【作者】 李琪；

【导师】 胡克；

【作者基本信息】 武汉大学 ， 内科学（呼吸内科）（专业学位）， 2017， 博士

【摘要】 目的:观察5 氮杂-2’-脱氧胞音(5-aza-2’-deoxycytidine,5-Aza-CdR)对肺腺癌SPC-A-1细胞RAR-β、WIF1、RASSF2A基因甲基化状态的影响,探讨5-Aza-CdR对RAR-β、WIF1、RASSF2A基因表达缺陷的调控机制。方法:分别用不同处理浓度及不同作用时间的5-Aza-CdR对体外培养的人肺腺癌SPC-A-1细胞株进行处理。细胞株依据不同处理浓度分为A组(未处理组)及B、C、D组(分别加入3、6、12 μmol/L的5-Aza-CdR),均于体外培养72h后待测。再依据不同作用时间分为24h、48h、72h、96h组,均加入12 μmol/L浓度的5-Aza-CdR。采用甲基化特异性PCR(MSP)方法检测4组细胞(A、B、C、D组)RAR-β、WIF1、RASSF2A基因的甲基化状态差异,RT-PCR法分别检测不同浓度组及不同时间组RAR-β、WIF1、RASSF2A基因mRNA表达水平的差异,Western blot分别检测不同浓度组及不同时间组RAR-β、WIF1、RASSF2A蛋白表达水平的差异。结果:(1)A组标本检测出RAR-β、WIF1、RASSF2A基因均为甲基化状态,B、C、D组检测出RAR-β、WIF1、RASSF2A均为去甲基化状态;(2)不同浓度组中,A组的RAR-β、WIF1、RASSF2A基因mRNA及蛋白表达水平均最低,D组mRNA及蛋白表达水平最高(p<0.05)。mRNA及蛋白表达水平随着5-Aza-CdR处理浓度升高而逐渐增强(p<0.05),mRNA与蛋白表达具有一定的浓度依赖性。此外,不同时间组中,72h组与96h组mRNA及蛋白表达水平差异无统计学意义(p>0.05),而在72h作用时间内,mRNA及蛋白表达水平随时间延长而逐渐增强(p<0.05),mRNA与蛋白表达具有一定的时间依赖性。结论:肺癌RAR-β、WIF1、RASSF2A基因因DNA高甲基化出现表达缺陷,甲基化是一个可逆转的过程,甲基化可作为肿瘤特异性治疗的靶点。5-Aza-CdR通过去甲基化作用可重新激活表达缺陷的RAR-β、WIF1、RASSF2A基因,从而使mRNA及蛋白表达水平明显上调,发挥抑癌作用。目的:探讨5-Aza-CdR对肺腺癌SPC-A-1细胞增殖及细胞凋亡的影响及可能的作用机制。方法:分别用不同处理浓度的5-Aza-CdR对体外培养的人肺腺癌SPC-A-1细胞株进行处理。分为A组(未处理组)及B、C、D组(分别加入3、6、12 μmol/L的5-Aza-CdR),均于加药培养72h后待测。Western blot检测4组细胞Bcl-2、Bax蛋白表达水平的差异。流式细胞仪检测4组细胞周期及细胞凋亡率的差异。Hoechst33258染色法观察不同浓度5-Aza-CdR对肺腺癌SPC-A-1细胞凋亡的影响。结果:(1)4组中,随着5-Aza-CdR的处理浓度增加,Bcl-2蛋白表达水平逐渐下降,而Bax蛋白表达水平逐渐升高(p<0.05),5-Aza-CdR量效有一定的相关性;(2)B、C、D加药组与未处理A组比较,细胞总体凋亡率均有显著性升高(P<0.05),D组细胞总体凋亡率最高(17.45±1.82)%(P<0.05)。随着5-Aza-CdR处理浓度的升高,处于G0/G1期的细胞比例明显升高(P<0.05),处于S期或G2/M期的细胞比例明显下降(P<0.05)。5-Aza-CdR对细胞周期比例及细胞凋亡的影响呈一定的浓度依赖性。(3)D组RAR-β、WWIF1、RASSF2A、Bax蛋白表达水平均与细胞凋亡率成正相关(P<0.05),Bcl-2蛋白表达水平与细胞凋亡率成负相关(P<0.05)。结论:5-Aza-CdR可干扰肺癌细胞周期,将细胞周期循环阻滞于G0/G1期,具有明显抑制细胞增殖的作用。5-Aza-CdR通过下调Bcl-2的表达及上调Bax的表达,并通过去甲基化作用上调RAR-β、WIF1、RASSF2A的表达,可诱导细胞凋亡,发挥抗癌效应。目的:探讨5-Aza-CdR对人肺癌移植瘤裸鼠的抑瘤作用及可能的作用机制。方法:构建人肺腺癌SPC-A-1细胞移植瘤裸鼠的模型,将接种成瘤的40只裸鼠随机分为模型组及治疗Ⅰ、Ⅱ、Ⅲ组,每组各10只。模型组裸鼠给予生理盐水灌胃,隔日一次,按裸鼠体质量计算,治疗Ⅰ、Ⅱ、Ⅲ工组裸鼠分别腹腔注射0,5 μg/g、1μg/g、2μg/g单位剂量的5-Aza-CdR注射液,隔日一次,治疗共持续4周。测定4组裸鼠肿瘤体积与体质量,计算抑瘤率,观察不同剂量的5-Aza-CdR对人肺癌裸鼠移植瘤的生长影响及对荷瘤裸鼠的毒副作用,MSP方法检测5-Aza-CdR治疗前后的移植瘤RAR-β、WIF1、RASSF2A甲基化状态的差异,Western blot技术检测4组裸鼠移植瘤RAR-β、WIF1、RASSF2A蛋白表达的差异。结果:(1)5-Aza-CdR治疗4周后,治疗Ⅰ、Ⅱ、Ⅲ组肿瘤平均体积和体质量与模型组比较均明显减小(P<0.05),且肿瘤平均体积和体质量随着5-Aza-CdR治疗剂量的增加而明显减小(P<0.05),抑瘤率随着5-Aza-CdR治疗剂量的增加而明显升高,抑瘤效应呈剂量依赖性。(2)模型组移植瘤标本检测出RAR-β、WIF1、RASSF2A基因均为甲基化状态,治疗Ⅰ、Ⅱ、Ⅲ组检测出RAR-β、WIF1、RASSF2A 均发生去甲基化。(3)模型组的 RAR-β、WIF1、RASSF2A蛋白表达水平在4组中最低,治疗Ⅲ组蛋白表达水平最高(p<0.05),蛋白表达水平随着5-Aza-CdR治疗剂量增加而逐渐升高(p<0.05),蛋白表达具有一定的剂量依赖性。结论:5-Aza-CdR能抑制人肺癌裸鼠移植瘤的生长,可能是通过去甲基化上调抑癌基因RAR-β、WIF1、RASSF2A的表达,进而诱导细胞凋亡来实现抑瘤作用。通过探索5-Aza-CdR的体内外抗肿瘤作用机制,可能为今后肺癌的临床综合治疗提供一种新的方法和理论依据。更多还原

【Abstract】 Objective:To observe the effects of 5-aza-2’-deoxycytidine(5-Aza-CdR)on the methylation status of RAR-β,WIF1 and RASSF2A in lung adenocarcinoma cell line SPC-A-1,and to investigate the regulatory mechanism of 5-Aza-CdR on the defective expression of RAR-β,WIF1 and RASSF2A gene.Methods:The human lung adenocarcinoma cell line SPC-A-1 was treated by 5-Aza-CdR with different concentration and different time.The cell line SPC-A-1 was divided into group A(untreated group)and group B,C,D(add 3,6,12 μmol/L 5-Aza-CdR,respectively)according to different concentration,and were all cultured in vitro for 72h.In addition,the cell line was divided into 24h,48h,72h and 96h