【作者】 孙志华；

【导师】 伍钢；

【作者基本信息】 华中科技大学 ， 肿瘤学， 2012， 博士

【摘要】 第一部分Geftinib耐药的肺腺癌H1975细胞株的特征鉴定目的：本研究的目的是为了验证肺腺癌H1975细胞株是否具有特征性的突变位点及其对Geftinib是否耐药。方法：肺腺癌H1975细胞进行基因测序明确是否含有T790M及L858R突变：MTT法进行肺腺癌H1975细胞Geftinib耐药性的鉴定。结果：基因测序表明H1975细胞株含有EGFR T790M及L858R突变,且对Geftinib的50%抑制浓度ICso值为39.176μM,符合Geftinib耐药细胞株的描述；结论：本实验用肿腺癌H1975细胞含有T790M及L858R突变,且对Geftinib耐药符合肺腺癌H975细胞株的特征。第二部分BEZ235体外对吉非替尼耐药的肺腺癌H1975细胞的作用及其机制研究目的：通过体外细胞实验,探讨PI3K/mTOR双靶点抑制剂BEZ235对Geftinib耐药的肿腺癌H1975细胞株增殖的影响及其对H1975细胞PI3K/Akt/mTOR信号通路的作用,试图明确在体外实验中BEZ235能否通过抑制PI3K/Akt/mTOR信号通路从而克服T790M突变导致的Gefitinib耐药。方法：MTT分析法初步明确BEZ235单用或与Geftinib联合对肺腺癌H1975细胞增殖抑制的影响；细胞划痕试验检测BEZ235和Geftinib的不同组合对H1975细胞迁移能力的影响；Transwell小室侵袭试验了解BEZ235和Geftinib不同组合对H1975细胞侵袭能力的影响；流式细胞术测定经BEZ235和Geftinib作用后各组H1975细胞周期分布及凋亡情况；RT-PCR法检测BEZ235和Geftinib对H1975细胞PIK3CA、mTOR, VEGF基因转录水平表达的影响；细胞免疫荧光法检测BEZ235和Geftinib对H1975细胞p-mTOR的表达定位,初步判定表达的变化；Western blot检测BEZ235和Geftinib对H1975细胞PI3K/Akt/mTOR信号通路蛋白AKT, p-AKT、S6、p-S6的影响。结果：BEZ235单独作用于H1975细胞的IC50为0.327μm,且有明显的浓度依赖性；与Geftinib联合后BEZ235的IC50值为0.073μM,与单用BEZ235有显著差异(P<0.05)。BEZ235与Geftinib联合对H1975细胞迁移和侵袭能力的影响比Geftinib单用差异有统计学意义(P<0.05)；BEZ235单用或与Gefitinib联合使H1975细胞的凋亡增加,与Geftinib组及空白组相比,差异有统计学意义(P<0.05),但G1期阻滞并没有差异性。BEZ235单用或与Geftinib联合在转录水平上促进PIK3CA的表达,抑制VEGF的表达,两药单用或联合对mTOR则均表现为抑制作用；Western blot表明BEZ235能抑制PI3K/Akt/mTOR信号通路上磷酸化的p-Akt、p-S6蛋白表达,而对总Akt及S6蛋白的表达无显著影响。结论：BEZ235单独作用于H1975细胞对其增殖有抑制作用,与Geftinib联合有协同性增殖抑制作用,可用于下一步实验；通过体外实验表明PI3K/mTOR双靶点抑制剂BEZ235能下调PI3K/Akt/mTOR信号通路上的蛋白磷酸化水平,有效的抑制PI3K/Akt/mTOR信号通路上肿瘤生长信号向下传递,从而克服EGFR T790M突变导致的Geftinib获得性耐药；BEZ235体外试验中能抑制人肺腺癌H1975细胞的迁移和侵袭能力,BEZ235能增加H1975细胞凋亡的比例,且随干预时间的延长有增加的趋第三部分BEZ235对BALB/c裸鼠H1975细胞移植瘤生长的作用及其机制研究目的：通过建立BALB/c裸鼠移植瘤模型,在体探讨BEZ235对Geftinib耐药肺腺癌H1975细胞BALB/c裸鼠移植瘤生长的影响及其对PI3K/Akt/mTOR信号通路的作用,以期明确BEZ235能否在体内也是通过抑制PI3K/Akt/mTOR信号通路从而克服Gefitinib耐药而抑制肿瘤的生长及有无抗血管生成作用。方法：32只BALB/c裸鼠分成四组,第一组为空白对照组(BLK),第二组为单用Gefitinib组(GEF),第三组为单用BEZ235组(BEZ),第四组为BEZ235与Gefitinib联合组(B+G),每组8只。BALB/c裸鼠H1975细胞移植瘤干预前后肿瘤生长曲线的绘制了解肿瘤生长有无差异；干预过程中比较BALB/c裸鼠各组成瘤时间和肿瘤体积有无差异性；干预后比较各组BALB/c裸鼠切取的肿瘤重量从而评价干预效果是否有差异；活体MRI成像从影像学方面了解干预与否对肿瘤生长的影响；通过H1975细胞皮下种植建立BALB/c裸鼠移植瘤模型,BALB/c裸鼠移植瘤的Western blot检测BEZ235和(或)Geftinib对H1975细胞表达磷酸化蛋白p-mTOR、VEGF的影响；各组BALB/c裸鼠切取肿瘤组织切片免疫组化检测VEGF. p-S6、CD31,了解p-S6表达有无差异,了解与肿瘤血管生成相关指标VEGF. CD31在肿瘤组织中表达的是否有差异,以明确在体内BEZ235是否有抗血管生成作用。结果：BALB/c裸鼠移植瘤研究表明BEZ235单用或与Gefitinib联合,BALB/c裸鼠H1975细胞移植瘤的生长延缓；活体MR成像、干预过程中测得的肿瘤体积及干预后切取的肿瘤重量与空白对照组、GEF单用组相比有显著性差异(P<0.01)；BALB/c裸鼠移植瘤Western blot表明体内BEZ235能抑制PI3K/Akt/mTOR信号通路上p-mTOR表达,VEGF在BEZ235单用或与Geftinib联合组是下降的；移植瘤免疫组化分析表明BEZ235单用或与Geftinib联合使BALB/c裸鼠肿瘤组织中的VEGF和CD31表达显著下降,以联合组下降最显著(P<0.01)。结论：通过移植瘤实验表明,BEZ235单用或与Geftinib联合干预后肿瘤的生长曲线显示BEZ235单用抑制肺腺癌H1975细胞移植瘤的生长,与Geftinib联合有协同作用；干预结束后肿瘤重量、活体MR成像得出同样的结果；在移植瘤模型中BEZ235能降低肿瘤组织中p-S6. CD31、VEGF的表达从而抑制H1975细胞移植瘤的血管生成。更多还原

【Abstract】 Part ⅠIdentification of characterization on lung adenocarcinoma H1975cell lines with gefitinib resistanceObjective:This study is to verify lung adenocarcinoma H975cell lines whether carried the T790M mutations and was resistant to gefitinib.Methods:The gefitinib resistant H1975cell lines was identified whether it carried the T790M and L858R mutations by gene sequencing. MTT assay was used to detect the effect of gefitinib to the proliferation of H1975cell lines to ascertain whether it