【作者】 郭芮伶；

【导师】 吴国明；

【作者基本信息】 第三军医大学 ， 内科学， 2008， 博士

【摘要】 缺氧是肺癌等实体肿瘤的主要微环境特征,其选择压力及其诱导的遗传变异是肿瘤细胞遗传异质性和遗传不稳定性的重要原因,可诱导肿瘤细胞表现出更具恶性的生物学表型、更强的侵袭和转移能力以及对化疗和放疗的不敏感性。DNA错配修复(MMR)在维持遗传稳定性中具有重要作用。已有研究表明,缺氧可能与MMR基因表达失活有关,但其机制尚不清楚。以往的研究多认为,基因水平的改变主要为基因的突变或缺失即遗传学层面上的改变,但随着基因表遗传学研究的加深,越来越多的人认识到基因的表达增高或降低与表遗传学水平上的改变如DNA甲基化等相关。但是,DNA甲基化等表遗传学水平上的改变是否参与了缺氧对MMR基因的调控尚需进一步探讨。同时,鉴于甲基化模式异常与肿瘤密切相关,而肿瘤细胞内可能存在迫使其发生甲基化失衡的压力,缺氧是否参与其中也值得关注。此外,甲基化作为一个可逆的调控过程,有望成为肿瘤干预新的切入点。已证实去甲基化药物5-Aza-CdR可恢复甲基化基因的表达,并在多种肿瘤中表现出一定的抗癌活性,但在小细胞肺癌中的应用目前尚未见报道。目的1.研究缺氧(3% O2)对人小细胞肺癌H446细胞生物学性状的影响。2.观察缺氧对错配修复基因MLH1、MSH2表达的影响,并探讨启动子甲基化在其表达调控中的作用。3.探讨缺氧与DNA甲基化的关系。4.研究甲基转移酶抑制剂5-Aza-CdR对H446细胞的抑制生长作用及其与缺氧的关系。方法1.观察缺氧对H446细胞生物学性状的影响光镜及透射电镜观察缺氧条件下H446细胞的形态学变化;MTT法检测缺氧条件下H446细胞的增殖能力及对多种化疗药物的半数抑制浓度; PI标记流式细胞术检测缺氧条件下细胞周期分布;自发光荧光仪检测缺氧条件下细胞内caspase-3/7活性;罗丹明123外排实验评估缺氧对P-gp介导的药物外排能力的影响;流式细胞仪及荧光显微镜检测缺氧对细胞内活性氧簇生成的影响。2.观察缺氧对MLH1、MSH2基因表达的影响RT-PCR检测MLH1、MSH2基因在mRNA水平上的表达;Western blot检测MLH1、MSH2基因在蛋白水平上的表达。3.探讨DNA甲基化在MLH1、MSH2基因表达下调中的作用及其与缺氧的关系MSP检测缺氧条件下MLH1、MSH2基因启动子甲基化状态的变化并测序鉴定;RT-PCR及Western blot检测5-Aza-CdR对缺氧条件下MLH1、MSH2基因表达的恢复作用;MS-AP-PCR检测缺氧条件下H446细胞基因组甲基化水平的变化。4.观察5-Aza-CdR对H446细胞的生长抑制作用及其与缺氧的关系MTT法检测细胞增殖能力及对多种化疗药物的半数抑制浓度;PI标记流式细胞术检测细胞周期分布;自发光荧光仪检测细胞内caspase-3/7活性;罗丹明123外排实验评估P-gp介导的药物外排能力;MS-AP-PCR检测细胞基因组甲基化水平。结果1.在缺氧条件下,H446细胞增殖能力显著减弱,胞浆内可见线粒体肿胀、细胞器空泡变、髓鞘样改变和核糖体增多,部分细胞内出现微腺腔;同时,H446细胞对VP-16、多柔比星等化疗药物的敏感性显著降低,Rh123外排效率显著增加(常氧:11.45±2.33,缺氧:17.25±1.46,P<0.05);随着缺氧时间的延长,H446细胞中S期细胞显著增加,G2期细胞显著减少(P<0.01),缺氧48h后细胞内casase-3/7活性显著增强。H446细胞内活性氧含量在缺氧早期显著增多,缺氧12h后显著减少(P<0.01)。2.缺氧状态下,H446细胞MLH1、MSH2基因在转录和翻译水平均显著性降低(P<0.01)。同时,随着缺氧时间延长,MSH2基因启动子直接由非甲基化状态转变为完全甲基化状态,所有待测CpG位点均发生甲基化,而MLH1基因启动子逐渐由非甲基化状态、部分甲基化状态转变为完全甲基化状态,只有部分位点发生了甲基化。5-Aza-CdR可使缺氧状态下H446细胞的MLH1、MSH2基因表达水平有所恢复,但去除5-Aza-CdR后基因表达再次下调。常氧及缺氧条件下H446细胞基因组甲基化水平存在显著差异,后者同时出现部分基因高甲基化和部分基因低甲基化。3.常氧条件下,5-Aza-CdR可显著降低H446细胞的增殖能力(呈剂量依赖性,P<0.01),诱导G1期阻滞(呈时间依赖性,P<0.05)和细胞凋亡(P<0.05)。同时,P-gp介导的药物外排效率增加,基因组DNA甲基化水平降低,但H446细胞对顺铂等化疗药物的敏感性无显著性变化。缺氧在抑制细胞增殖、诱导G1期阻滞和降低基因组DNA甲基化水平等方面与5-Aza-CdR具有协同作用。结论1.缺氧状态下,人小细胞肺癌H446细胞生物学性状发生显著改变:细胞增殖能力减弱、大量细胞阻滞在S期、凋亡潜能细胞减少、药物外排效率增强,并对化疗药物的敏感性降低。2.缺氧状态下,H446细胞错配修复基因MLH1、MSH2基因的表达显著降低,启动子甲基化可能是其表达下调的重要机制之一。3.缺氧可能是造成肿瘤细胞甲基化异常的压力之一。4.在常氧及缺氧状态下,5-Aza-CdR均可显著抑制H446细胞生长,错配修复基因表达恢复、诱导G1期阻滞和细胞凋亡可能是其抗瘤活性的主要机制,而缺氧具有部分协同作用。更多还原

