EGFR-TKIs在非小细胞肺癌中获得性耐药机制和逆转研究

发布时间：2018-05-07 00:43

本文选题：非小细胞肺癌 + EGFR-TKIs　；参考：《天津医科大学》2017年博士论文

【摘要】：肺癌已是我国因肿瘤致死的首要病因。传统的治疗包括手术,放疗,化疗均缺乏特异性。靶向治疗的兴起大大改善了肺癌患者生存时间和生存质量。然而获得性耐药的发生困扰着靶向治疗的进一步临床应用。因此阐明获得性耐药的分子机制,并在此基础上寻找逆转分子靶向药物获得性耐药的方法,进一步深入研究获得性耐药的相关分子生物学分子机制,成为本论文研究的主要目的。首先根据实验室前期的对吉非替尼敏感细胞PC-9和三株吉非替尼耐药细胞PC-9/AB2,PC-9/AB11,PC-9/BB4的表达谱芯片分析研究结果示:与PC-9细胞相比,三株耐药细胞在细胞周期相关通路Rb-E2Fs信号通路的变化尤为突出。本课题中在收集的临床标本中的免疫组化染色(IHC)证明了肺腺癌与Rb-E2Fs信号通路相关。并且进一步用western-blot证明相较于正常支气管上皮细胞B2B,肺腺癌细胞PC-9,PC-9/AB2和HCC-827细胞周期相关蛋白表现为不同种类和程度的表达异常。因此肺癌的发生和吉非替尼获得性耐药与细胞周期调控异常相关。其次,在吉非替尼敏感细胞(PC-9)和吉非替尼耐药细胞(PC-9/AB2)中应用MTT,EdU,细胞周期和细胞凋亡实验,试探性的将细胞周期抑制剂PD0332991与吉非替尼联合应用逆转其获得性耐药。MTT中,联合用药组细胞的存活率明显低于单药处理组;EdU增殖实验,联合用药组的增值率分别为3.7%(PC-9)和2.1%(PC-9/AB2),明显低于单药处理组;细胞凋亡试验,联合用药组的凋亡率为23.66%(PC-9/AB2),明显高于吉非替尼组的13.2%;细胞周期实验中联合用药组G0/G1抑制率分别为30.6%(PC-9)和23.75%(PC-9/AB2),明显高于各单药处理组。运用基因芯片分析,real-time PCR,western-blot证明细胞周期抑制剂PD 0332991与吉非替尼联合应用逆转吉非替尼的获得性耐药与Rb-E2Fs信号通路的细胞周期密切相关。体内实验,PC-9/AB2裸鼠皮下成瘤模型中PD 0332991与吉非替尼联合用药组,肿瘤缩小的速度及百分比均明显快于其他单药处理组。免疫组化染色联合用药组肿瘤中Ki-67阳性率为3%,TUNNEL阳性率为67%,CD34阳性率为4%,明显较单药处理组和对照组有意义。在临床中选取3位接受吉非替尼治疗进展的患者,在服用吉非替尼的同时,增加PD 0332991的服用。3周后两位患者的病情得到了很好的控制,一位患者头颅转移灶大部分消失,并且骨转移导致的骨痛症状消失。最后,为了进步揭示细胞周期调控产生获得性的耐药和肺癌发生的机制,我们研究了lnc RNA HOATIR在肺癌中的作用。首先在收集的67对肺癌术后组织的基因水平分析发现:肿瘤组织中HOTAIR的表达率为100%,而相对应的癌旁组织的表达率只有42.65%。并且其表达高低与肿瘤的病理分化程度呈反比。证明了HOATIR与肺癌的相关性。进一步的细胞实验中:过表达HOTAIR,细胞周期处在G1期的细胞比例下降,而S期比例升高。EdU增殖实验证明了细胞增值率较对照组平均增加了20%,侵袭率平均增加了30%,转移率增加了50%。而敲低HOTAIR的表达依然有意义。Western-blot和TOP/FOP荧光素酶报告实验证明HOTAIR功能的发挥与Rb-E2Fs信号通路,EMT,β-catenin细胞通路相关。裸鼠原位种植模型,进一步证明了细胞实验的结果。本课题的研究明确了肺腺癌EGFR-TKIs获得性耐药的发生与Rb-E2Fs信号通路异常导致细胞周期的调控异常相关,并且应用CDK4/6抑制剂PD 0332991可以增强吉非替尼的抑瘤作用,逆转吉非替尼的获得性耐药。进一步对肺癌细胞周期的调控异常机制研究中,发现lnc RNA HOTAIR参与肺癌的细胞周期调控,细胞增殖调控,细胞侵袭转移调控,这种调控作用同样与Rb-E2Fs信号通路,EMT,β-catenin细胞通路相关,其可作为新一代细胞周期抑制剂的研究靶点。
[Abstract]:Lung cancer is the leading cause of death for cancer in China. Traditional treatments include surgery, radiotherapy and chemotherapy. The emergence of targeted therapy greatly improves the survival time and quality of life of the lung cancer patients. However, the occurrence of sexual resistance has plagued the further application of targeted therapy. On the basis of this, it is the main purpose of this study to search for the methods of reversing molecular targeting drug resistance, and to further study the molecular mechanism of acquired resistance. First, PC-9 and three gefitinib resistant cells PC-9/AB2, PC-9/A B11, PC-9/BB4 expression spectrum chip analysis showed that the change of Rb-E2Fs signal pathway in the cell cycle related pathway of three resistant cells was particularly prominent compared with PC-9 cells. In this subject, immunohistochemical staining (IHC) in the collected clinical specimens showed that the lung adenocarcinoma was associated with the Rb-E2Fs signaling pathway and further used western-blo. T showed that the PC-9, PC-9/AB2, and HCC-827 cell cycle related proteins of lung adenocarcinoma cells were expressed in different types and degrees of expression compared to normal bronchial epithelial cells. Therefore, the occurrence of lung cancer and gefitinib acquired resistance were associated with the regulation of cell cycle. Secondly, in gefitinib sensitive cells (PC-9) and gefitinib. In drug cells (PC-9/AB2), MTT, EdU, cell cycle and apoptosis experiments were used to reverse the combination of cell cycle inhibitor PD0332991 and gefitinib in the drug resistant.MTT. The survival rate of the combined drug group was significantly lower than that of the single drug group; the