was resistant to gefitinib.Results:Gene sequencing showed that the H1975cell lines contained the EGFR T790M and L858R mutations. IC50value of gefitinib alone in H1975cell lines was39.176μM, that was consistent with the describes of gefitinib acquired resistant cell lines. In the cell proliferation experiments.Conclusion:The lung adenocarcinoma H975cell lines contain the T790M and L858R mutations and conform with the characteristics of the cell lines that is resistant to gefitinib. Part IIEffect and mechanism of dual-target inhibitor BEZ235on lung adenocarcinoma H1975cell lines in virtoObjective:Through cell experiments, the sensitivity of the H1975cell lines to dual target PI3K/mTOR inhibitor BEZ235was preliminarily assessed. The effect of the dual-target inhibitor BEZ235to lung adenocarcinoma H975cell lines with gefitinib resistance and its role to PI3K/Akt/mTOR signaling pathway in H975cell lines, and try to clarify whether BEZ235can inhibit PI3K/Akt/mTOR signaling pathway and overcome gefitinib resistance caused by the EGFR T790M mutation.Methods:The inhibiting effect of BEZ235alone or combation with gefitinib on the proliferation of lung adenocarcinoma H975cells were studied. The1975cells were devided into four groups treated with DMSO as control, BEZ235, gefitinib and combin-ation with BEZ235and gefitinib respectively. The influence of different combinations of BEZ235and gefitinib on H975cell migration was evaluated through cell scratch test. Transwell chamber invasion test is used to assess the effect of different combinations with BEZ235and gefitinib to the ability of H1975cell invasion. Flow cytometry was employed to measure the distribution of cell cycle and apoptosis of H975cell intervened by BEZ235and(or) gefitinib. The transcrip levels of PIK3CA, mTOR and VEGF were measured by reverse transcription-PCR assay in H975cell lines intervened by BEZ235and(or) gefitinib. Timmunofluorescence and western blotting assay were applied to detect the expressing level of phosphorylated protein in the PI3K/Akt/mTOR signaling pathway,such as p-mTOR, p-Akt p-S6and,as well as Akt and S6.Results:The IC50value of BEZ235alone was0.327μM, and showed a concentration-dependent. When it was combined with gefitinib, the IC50value was0.073μ.M, there were significant differences between the two groups(P<0.05). The abilities of migration and invasion of H1975cell lines that were treated BEZ235alone or combination with gefitinib were inhibited to be compared with gefitinib alone, the difference was statistically significant, but no significant difference in G1phase arrest between the four groups. The expression of PIK3CA were up-regulated and that of VEGF were inhibited at the transcriptional level by BEZ235alone or combination with gefitinib, while the expression of mTOR was down-regulated either BEZ235or gefitinib. Western blot showed that BEZ235could inhibit the phosphorylation levels of p-Akt and p-S6in PI3K/Akt/mTOR signaling pathway, while the expression of total protein were not significant down-regulated as Akt and S6in any group.Conclusion:The dual inhibitor BEZ235may inhibit the proliferation of H1975cell lines, and combine with gefitinib to show a synergistic effect on proliferation inhibition. BEZ235can