M3受體亞型在NSCLC細胞生長增殖、侵襲和遷移中的作用及機制研究
發(fā)布時間：2018-08-13 20:01
【摘要】：肺癌是發(fā)病率和死亡率增長最快,對人群健康和生命威脅最大的惡性腫瘤之一。肺癌最常見的組織學分類為非小細胞肺癌(non-small cell lung cancer,NSCLC)和小細胞肺癌(small cell lung cancer,SCLC),其中80%以上的肺癌病例屬于非小細胞肺癌。非小細胞肺癌又可分為鱗狀細胞癌(25-30%),腺癌(35-40%)和大細胞癌(10-15%)。早期非小細胞肺癌治療主要以手術(shù)切除為主,常規(guī)化療是聯(lián)合應用鉑類藥物(順鉑、卡鉑)與抗代謝藥、抗微管藥或抗拓撲異構(gòu)酶藥,由于毒副反應以及預后不良,仍不能有效提高患者的5年生存率,近年來藥物靶向治療(主要包括以表皮生長因子受體為靶點的酪氨酸激酶抑制劑或單抗,和以肺癌血管生成為靶點的血管內(nèi)皮生長因子抑制劑或單抗)以及針對患者自身免疫系統(tǒng)的免疫刺激單抗(免疫檢查點阻斷劑,包括Ipilimumab,Nivolumab,MK-3475等)在特定人群內(nèi)取得了可喜進展,引發(fā)了人們對肺癌發(fā)病機制的深入思考和多方位作用靶點的研究,針對其具體的發(fā)病機制探索發(fā)展新的治療方法將有望推進肺癌個體化治療的進程。乙酰膽堿(ACh)是由神經(jīng)細胞合成的經(jīng)典神經(jīng)遞質(zhì),通過激活乙酰膽堿受體(ACh R)的兩個家族成員——毒蕈堿型(m ACh R)和煙堿型(n ACh R)膽堿受體發(fā)揮作用。越來越多的研究表明,除神經(jīng)細胞外,多種腫瘤細胞均可合成和分泌乙酰膽堿作為一種生長因子,參與細胞的生長增殖,抵抗凋亡,血管發(fā)生,轉(zhuǎn)移侵襲,促進癌癥的進程。這種非神經(jīng)元膽堿能系統(tǒng)極有可能是調(diào)控腫瘤發(fā)展進程的另一個重要的信號系統(tǒng)。許多實驗室的工作表明,在惡性腫瘤表達的多種m ACh R亞型中,與正常組織相比M3受體表達顯著上調(diào),且與腫瘤的預后不良相關(guān)。有研究表明,M3受體選擇性拮抗劑能夠抑制小細胞肺癌細胞增殖并降低裸鼠移植瘤MAPK磷酸化水平,為肺癌的預防和治療提供了新的藥物研發(fā)策略。有研究報道,在正常角質(zhì)化細胞、黑色素瘤細胞、M受體轉(zhuǎn)染的CHO細胞及SCLC細胞上,激活M1或M3受體可改變細胞黏附分子的活性,可能與腫瘤的轉(zhuǎn)移與浸潤相關(guān)。因此有關(guān)M3受體的研究受到極大關(guān)注,成為相關(guān)研究領(lǐng)域的熱點。本研究的重點是觀察M3受體拮抗劑對2種NSCLC細胞生長增殖、運動和遷移中的影響,并探索M3受體參與非小細胞肺癌發(fā)展進程的信號通路和關(guān)鍵分子,預期研究結(jié)果將有助于確認M3受體是否可作為抗腫瘤靶標,將為確立膽堿能受體系統(tǒng)在腫瘤生長調(diào)控中的地位提供實驗依據(jù)。實驗方法如下:(1)以CCK-8法測定M3受體拮抗劑、激動劑以及PKC抑制劑對體外培養(yǎng)的NSCLC細胞增殖的影響。(2)應用激光共聚焦顯微鏡監(jiān)測激動劑單獨給藥以及拮抗劑和激動劑伴隨給藥NSCLC細胞內(nèi)游離鈣離子的動態(tài)變化。(3)傷口愈合實驗檢測M3受體拮抗劑對NSCLC細胞遷移能力的影響。(4)Transwell侵襲實驗檢測M3受體拮抗劑對NSCLC細胞侵襲能力的影響。(5)利用脂質(zhì)體介導的si RNA轉(zhuǎn)染技術(shù)敲減M3受體并檢測敲減M3受體后對NSCLC細胞生長活力的影響。(6)以Western蛋白印跡法檢測M3受體拮抗劑對NSCLC細胞增殖信號通路(Akt、MAPK)、細胞周期調(diào)節(jié)蛋白(cyclin D1、CDK4、p21)以及遷移相關(guān)分子(MMP-2)蛋白表達水平的影響。實驗結(jié)果如下:(1)本研究首先觀察M3受體拮抗劑在(1.25μM~80μM)劑量范圍內(nèi)對NSCLC細胞生長增殖的影響,實驗結(jié)果顯示,R2-8018與達非那新體外能夠濃度依賴性地抑制H1299細胞和H460細胞的增殖,其中R2-8018抑制H1299細胞增殖的效果更明顯,作用48 h的IC50值為10.6μmol/L。(2)外源性補充激動劑卡巴膽堿和氯化乙酰膽堿無法促進H1299細胞和H460細胞增殖。(3)轉(zhuǎn)染M3 si RNA 72 h之后檢測檢測敲減效率,western蛋白印跡法結(jié)果顯示,轉(zhuǎn)染M3 si RNA顯著降低M3受體的表達(p0.01);CCK-8法48 h和72 h檢測結(jié)果顯示,敲減M3受體,H1299細胞增殖明顯被抑制,并且隨si RNA作用時間延長,抑制作用更顯著(48 h:p0.5,72 h:p0.01)。結(jié)果說明,敲減M3受體能夠抑制H1299細胞增殖。(4)激光共聚焦顯微鏡監(jiān)測細胞內(nèi)鈣離子的動態(tài)變化,30μM的卡巴膽堿能夠引起H1299細胞鈣信號增強,而給予拮抗劑達非那新預處理30 min的H1299細胞30μM卡巴膽堿則觀察不到鈣信號的增強,達非那新能夠完全阻斷卡巴膽堿引起的細胞鈣信號增強。(5)蛋白激酶C抑制劑Staurosporine體外對H1299細胞存活具有明顯的抑制作用,并且R2-8018可濃度依賴性地降低H1299細胞PKC-α蛋白表達(10μM:p0.01,20μM:p0.01)。(6)R2-8018作用H1299細胞24 h,可顯著抑制H1299細胞的遷移和侵襲能力,并濃度依賴性地下調(diào)基質(zhì)金屬蛋白酶MMP-2蛋白表達水平。(7)達非那新下調(diào)H1299細胞Akt、GSK3β和cyclin D1蛋白表達水平。(8)R2-8018上調(diào)H1299細胞p21蛋白表達水平。結(jié)論:應用M3受體拮抗劑或敲減M3受體能夠體外抑制非小細胞肺癌細胞增殖,作為一種G蛋白偶聯(lián)受體,拮抗M3受體我們可以觀察到H1299細胞鈣離子釋放受到抑制以及蛋白激酶C活性降低,說明拮抗M3受體可能通過其經(jīng)典的G蛋白偶聯(lián)信號發(fā)揮細胞生長抑制作用;M3受體拮抗劑能降低NSCLC細胞Akt、GSK3β、cyclin D1、CDK4蛋白表達,增加p21蛋白表達,其涉及機制可能為拮抗M3受體可以通過抑制Akt磷酸化,減輕其對下游GSK3β磷酸化,進而增強GSK3β活性,促進對cyclin D1的降解,抑制cyclin D1-CDK4復合物的形成,并且上調(diào)p21表達,使細胞無法順利進入S期而發(fā)生G0/G1期阻滯,最終導致細胞增殖受到抑制,此外,M3受體拮抗劑還可能通過抑制MAPK信號通路的激活而產(chǎn)生抑制細胞增殖的作用;M3受體拮抗劑下調(diào)基質(zhì)金屬蛋白酶MMP-2蛋白表達,抑制NSCLC細胞的遷移和侵襲能力。