added value of the combined drug group was 3.7% (PC-9), respectively. And 2.1% (PC-9/AB2), obviously lower than the single drug treatment group; apoptosis test, the apoptosis rate of combined drug group was 23.66% (PC-9/AB2), obviously higher than that of gefitinib group 13.2%. The inhibition rate of G0/G1 in the combined drug group was 30.6% (PC-9) and 23.75% (PC-9/AB2) in the cell cycle experiment, which was significantly higher than that of the single drug treatment group. The use of gene chip analysis, real- Time PCR, Western-blot demonstrated that cell cycle inhibitor PD 0332991 combined with gefitinib in combination with gefitinib reversal of acquired resistance to gefitinib is closely related to the cell cycle of the Rb-E2Fs signaling pathway. In vivo experiments, the combined use of PD 0332991 and gefitinib in the subcutaneous tumor model of PC-9/AB2 nude mice, the rate and percentage of tumor reduction are all clear The Ki-67 positive rate was 3%, the positive rate of TUNNEL was 3%, the positive rate of TUNNEL was 67%, and the positive rate of CD34 was 4%, which was significantly higher than that of the single drug treatment group and the control group. In the clinic, the patients who received gefitinib, while taking gefitinib, increased the use of PD 0332991. 3 weeks later, the two patients were well controlled. A majority of the head metastases disappeared and the bone pain caused by bone metastases disappeared. Finally, we studied the role of LNC RNA HOATIR in lung cancer in order to improve the mechanism of cell cycle regulation to produce acquired resistance and lung cancer. The 67 gene level analysis of the tissue after lung cancer showed that the expression rate of HOTAIR in the tumor tissue was 100%, while the expression rate of the corresponding para cancerous tissue was only 42.65%. and its expression was inversely proportional to the degree of pathological differentiation of the tumor. The correlation between HOATIR and lung cancer was demonstrated. In the further cell experiment, the expression of HOTAIR and the cell cycle were overexpressed. The proportion of cells in the period of G1 phase decreased, and the increase of.EdU proliferation in S phase showed that the rate of cell increment was increased by 20% compared with that of the control group, the average invasion rate increased by 30%, the transfer rate increased by 50%. and the expression of the knockdown HOTAIR was still meaningful.Western-blot and TOP/FOP luciferase reporter experiment proved HOTAIR function and Rb-E2Fs The signal pathway, EMT, beta -catenin pathway related. Nude mouse in situ implantation model, further demonstrated the results of cell experiment. This study confirmed that the occurrence of EGFR-TKIs acquired resistance in lung adenocarcinoma is associated with abnormal regulation of cell cycle caused by abnormal Rb-E2Fs signaling pathway, and the application of CDK4/6 inhibitor PD 0332991 can be enhanced. The antitumor effect of gefitinib reverses the acquired resistance to gefitinib. Further research on the regulation of abnormal mechanism of lung cancer cell cycle, it is found that LNC RNA HOTAIR participates in the regulation of cell cycle of lung cancer, regulation of cell proliferation, and regulation of cell invasion and metastasis. This regulation is identical to the Rb-E2Fs signaling pathway, EMT, and beta -catenin cell pathway It can be used as a new research target for cell cycle inhibitors.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R734.2

【参考文献】