inhibit the proliferation of H1975cell lines that are resistant to gefitinib through down-regulating the phosphorylated protein in PI3K/Akt/mTOR signal pathways, and can effectively inhibit tumor growth signals are passed to downstream of PI3K/Akt/mTOR signal pathway, and can overcome the gefitinib acquired resistance caused by EGFR T790M mutation. BEZ235can inhibit the abilities of migration and invasion of human lung adenocarcinoma H1975cell lines and increase the proportion of apoptotic and necrotic cells in vitro. Part ⅢEffect and mechanism of BEZ235on xenograft of lung adenocar-cinoma H1975cell lines in BALB/c nude miceObjective:Through xenograft models of BALB/c nude mice,we explored the effect of the PI3K/mTOR dual-target inhibitor BEZ235to lung adenocarcinoma H1975cell line with gefitinib resistance and its role to PI3K/Akt/mTOR signaling pathway in H1975cell lines, and try to clarify whether BEZ235can inhibit PI3K/Akt/mTOR signaling pathway and overcome gefitinib resistance caused by the EGFR T790M mutation and suppressed both tumor growth and angiogenesis in BALB/c nude mice.Methods:32nude mice were devided into four groups(N=8) treated with DMSO as control, gefitinib, BEZ235and combination with BEZ235and gefitinib respectively. Xenograft model of BALB/c nude mice were established by H1975cell lines transplanted subcutaneously. Western blot assay of xenograft intervened by BEZ235and(or) gefitinib were applied to detect the expressing level of phosphorylated protein p-mTOR,as well as VEGF. The growth curve of transplanted tumor in BALB/c nude mice were drawn to compare whether the tumor growth were different in four groups. When BALB/c nude mice were treated by BEZ235and/or gefitinib, the time to tumor formation and tumor volume were compared to decide whether they were different. After the intervention was finished, the tumor weight of BALB/c nude mice were evaluated to decide the effect of intervention whether there are differences in four groups. MR imaging was used to evaluate whether tumor growth is affected by the intervention or no. The expression of VEGF and CD31in tumor tissues of xenograft in BALB/c nude were detected by IHC to decide whether BEZ235could inhibit angiogenesis.Results:The study of xenograft in BALB/c nude mice have shown that the tumor growth was inhibited, when BALB/c nude mice were treated by BEZ235alone or combination with gefitinib, the combination group was more significant. The volume and weight of tumor were compared with both control and GEF groups were significant difference (P<0.01). Western blot assay of tumor tissue in BALB/c nude mice showed that the expression of p-mTOR and VEGF were down-regulated in BEZ235alone or combination with gefitinib, which was similar with the results in vitro. Immunohisto-chemical assay showed that the expression of p-S6、VEGF and CD31in tumor tissue were significantly decreased in BEZ235alone or combination with gefitinib.Conclusion:BEZ235can down-regulate the expression of CD31and VEGF, and thus inhibit tumor angiogenesis in xenograft of H1975cell lines in BALB/c nude mice, and can inhibit the transplanted tumor growth of H1975cell lines in BALB/c nude and has synergistic effect with gefitinib in vivo.更多还原