本文選題：乙酰膽堿 + MACC1　； 參考：《南方醫(yī)科大學(xué)》2016年碩士論文

【摘要】：研究背景：胃癌是世界上常見的惡性腫瘤之一,在全球癌癥總發(fā)病率中排第五位,在癌癥死亡原因中排第三位,其中以中國的發(fā)病率最高。胃癌發(fā)病率在我國男性中排第二位,在女性中排第三位,雖然近年來我國胃癌的發(fā)病率有下降趨勢,但是發(fā)病人數(shù)依然較多,2015年我國估計有67.91萬人診斷出胃癌,有49.8萬人死于胃癌。早期胃癌的治療主要以手術(shù)為主,但是絕大多數(shù)胃癌往往到了晚期才被確診,晚期胃癌只能采取放化療、靶向藥物等治療方法,療效往往不理想。晚期胃癌往往伴隨廣泛浸潤或遠(yuǎn)處轉(zhuǎn)移,因此研究胃癌轉(zhuǎn)移機制有助于進(jìn)一步理解晚期胃癌的惡性生物學(xué)行為及發(fā)現(xiàn)新的治療新策略。腫瘤轉(zhuǎn)移是指腫瘤細(xì)胞脫離原發(fā)病灶在體內(nèi)播散,并在其他部位定植生長形成繼發(fā)腫瘤病灶。上皮間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition, EMT)被認(rèn)為是惡性腫瘤轉(zhuǎn)移的起始步驟。在EMT中,非運功的、有極性的、細(xì)胞連接緊密的上皮來源腫瘤細(xì)胞,轉(zhuǎn)化成為非極性的、運動的、侵襲性的間質(zhì)性細(xì)胞。發(fā)生EMT的腫瘤細(xì)胞,其分子標(biāo)志發(fā)生了顯著的變化,例如,上皮細(xì)胞-細(xì)胞粘附分子E-鈣黏蛋白表達(dá)下降,而間質(zhì)細(xì)胞的一些表型的表達(dá)則會上調(diào),如波形蛋白、纖連蛋白。另外,腫瘤細(xì)胞也可改造細(xì)胞外基質(zhì)(extracellular matrix, ECM)促進(jìn)侵襲和轉(zhuǎn)移,其中各種基質(zhì)金屬蛋白酶(matrix metalloproteinase, MMP)的分泌發(fā)揮了重要作用。腫瘤微環(huán)境在腫瘤發(fā)生、發(fā)展及轉(zhuǎn)移中的重要作用近年來受到了高度的重視。神經(jīng)是腫瘤微環(huán)境的重要組成部分,雖然已經(jīng)有研究證實腫瘤可以沿神經(jīng)生長,然后通過神經(jīng)途徑播散,即嗜神經(jīng)侵襲(perineural invasion, PNI),但是神經(jīng)在腫瘤發(fā)生發(fā)展中所發(fā)揮的主動性作用卻很少受到關(guān)注。早在19世紀(jì)40年代,就已經(jīng)有研究探索了去除腫瘤神經(jīng)支配所帶來的效果,但是并未引起研究者重視神經(jīng)對腫瘤的作用。2013年,Magnon等人發(fā)表的研究使研究者們重新關(guān)注到神經(jīng)對于腫瘤的作用。他們揭示了自主神經(jīng)系統(tǒng)對小鼠前列腺癌發(fā)生發(fā)展至關(guān)重要,其中,去除交感神經(jīng)系統(tǒng)(sympathetic nervous system, SNS)主要在腫瘤發(fā)生的早期抑制生長,而去除副交感神經(jīng)系統(tǒng)(parasympathetic nervous system, PNS)主要在腫瘤發(fā)展的后期抑制轉(zhuǎn)移。