本文選題：非小細胞肺癌 + EGFR-TKIs��； 參考：《天津醫(yī)科大學》2017年博士論文

【摘要】：肺癌已是我國因腫瘤致死的首要病因。傳統(tǒng)的治療包括手術,放療,化療均缺乏特異性。靶向治療的興起大大改善了肺癌患者生存時間和生存質量。然而獲得性耐藥的發(fā)生困擾著靶向治療的進一步臨床應用。因此闡明獲得性耐藥的分子機制,并在此基礎上尋找逆轉分子靶向藥物獲得性耐藥的方法,進一步深入研究獲得性耐藥的相關分子生物學分子機制,成為本論文研究的主要目的。首先根據實驗室前期的對吉非替尼敏感細胞PC-9和三株吉非替尼耐藥細胞PC-9/AB2,PC-9/AB11,PC-9/BB4的表達譜芯片分析研究結果示:與PC-9細胞相比,三株耐藥細胞在細胞周期相關通路Rb-E2Fs信號通路的變化尤為突出。本課題中在收集的臨床標本中的免疫組化染色(IHC)證明了肺腺癌與Rb-E2Fs信號通路相關。并且進一步用western-blot證明相較于正常支氣管上皮細胞B2B,肺腺癌細胞PC-9,PC-9/AB2和HCC-827細胞周期相關蛋白表現為不同種類和程度的表達異常。因此肺癌的發(fā)生和吉非替尼獲得性耐藥與細胞周期調控異常相關。其次,在吉非替尼敏感細胞(PC-9)和吉非替尼耐藥細胞(PC-9/AB2)中應用MTT,EdU,細胞周期和細胞凋亡實驗,試探性的將細胞周期抑制劑PD0332991與吉非替尼聯合應用逆轉其獲得性耐藥。MTT中,聯合用藥組細胞的存活率明顯低于單藥處理組;EdU增殖實驗,聯合用藥組的增值率分別為3.7%(PC-9)和2.1%(PC-9/AB2),明顯低于單藥處理組;細胞凋亡試驗,聯合用藥組的凋亡率為23.66%(PC-9/AB2),明顯高于吉非替尼組的13.2%;細胞周期實驗中聯合用藥組G0/G1抑制率分別為30.6%(PC-9)和23.75%(PC-9/AB2),明顯高于各單藥處理組。運用基因芯片分析,real-time PCR,western-blot證明細胞周期抑制劑PD 0332991與吉非替尼聯合應用逆轉吉非替尼的獲得性耐藥與Rb-E2Fs信號通路的細胞周期密切相關。體內實驗,PC-9/AB2裸鼠皮下成瘤模型中PD 0332991與吉非替尼聯合用藥組,腫瘤縮小的速度及百分比均明顯快于其他單藥處理組。免疫組化染色聯合用藥組腫瘤中Ki-67陽性率為3%,TUNNEL陽性率為67%,CD34陽性率為4%,明顯較單藥處理組和對照組有意義。在臨床中選取3位接受吉非替尼治療進展的患者,在服用吉非替尼的同時,增加PD 0332991的服用。3周后兩位患者的病情得到了很好的控制,一位患者頭顱轉移灶大部分消失,并且骨轉移導致的骨痛癥狀消失。最后,為了進步揭示細胞周期調控產生獲得性的耐藥和肺癌發(fā)生的機制,我們研究了lnc RNA HOATIR在肺癌中的作用。首先在收集的67對肺癌術后組織的基因水平分析發(fā)現:腫瘤組織中HOTAIR的表達率為100%,而相對應的癌旁組織的表達率只有42.65%。并且其表達高低與腫瘤的病理分化程度呈反比。證明了HOATIR與肺癌的相關性。進一步的細胞實驗中:過表達HOTAIR,細胞周期處在G1期的細胞比例下降,而S期比例升高。EdU增殖實驗證明了細胞增值率較對照組平均增加了20%,侵襲率平均增加了30%,轉移率增加了50%。而敲低HOTAIR的表達依然有意義。Western-blot和TOP/FOP熒光素酶報告實驗證明HOTAIR功能的發(fā)揮與Rb-E2Fs信號通路,EMT,β-catenin細胞通路相關。裸鼠原位種植模型,進一步證明了細胞實驗的結果。本課題的研究明確了肺腺癌EGFR-TKIs獲得性耐藥的發(fā)生與Rb-E2Fs信號通路異常導致細胞周期的調控異常相關,并且應用CDK4/6抑制劑PD 0332991可以增強吉非替尼的抑瘤作用,逆轉吉非替尼的獲得性耐藥。進一步對肺癌細胞周期的調控異常機制研究中,發(fā)現lnc RNA HOTAIR參與肺癌的細胞周期調控,細胞增殖調控,細胞侵襲轉移調控,這種調控作用同樣與Rb-E2Fs信號通路,EMT,β-catenin細胞通路相關,其可作為新一代細胞周期抑制劑的研究靶點。
[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.

【學位授予單位】：天津醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R734.2

【參考文獻】

相關期刊論文 前1條

1 Dao-Hong Chen;Xiao-Shi Zhang;;Targeted therapy: resistance and re-sensitization[J];Chinese Journal of Cancer;2015年11期

，

本文編號：1854666