groups according to different time,and were all added with 12 μmol/L concentration of 5-Aza-CdR.Differences in RAR-β,WIF1 or RASSF2A gene methylation status among the four groups(A,B,C,D)were detected by methylation-specific PCR(MSP).Differences in mRNA expression and protein expression of RAR-β,WIF1 or RASSF2A gene among the different concentrations or different time groups were detected by RT-PCR and Western blot,respectively.Results:(1)RAR-P,WIF1 and RASSF2A gene methylation was observed in group A,while demethylation of RAR-β,WIF1 and RASSF2A gene was observed in the other 3 groups.(2)Among the different concentrations groups,mRNA and protein expression levels of RAR-β,WIF1 or RASSF2A gene in the group A were the lowest,while the mRNA and protein expression levels in the group D were the highest(p<0.05).The expression level of mRNA and protein increased with the concentration of 5-Aza-CdR(p<0.05),and mRNA or protein expression had a certain concentration dependence.Furthermore,there was no significant difference in mRNA and protein expression between 72h group and 96h group(p>0.05).However,the expression level of mRNA and protein increased gradually with the prolongation of time in 72h(p<0.05),and mRNA or protein expression had a certain time dependence.Conclusion:The expression of RAR-β,WIF1 and RASSF2A gene in lung cancer was defective due to hypermethylation of DNA,and methylation is a reversible process,which can be used as a target for tumor specific therapy.5-Aza-CdR can reactivate the expression of defective RAR-β,WIF1 and RASSF2A gene via its demethylation effect,and cause the expression of mRNA and protein be up-regulated,and thus plays a role in tumor inhibition.Objective:To investigate the effects of 5-Aza-CdR on cell proliferation and apoptosis of lung adenocarcinoma cell line SPC-A-1 and its possible mechanism.Methods:The human lung adenocarcinoma cell line SPC-A-1 was treated by 5-Aza-CdR with different concentration.The cell line SPC-A-1 was divided into group A(untreated group)and group B,C,D(add 3,6,12 μmol/L 5-Aza-CdR,respectively)according to different concentration,and were all cultured in vitro for 72h.Differences in protein expression of Bcl-2 and Bax among the four groups were detected by Western blot.Then differences in cell cycle and apoptosis rate among the four groups were detected by flow cytometry.Moreover,Hoechst33258 staining was used to observe the effect of 5-Aza-CdR with different concentration on the apoptosis of lung adenocarcinoma cell line SPC-A-1.Results:(1)Among the four groups,the expression level of Bcl-2 protein decreased gradually with the increase of concentration of 5-Aza-CdR,while the expression level of Bax protein increased gradually with the concentration of 5-Aza-CdR(p<0.05).The doses of 5-Aza-CdR were correlated with the effects.(2)Overall apoptosis rates of in 5-Aza-CdR treatment groups were significantly increased compared with group A(P<0.01),and the overall apoptosis rate in the group D was the highest among the four groups(17.45±1.82)%(P<0.05).The proportion of cells in G0/G1 phase was significantly increased with the concentration of 5-Aza-CdR,while the proportion of cells in S phase or G2/M phase decreased significantly with the increase of concentration of 5-Aza-CdR(p<0.05).The effect of 5-Aza-CdR on the ratio of cell cycle and apoptosis was concentration dependent.