本文選題：M2 + mAChR　； 參考：《上海交通大學(xué)》2015年博士論文

【摘要】：眾所周知,膽堿受體(AChR)在介導(dǎo)遞質(zhì)乙酰膽堿(acetylcholine,ACh)神經(jīng)信號傳遞過程中起著關(guān)鍵作用。越來越多研究顯示非神經(jīng)源性ACh作為一種局部信號分子或細(xì)胞因子以自分泌或旁分泌方式參與多種細(xì)胞的生物學(xué)功能調(diào)控,微環(huán)境中ACh與腫瘤細(xì)胞的惡性表型(包括過度增殖、轉(zhuǎn)移、耐藥及腫瘤干細(xì)胞)調(diào)控備受學(xué)術(shù)界關(guān)注。ACh作為配體既可作用于門控通道型煙堿型膽堿受體(nAChR),又可作用于G-蛋白耦聯(lián)型M1組(M1、M3、M5亞型)和M2組(M2、M4亞型)毒蕈堿型膽堿受體(mAChR)。相同的信號分子ACh作用于不同類型受體及其亞型,其受體水平和受體后信號通路水平上生物學(xué)功能互作和整合是ACh調(diào)控腫瘤細(xì)胞中極為重要的科學(xué)問題。由于吸煙是肺癌高危因素,煙草主要成分尼古丁(煙堿)激活nAChR調(diào)控肺癌細(xì)胞的研究較為深入,M1組mAChR對肺癌細(xì)胞增殖和上皮間質(zhì)轉(zhuǎn)化(epithelial to mesenchymal transition,EMT)體外作用也有研究,而M2組mAChR對肺癌惡性表型的體內(nèi)外調(diào)控作用迄今尚未見報道。肺癌靶向治療業(yè)已獲得重大進(jìn)展,但獲得性耐藥仍然是治療難題,腫瘤干細(xì)胞學(xué)說認(rèn)為肺癌耐藥與腫瘤干細(xì)胞密切相關(guān)。膽堿受體在分子靶向藥物的耐藥和肺癌干細(xì)胞的干性維持的作用值得進(jìn)一步探討。為此,本課題重點圍繞M2型mAChR在體內(nèi)和體外水平上對非小細(xì)胞肺癌(NSCLC)增殖、轉(zhuǎn)移以及耐藥和肺癌干細(xì)胞的調(diào)控作用及其信號途徑開展研究。首先,采用CCK-8、RTCA及Western blot等方法檢測NSCLC細(xì)胞A549、PC9中非神經(jīng)源性膽堿能系統(tǒng)的存在。通過mAChR各亞型選擇性拮抗劑對NSCLC細(xì)胞的增殖和調(diào)控MAPK和AKT信號通路作用,揭示M2 mAChR起到明顯正向調(diào)控作用。M2 mAChR shRNA方法進(jìn)一步證實其作用和信號途徑。荷瘤實驗表明,M2 mAChR拮抗劑美索曲明(Methoctramine,MT)和M2R-shRNA均顯著延緩A549細(xì)胞的體內(nèi)生長。其次,采用Western blot、transwell、免疫熒光等方法考察了MT及M2 mAChR shRNA抑制NSCLC細(xì)胞A549和PC9發(fā)生EMT、侵襲和遷移過程。采用MT明顯逆轉(zhuǎn)NSCLC細(xì)胞A549體內(nèi)發(fā)生EMT并減少轉(zhuǎn)移灶的形成。MT及M2 mAChR siRNA可顯著抑制腫瘤轉(zhuǎn)移相關(guān)的NFκB信號通路,及其下游轉(zhuǎn)錄因子snail和zeb1表達(dá)。再次,建立耐EGFR靶向藥物吉非替尼(Gefitinib,Ge)的NSCLC細(xì)胞株P(guān)C9-GR和干性標(biāo)志CD221陽性的NSCLC干細(xì)胞CD221+-SPC-A1,M2 mAChR拮抗劑逆轉(zhuǎn)PC9-GR細(xì)胞EMT,CD221+-SPC-A1細(xì)胞中M2 mAChR表達(dá)明顯升高(p0.01),且伴有EMT的變化(相較于CD221-SPC-A1細(xì)胞)。本課題首次揭示了M2 mAChR在體內(nèi)外均可對NSCLC增殖、EMT、遷移、侵襲起到的正向調(diào)控作用,并闡明了增殖作用由PI3K/AKT、MEK/ERK信號通路介導(dǎo),EMT、遷移和侵襲過程由NFκB/snail/zeb1信號通路介導(dǎo);M2 mAChR可能在NSCLC耐藥產(chǎn)生和干細(xì)胞的干性維持起到一定調(diào)控作用。上述研究成果為非神經(jīng)源性膽堿能系統(tǒng)對肺癌惡性表型的調(diào)控機(jī)制闡明和研發(fā)M2亞型高選擇性拮抗藥潛在應(yīng)用提供理論依據(jù)。
[Abstract]:It is well known that acetylcholine receptor (AChR) plays a key role in the transmission of acetylcholine (acetylcholine). More and more studies have shown that non-neurogenic ACh, as a local signal molecule or cytokine, participates in the regulation of biological functions of many kinds of cells in an autocrine or paracrine manner. ACh is associated with malignant phenotypes (including hyperproliferation) of tumor cells in microenvironment. The regulation of metastasis, drug resistance and tumor stem cell) has attracted the attention of academia. Ach as ligand can not only act on the gated channel type nicotinic choline receptor (nAChRN), but also on the G protein coupled type M1 group (M 1, M 3, M 3, M 5 subtype) and M2 group, the M 2N M 4 subtype of muscarinic receptor, muscarinic receptor mAChRN. The same signal molecule ACh acts on different types of receptors and their subtypes. The interaction and integration of biological functions at the level of receptor and postreceptor signaling pathway is an extremely important scientific issue in the regulation of tumor cells by ACh. Because smoking is a high risk factor for lung cancer, the effect of nicotine (nicotine) activating nAChR on lung cancer cells in M1 group was also studied