[Abstract]:Lung cancer is one of the malignant tumors with the fastest increase in morbidity and mortality and the greatest threat to human health and life. The most common histological classification of lung cancer is non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). More than 80% of lung cancer cases belong to non-small cell lung cancer. Cellular lung cancer can also be divided into squamous cell carcinoma (25-30%), adenocarcinoma (35-40%) and large cell carcinoma (10-15%). Surgical resection is the main treatment for early non-small cell lung cancer. Conventional chemotherapy is the combination of platinum drugs (cisplatin, carboplatin) and antimetabolic drugs, antimicrotubule drugs or antitopoisomerase drugs, which are still not available due to adverse reactions and poor prognosis. Drug-targeted therapy (mainly tyrosine kinase inhibitors or monoclonal antibodies targeting epidermal growth factor receptors, and vascular endothelial growth factor inhibitors or monoclonal antibodies targeting angiogenesis of lung cancer) and immunostimulatory monoclonal antibodies targeting the patient's autoimmune system (immunoassays) have been effective in improving 5-year survival. Point blockers, including Ipilimumab, Nivolumab, MK-3475 and so on, have made gratifying progress in a specific population, which has triggered in-depth consideration of the pathogenesis of lung cancer and multi-directional target research. Exploring and developing new therapies for the specific pathogenesis of lung cancer will hopefully promote the process of individualized treatment. Acetylcholine (ACh) Acetylcholine receptor (ACh R) and nicotinic choline receptor (NChR), two family members of acetylcholine receptor (ACh R), are classical neurotransmitters synthesized by nerve cells. More and more studies have shown that besides nerve cells, many tumor cells can synthesize and secrete acetylcholine as a growth factor. This non-neuronal cholinergic system is likely to be another important signaling system that regulates the development of tumors. Many laboratory studies have shown that M3 is more likely to be involved in the expression of various subtypes of M ACh R in malignant tumors than in normal tissues. Receptor expression is significantly up-regulated and is associated with poor prognosis. Studies have shown that M3 receptor selective antagonists can inhibit the proliferation of small cell lung cancer cells and reduce MAPK phosphorylation levels in nude mice xenografts, providing a new drug development strategy for the prevention and treatment of lung cancer. Activation of M1 or M3 receptors on cells, M receptor transfected CHO cells and SCLC cells may alter the activity of cell adhesion molecules, which may be related to tumor metastasis and invasion. It is expected that the results will help to confirm whether M3 receptor can be used as an anti-tumor target and provide experimental evidence for the role of cholinergic receptor system in tumor growth regulation. The effects of M3 receptor antagonists, agonists and PKC inhibitors on the proliferation of cultured NSCLC cells were measured by CCK-8 method. (2) The dynamic changes of intracellular free calcium in NSCLC cells treated with agonists alone and with antagonists and agonists were monitored by laser confocal microscopy. (3) Wound healing test was used to detect the effects of M3 receptor antagonists on N. The effect of M3 receptor antagonist on the invasion of NSCLC cells was detected by Transwell invasion assay. (5) The effect of M3 receptor antagonist on the growth of NSCLC cells was detected by liposome-mediated Si RNA