R和Mel一l四种基因在小细胞肺癌细胞株NCI一H446中均有比较高的表达，三种AS一PS一ODN作用后都能显著下调自身基因的表达，bcl一2 AS一PS一ODN能使自身基因表达下调62.58%、IL一6AS一PS一ODN能使自身基因表达下调84.07%、IL一6R AS一PS一ODN能使自身基因表达下调60.31%。bcl一2 AS一PS一ODN和IL一6 AS一PS一ODN作用细胞后除了能下调自身基因表达外，还伴有其它基因表达的改变，bel一ZAS一PS一ODN作用后IL一6表达上调74.3%，IL一6 AS一PS一ODN作用后有bc卜2表达下调32.2%和IL一6R表达上调65.8%，而IL一6R AS一PS一ODN作用后其它基因的改变很小。三种反义寡核昔酸作用后，Mcl一1基因表达的变化都不明显。 结论1、bcl一2 AS一PS一ODN和IL一6 AS一PS一ODN通过下调抗凋亡基因的表达来 诱导凋亡、抑制细胞增殖。 2、AS一PS一ODN联合作用效果较单独作用更佳。 3、IL一6与bcl一2处于同一调控链上，IL一6属上游调控基因，能够调控bcl一2 的表达，同时bel一2亦能通过负反馈调控IL一6的表达。更多还原

【Abstract】 Objective The bcl-2 gene is a critical regulator of the cell death process, and it encodes a 26DK Bcl-2 protein that promotes cell survival by blocking programmed cell death(apoptosis). In small cell lung cancer, up to 90% of tumors and cell lines overexpress bcl-2.The interleukin-6 (IL-6), a cytokine, plays a important role by combining to interleukin-6 receptor (IL-6R). The different studies have showed that IL-6 expression is related to occurrence, progression and metastasis of lung cancer. Antisense technique is the highlight with its effectiveness, specificity and convenience. This subject aimed to investigate separate as well as combination effect of three antisense phosphorothioate oligodeoxynucleotides (AS-PS-ODNs), i.e. bcl-2 AS-PS-ODN, IL-6 AS-PS-ODN and IL-6R AS-PS-ODN, on the proliferation and apoptosis of small cell lung cancer cell line NCI-H446, and on mRNA expression level of bcl-2,IL-6,IL-6R and Mcl-1 in NCI-H446 cell line .Methods Proliferations of cell line NCI-H446 treated with different group of AS-PS-ODNs were evaluated by growth curve and cell clone formation rate test. DNA content assay and cell apoptosis detection kit were used to assessed the apoptosis by flow cytometry. Semi-quantitative reverse transcription-PCR was performed to detect mRNA expression level of bcl-2, IL-6, IL-6R and Mcl-1 in NCI-H446 cell line. Results 1, All groups of AS-PS-ODNs could inhibit proliferation of NCI-H446 cell line. After treatment with AS-PS-ODNs in 5umol/L for 48 hours , inhibition rate of cell proliferation is 15.46% in group of bcl-2 AS-PS-ODN, 22.79% in group of IL-6 AS-PS-ODN 22.79% , 19.85% in group of IL-6R AS-PS-ODN, 41.91 % in combined group of bcl-2 AS-PS-ODN and IL-6 AS-PS-ODN respectively, significantly higher than that in control or (N)S-PS-ODN (P<0. 05). While NCI-H446 cell line being treated with AS-PS-ODNs in 10mol/L for 7 days ,the cell clone formation rate was 13.25% inbcl-2 AS-PS-ODN, 7.00% in IL-6 AS-PS-ODN 22.79%, 1.63% in combined group of bcl-2 AS-PS-ODN and IL-6 AS-PS-ODN and 11.88% in IL-6R AS-PS-ODN respectively, also significantly lower than that in control or (N)S-PS-ODN (P<0. 01). All groups of AS-PS-ODN could induced apoptosis . The result detected by cell apoptosis detection kit demonstrated that the apoptosis rate is less than 32.0% in group of single AS-PS-ODN, and 64.7% in combined group of bcl-2 AS-PS-ODN and IL-6 AS-PS-ODN. The difference is also significant as comparing to control or (N)S-PS-ODN (P< 0. 01). Consistently, DNA content assay displayed that the apoptosis rate is less than 23.5% in group of single AS-PS-ODN, and 36.4% in combined group of bcl-2 AS-PS-ODN and IL-6 AS-PS-ODN.2, Semi-quantitative reverse transcription-PCR analysis revealed that expressions of bcl-2,IL-6 , IL-6R and Mcl-1 are quite high in NCI-H446 cell line. Three AS-PS-ODNs could obviously down-regulated expression.of itself mRNA, 62.58% in bcl-2 AS-PS-ODN, 84.07% in IL-6 AS-PS-ODN 22.79% and 60.31% in IL-6R AS-PS-ODN. Moreover, bcl-2 AS-PS-ODN could still up-regulate IL-6 expression by 74.3 % .while IL-6 AS-PS-ODN could down-regulate bcl-2 expression by 32.2 % accompanying with increased IL-6R expression by 65.8 % . However, IL-6R AS-PS-ODN could regulate a little to other gene and Mcl-1 expression could not be found in three AS-PS-ODNs group.Conclusion 1. bcl-2,IL-6 and IL-6R AS-PS-ODNs inhibit proliferation and viability of NCI-H446 and induce apoptosis by down-regulating expression of anti-apoptosis genes. 2. Combination effect of AS-PS-ODNs was better than single AS-PS-ODN. 3. IL-6 can regulate bcl-2 expression because it is upstream, and bcl-2 can also regulateIL-6 expression by feedback..更多还原