in vitro, and the effects of mAChR on the proliferation and epithelial interstitial transition of lung cancer cells in vitro were also studied. However, the regulation of M 2 mAChR on malignant phenotype of lung cancer in vivo and in vitro has not been reported. Significant progress has been made in targeted treatment of lung cancer, but acquired drug resistance is still a difficult problem. The theory of cancer stem cells holds that drug resistance of lung cancer is closely related to cancer stem cells. The role of choline receptor in molecular targeting drug resistance and dry maintenance of lung cancer stem cells deserves further study. Therefore, this study focuses on the proliferation, metastasis, drug resistance and regulation of lung cancer stem cells by M2 mAChR in vivo and in vitro. Firstly, the presence of non-neurogenic cholinergic system in NSCLC cells A549 / PC9 was detected by CCK-8 RTCA and Western blot. Through the selective antagonists of various subtypes of mAChR on the proliferation of NSCLC cells and the regulation of MAPK and AKT signaling pathway, it was revealed that M2 mAChR played a significant positive regulatory role. M2 mAChR shRNA method further confirmed its role and signal pathway. Tumor-bearing experiment showed that metotrimine mAChR antagonist metotrimine and M2R-shRNA significantly retarded the growth of A549 cells in vivo. Secondly, the inhibition of NSCLC cells A549 and PC9 by MT and M2 mAChR shRNA was investigated by Western blottertranswell and immunofluorescence. MT significantly reversed the occurrence of EMT in A549 cells of NSCLC and reduced the formation of metastatic foci. MT and M2 mAChR siRNA significantly inhibited the expression of NF 魏 B signaling pathway associated with tumor metastasis and its downstream transcription factors snail and zeb1. Thirdly, NSCLC cell line PC9-GR and NSCLC stem cell CD221 -SPC-A1 + M2 mAChR antagonist, which was resistant to EGFR targeting gefitinibor Ge), reversed the expression of M2 mAChR in PC9-GR cell line EMTT CD221 -SPC-A1, and was accompanied by EMT changes (compared with CD221-SPC-A1 cell line). In this study, we first revealed the positive regulation of M2 mAChR on the proliferation, migration and invasion of NSCLC in vivo and in vitro. The results showed that the proliferation was mediated by the PI3K / AKT / MEK / ERK signaling pathway, and the migration and invasion was mediated by NF 魏 B/snail/zeb1 signaling pathway, which may play a role in the production of drug resistance of NSCLC and the dry maintenance of stem cells. These results provide theoretical basis for the regulation mechanism of non-neurogenic cholinergic system on malignant phenotype of lung cancer and the potential application of high selectivity antagonist of M2 subtype.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R734.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 Guo-Xiao Liu;Hong-Qing Xi;Xiao-Yan Sun;Bo Wei;;Role of periostin and its antagonist PNDA-3 in gastric cancer metastasis[J];World Journal of Gastroenterology;2015年09期

2 趙承孝;楊澤;;Twist2的生物學(xué)功能及其分子機(jī)制[J];遺傳;2015年01期

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本文編號：1943914