transfection and the effect of M3 receptor knockdown on the proliferation of NSCLC cells was detected. (6) Western blotting was used to detect the effect of M3 receptor antagonist on the NSCLC fine cells. The results were as follows: (1) The effects of M3 receptor antagonists on the proliferation of NSCLC cells in the dose range of (1.25 to 80 mu) were observed. The results showed that R2-8018 and R2-8018 were non-specific. Naoxin inhibited the proliferation of H1299 cells and H460 cells in a concentration-dependent manner in vitro, and R2-8018 inhibited the proliferation of H1299 cells more significantly, with the IC50 value of 10.6 micromol/L at 48 h. (2) Exogenous agonists carbachol and acetylcholine chloride could not promote the proliferation of H1299 cells and H460 cells. (3) M3 Si RNA transfection 72 h later. Western blot showed that M3 Si RNA significantly decreased the expression of M3 receptor (p0.01); CCK-8 assay showed that M3 receptor knockdown significantly inhibited the proliferation of H1299 cells, and the inhibitory effect was more significant (48 h: p0.5, 72 h: p0.01). Receptors inhibited the proliferation of H1299 cells. (4) Laser confocal microscopy was used to monitor the dynamic changes of intracellular calcium. Ca2+ signal was enhanced in H1299 cells by carbachol at 30 mu M, but not by carbachol at 30 mu M in H1299 cells pretreated with daphenazine for 30 min. (5) Staurosporine, a protein kinase C inhibitor, significantly inhibited the survival of H1299 cells in vitro, and R2-8018 decreased the expression of PKC-alpha protein in H1299 cells in a concentration-dependent manner (10 mu M: p0.01, 20 mu M: p0.01). (6) R2-8018 significantly inhibited the migration of H1299 cells 24 hours after treatment with R2-8018. Transfer and invasion, and concentration-dependent down-regulation of MMP-2 protein expression. (7) Daphenazine down-regulated the expression of Akt, GSK3 beta and cyclin D1 protein in H1299 cells. (8) R2-8018 up-regulated the expression of p21 protein in H1299 cells. Cell proliferation, as a G-protein-coupled receptor, antagonizes M3 receptor. We can observe the inhibition of calcium release and the decrease of protein kinase C activity in H1299 cells, suggesting that antagonizing M3 receptor may inhibit cell growth through its classical G-protein-coupled signal; M3 receptor antagonist can reduce Akt, GSK3beta, cyclin D1 in NSCLC cells. The expression of CDK4 protein and the expression of p21 protein may be related to the antagonism of M3 receptor by inhibiting Akt phosphorylation, reducing its phosphorylation to downstream GSK3 beta, thereby enhancing the activity of GSK3 beta, promoting the degradation of cyclin D1, inhibiting the formation of cyclin D1-CDK4 complex, and up-regulating the expression of p21, which prevents the cells from entering S phase smoothly. In addition, M3 receptor antagonists may inhibit cell proliferation by inhibiting the activation of MAPK signaling pathway. M3 receptor antagonists down-regulate the expression of MMP-2 protein and inhibit the migration and invasion of NSCLC cells.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R734.2
本文編號：2182029