【Abstract】 Hypoxia is a common microenvironmental characteristic of human solid tumors including lung cancer. The selective pressure and induced mutations of hypoxia may be the strong driving force for genetic heterogeneity and instability, which contribute to a poor prognosis due to tumor progression towards a more malignant phenotype, with increased metastatic potential, and an increased resistance to treatment.Some evidence has indicated that hypoxia is related to inactive MMR, which plays an important role in maintaining genetic stability, but the mechanism is still unclear. It was previously thought that gene changes mainly meant mutation or deletion, namely at genetics level, but with recent progress of epigenetics, more and more people realized that the regulation of gene expression also was closely related to changes at epigenetics level such as DNA methylation, even more critical. Therefore, it is worth further exploring the relationship between DNA methylation and mechnism of hypoxia on MMR genes regulation.Furthermore, abnormal methylation patterns is closely related to cancer and some forces in cancer cells involved in. The relationship between hypoxia and methylation deserved attention.In conclusion, methylation is a reversible regulation program, which may become a new strategy of gene therapy for tumor. The treatment of demethylation drugs, such as 5-Aza-CdR, can restore the tumor suppressor gene and DNA repair gene expression and shows a certain antitumor activity in several tumors, but not mentioned in SCLC.Objective1. To observe the effect of hypoxia on biological characteristics of human SCLC cell line H446.2. To investigate the expression and the role of promoter methylation of DNA mismatch repair genes MLH1 and MSH2 in H446 cells under hypoxic condition.3. To study the growth inhibition effect of 5-Aza-CdR on H446 cells and the relationship with hypoxia.Materials and methods1. Light microscope and TEM were used to observe cell morphological changes. MTT was used to determine the proliferation and the 50% inhibitory concentration(IC50) for different drugs. FACs was emplored to detect the efficacy of drug exclusion and cell cycle. The caspase-3/7 activity and generation of ROS was assessed by luminometer, flow cytometry and fluorescence microscope, respectively.2. RT-PCR and Western blot were applied to detect MLH1 and MSH2 expression in human SCLC cell line H446 at the mRNA and the protein level, respectively, under the hypoxic condition and after 5-Aza-CdR treatment. Meanwhile, methylation-specific PCR(MSP) was used to determine promoter methylation of MLH1 and MSH2. MS-AP-PCR was emplored to detect the genomic methylation level of H446 cells under hypoxic condition.3. The proliferation, IC50, cell cycle caspase-3/7 activity, Rh123 exclusion efficacy and genomic methylation level of H446 cells with 5-Aza-CdR treatment were analyzed as above.Result1. Under hypoxic condition, the proliferation of H446 cell was decreased significantly. Mitochondria swelling,organelle bubbing and myelinogenesis were observed in H446 cells, even microadencavity emerged in some cells. Meanwhile, H446 cells were more resisted to VP-16 and doxorubicin (P<0.05) under hypoxic condition. Rh123 exclusion efficacy of H446 cells significantly increased (P<0.05). Hypoxia induced the arrest of H446 cells in S phase in the time manner. Moreover, the G2 phase cells decreased, finally disappeared after 48h, but the caspase-3/7 activity did not increase until 48h. FACs showed that ROS increased at first, but significantly decreased after 12h.2. The expression of MLH1 and MSH2 in H446 cells significantly decreased both at the mRNA and the protein level under the hypoxic condition. Meanwhile, the promoter methylation status changed with time of hypoxia: MSH2 gene promoter directly changed from unmethlation to complete methylation and all the CpG sites occurred methlation. Nevertheless, MLH1 gene promoter gradually transformed unmethylation into partial methylation, finally methylation completely and only part of CpG sites occurred methlation. 5-Aza-CdR treatment led to the restoration of MLH1 and MSH2 expression, while, both MLH1 and MSH2 were down-regulated again after removing 5-Aza-CdR. Some genes high-methylation and some genes low-methylation both appeared in H446 cells under hypoxic condition, which was different from H446 cells under normoxic condition.3. Under normoxic condition, 5-Aza-CdR could significantly decrease the proliferation of H446 cells in dose-indepentent manner, induce G1 phase arrest and apoptosis in time-independent manner. Simultaneously, Rh123 exclusion efficacy was increased, genomic methylation level was decreased, while the sensitivities to drugs were not affected. Hypoxia has a synergistic effect on growth inhibition, G1 phase arrest and genomic methylation level reduction.Conclusion1. Hypoxia could inhibit growth, induce S phase arrest, increase the efficacy of drug exclusion and decrease apoptotic potential cells, meanwhile, decrease the sensitivities of H446 cells to chemotherapeitic drugs. This shows that hypoxia may regulate the biological characteristics of tumor cells and its process through a variety of mechanisms.2. The promoter methylation of MLH1 and MSH2 may play an important role in its defective expression in H446 cells under the hypoxic conditon.3. Hypoxia might be one of forces inducing abnormal methylation of tumor cells.4. 5-Aza-CdR could inhibit cell growth, restore mismatch repair genes expression, induce G1 phase arrest and apoptosis, which might be important in antitumor effect of 5-Aza-CdR on H446 cell. Hypoxia has a certain synergy.更多还原