(3)The expression levels of RAR-P,WIFI1 RASSF2A and Bax protein in group D were positively correlated with the apoptosis rate(p<0.05).Meanwhile,the expression level of Bcl-2 protein was negatively correlated with the apoptosis rate(p<0.05).Conclusion:5-Aza-CdR can interfere with the cell cycle of lung cancer and arrest the cell cycle in G0/G1 phase,thus it can inhibit cell proliferation of lung cancer.5-Aza-CdR can down-regulate the expression of Bcl-2 and up-regulate the expression of Bax,and up-regulate the expression of RAR-β,WIF1 and RASSF2A by demethylation,therefore it can induce apoptosis and exert anticancer effect.Objective:To investigate the antitumor effect of 5-Aza-CdR on human lung cancer xenograft in nude mice and its possible mechanism.Methods:Nude mice model with human lung adenocarcinoma cell line SPC-A-1 transplanted was established.A total of 40 nude mice with lung adenocarcinoma xenografted tumor were randomly divided into the model group,treatment Ⅰ,Ⅱ andⅢ groups with 10 mice in each group.After model establishment,mice in the model group were given normal saline by lavage,qod.Meanwhile,according to the nude mice body weight,treatment Ⅰ,Ⅱ and Ⅲ groups were injected intraperitoneally with 0.5μg/g,1μg/g,and 2μg/g unit dose of 5-Aza-CdR injection,respectively,qod.The treatment lasted for 4 weeks.The tumor volume and body weight were measured in 4 groups,and the tumor inhibition rate was calculated.We observed the effects of different doses of 5-Aza-CdR on the growth of human lung cancer xenograft in nude mice and its side effects.Differences in RAR-β,WIF1 or RASSF2A gene methylation status among the four groups were detected by methylation-specific PCR(MSP).Then,differences in protein expression of RAR-β,WIF1 or RASSF2A in vivo among the four groups were detected by Western blot.Results:(1)After 4 weeks of 5-Aza-CdR treatment,the mean tumor volume and weight of Ⅰ,Ⅱ and Ⅲ groups were significantly decreased compared with the model group(P<0.05).Meanwhile,the mean tumor volume and weight decreased significantly with the increase of 5-Aza-CdR dose(P<0.05),and the tumor inhibition rate was significantly increased with the increase of 5-Aza-CdR dose.Tumor inhibition effect was dose dependent.(2)RAR-β,WIFI and RASSF2A gene methylation was observed in the model group,while demethylation of RAR-β,WIF1 and RASSF2A gene was observed in the other 3 groups.(3)The protein expression levels of RAR-β,WIF1 and RASSF2A in the model group were the lowest among the 4 groups,while the levels of protein expression in treatment III group were the highest(p<0.05).The expression levels of protein increased with the dose of 5-Aza-CdR(p<0.05),and protein expression had a certain dose dependence.Conclusion:5-Aza-CdR can suppress the growth of human lung cancer xenograft in nude mice,furthermore it plays a role in tumor inhibition via up-regulation of the expression of RAR-β,WIF1 and RASSF2A and apoptosis inducement.It may provide a new method and theoretical basis for clinical treatment of lung cancer in the future to explore the mechanism of anti-tumor effect of 5-Aza-CdR in vivo and in vitro.更多还原