[Abstract]:Lung cancer is one of the malignant tumors with the fastest increase in morbidity and mortality and the greatest threat to human health and life. The most common histological classification of lung cancer is non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). More than 80% of lung cancer cases belong to non-small cell lung cancer. Cellular lung cancer can also be divided into squamous cell carcinoma (25-30%), adenocarcinoma (35-40%) and large cell carcinoma (10-15%). Surgical resection is the main treatment for early non-small cell lung cancer. Conventional chemotherapy is the combination of platinum drugs (cisplatin, carboplatin) and antimetabolic drugs, antimicrotubule drugs or antitopoisomerase drugs, which are still not available due to adverse reactions and poor prognosis. Drug-targeted therapy (mainly tyrosine kinase inhibitors or monoclonal antibodies targeting epidermal growth factor receptors, and vascular endothelial growth factor inhibitors or monoclonal antibodies targeting angiogenesis of lung cancer) and immunostimulatory monoclonal antibodies targeting the patient's autoimmune system (immunoassays) have been effective in improving 5-year survival. Point blockers, including Ipilimumab, Nivolumab, MK-3475 and so on, have made gratifying progress in a specific population, which has triggered in-depth consideration of the pathogenesis of lung cancer and multi-directional target research. Exploring and developing new therapies for the specific pathogenesis of lung cancer will hopefully promote the process of individualized treatment. Acetylcholine (ACh) Acetylcholine receptor (ACh R) and nicotinic choline receptor (NChR), two family members of acetylcholine receptor (ACh R), are classical neurotransmitters synthesized by nerve cells. More and more studies have shown that besides nerve cells, many tumor cells can synthesize and secrete acetylcholine as a growth factor. This non-neuronal cholinergic system is likely to be another important signaling system that regulates the development of tumors. Many laboratory studies have shown that M3 is more likely to be involved in the expression of various subtypes of M ACh R in malignant tumors than in normal tissues. Receptor expression is significantly up-regulated and is associated with poor prognosis. Studies have shown that M3 receptor selective antagonists can inhibit the proliferation of small cell lung cancer cells and reduce MAPK phosphorylation levels in nude mice xenografts, providing a new drug development strategy for the prevention and treatment of lung cancer. Activation of M1 or M3 receptors on cells, M receptor transfected CHO cells and SCLC cells may alter the activity of cell adhesion molecules, which may be related to tumor metastasis and invasion. It is expected that the results will help to confirm whether M3 receptor can be used as an anti-tumor target and provide experimental evidence for the role of cholinergic receptor system in tumor growth regulation. The effects of M3 receptor antagonists, agonists and PKC inhibitors on the proliferation of cultured NSCLC cells were measured by CCK-8 method. (2) The dynamic changes of intracellular free calcium in NSCLC cells treated with agonists alone and with antagonists and agonists were monitored by laser confocal microscopy. (3) Wound healing test was used to detect the effects of M3 receptor antagonists on N. The effect of M3 receptor antagonist on the invasion of NSCLC cells was detected by Transwell invasion assay. (5) The effect of M3 receptor antagonist on the growth of NSCLC cells was detected by liposome-mediated Si RNA transfection and the effect of M3 receptor knockdown on the proliferation of NSCLC cells was detected. (6) Western blotting was used to detect the effect of M3 receptor antagonist on the NSCLC fine cells. The results were as follows: (1) The effects of M3 receptor antagonists on the proliferation of NSCLC cells in the dose range of (1.25 to 80 mu) were observed. The results showed that R2-8018 and R2-8018 were non-specific. Naoxin inhibited the proliferation of H1299 cells and H460 cells in a concentration-dependent manner in vitro, and R2-8018 inhibited the proliferation of H1299 cells more significantly, with the IC50 value of 10.6 micromol/L at 48 h. (2) Exogenous agonists carbachol and acetylcholine chloride could not promote the proliferation of H1299 cells and H460 cells. (3) M3 Si RNA transfection 72 h later. Western blot showed that M3 Si RNA significantly decreased the expression of M3 receptor (p0.01); CCK-8 assay showed that M3 receptor knockdown significantly inhibited the proliferation of H1299 cells, and the inhibitory effect was more significant (48 h: p0.5, 72 h: p0.01). Receptors inhibited the proliferation of H1299 cells. (4) Laser confocal microscopy was used to monitor the dynamic changes of intracellular calcium. Ca2+ signal was enhanced in H1299 cells by carbachol at 30 mu M, but not by carbachol at 30 mu M in H1299 cells pretreated with daphenazine for 30 min. (5) Staurosporine, a protein kinase C inhibitor, significantly inhibited the survival of H1299 cells in vitro, and R2-8018 decreased the expression of PKC-alpha protein in H1299 cells in a concentration-dependent manner (10 mu M: p0.01, 20 mu M: p0.01). (6) R2-8018 significantly inhibited the migration of H1299 cells 24 hours after treatment with R2-8018. Transfer and invasion, and concentration-dependent down-regulation of MMP-2 protein expression. (7) Daphenazine down-regulated the expression of Akt, GSK3 beta and cyclin D1 protein in H1299 cells. (8) R2-8018 up-regulated the expression of p21 protein in H1299 cells. Cell proliferation, as a G-protein-coupled receptor, antagonizes M3 receptor. We can observe the inhibition of calcium release and the decrease of protein kinase C activity in H1299 cells, suggesting that antagonizing M3 receptor may inhibit cell growth through its classical G-protein-coupled signal; M3 receptor antagonist can reduce Akt, GSK3beta, cyclin D1 in NSCLC cells. The expression of CDK4 protein and the expression of p21 protein may be related to the antagonism of M3 receptor by inhibiting Akt phosphorylation, reducing its phosphorylation to downstream GSK3 beta, thereby enhancing the activity of GSK3 beta, promoting the degradation of cyclin D1, inhibiting the formation of cyclin D1-CDK4 complex, and up-regulating the expression of p21, which prevents the cells from entering S phase smoothly. In addition, M3 receptor antagonists may inhibit cell proliferation by inhibiting the activation of MAPK signaling pathway. M3 receptor antagonists down-regulate the expression of MMP-2 protein and inhibit the migration and invasion of NSCLC cells.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R734.2
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