2014年Zhao等人發(fā)表的另一篇文獻(xiàn)報道,在小鼠胃癌模型中,手術(shù)或藥物去除胃神經(jīng)支配(即迷走神經(jīng)切斷術(shù)或局部注射神經(jīng)毒性藥物)能顯著降低胃癌發(fā)生概率及胃癌進(jìn)展速度。迷走神經(jīng)末梢可以合成和釋放乙酰膽堿,乙酰膽堿及其M3型膽堿受體已被報道參與了肺癌、肝癌、結(jié)腸癌及前列腺癌等的生長與轉(zhuǎn)移,但是其對胃癌轉(zhuǎn)移的作用及機制尚不明確。結(jié)腸轉(zhuǎn)移相關(guān)基因-1(metastasis-associated in colon cancer-1, MACC1)是2008年在結(jié)腸癌中首先發(fā)現(xiàn)的一個促癌基因。Stein等人發(fā)現(xiàn)MACC1在腸結(jié)癌組織中的表達(dá)要高于正常結(jié)腸粘膜上皮,且是結(jié)腸癌病人的一個獨立預(yù)后因素。2013年我們實驗團(tuán)隊證實了MACC1在胃癌組織中高表達(dá)并與胃癌病人分期及預(yù)后相關(guān)。MACC1促進(jìn)肝細(xì)胞生長因子受體c-Met信號并誘導(dǎo)胃癌細(xì)胞EMT、促進(jìn)MMP2及MMP9的表達(dá),從而促進(jìn)胃癌生長、侵襲及轉(zhuǎn)移。并且在糖剝奪情況下,胃癌細(xì)胞可通過磷酸化的腺苷酸活化蛋白激酶(AMP-activated protein kinase, AMPK)上調(diào)MACC1, MAAC1通過瓦伯格效應(yīng)抵抗胃癌細(xì)胞代謝應(yīng)激所帶來的損傷,從而保持胃癌細(xì)胞的存活。我們通過生物信息學(xué)的方法發(fā)現(xiàn)胃癌中磷酸化的AMPK水平與M3型膽堿受體的編碼基因CHRM3 (M3型膽堿受體的編碼基因)的表達(dá)呈正相關(guān)。我們推測,乙酰膽堿作用于M3型膽堿受體后,可能通過磷酸化的AMPK促進(jìn)MACC1的表達(dá),并且MACC1在乙酰膽堿促胃癌轉(zhuǎn)移過程中發(fā)揮了介導(dǎo)作用。因此本研究旨在探索乙酰膽堿對人胃癌細(xì)胞侵襲／遷移及EMT的影響,闡明M3型膽堿受體/AMPK/MACC1信號通路及其在人胃癌細(xì)胞侵襲／遷移及EMT中的作用。研究假設(shè)：1.乙酰膽堿促進(jìn)人胃癌細(xì)胞侵襲／遷移及EMT；2.M3型膽堿受體介導(dǎo)乙酰膽堿促人胃癌細(xì)胞侵襲／遷移及EMT的作用；3.乙酰膽堿作用于M3型膽堿受體后,通過AMPK上調(diào)MACC1的表達(dá),且MACC1在乙酰膽堿促人胃癌細(xì)胞侵襲／遷移及EMT中發(fā)揮重要的中間信號作用。研究內(nèi)容：1.篩選M3型膽堿受體高表達(dá)的胃癌細(xì)胞株分別提取BGC-803、BGC-823、MGC-803、MKN-28、MKN-45、SGC-7901人胃癌細(xì)胞的RNA,逆轉(zhuǎn)錄成cDNA后,實時定量PCR檢測各細(xì)胞株M3型膽堿受體的mRNA表達(dá)情況,并選取表達(dá)量最高的兩株細(xì)胞進(jìn)行后續(xù)實驗。2.乙酰膽堿對胃癌細(xì)胞侵襲／遷移及EMT的影響將10uM濃度的乙酰膽堿添加到MKN-45及MGC-803胃癌細(xì)胞的培養(yǎng)基,分別培養(yǎng)0h、24h及48 h,用帶有基質(zhì)膠的Transwell侵襲實驗及不帶基質(zhì)膠的Transwell遷移實驗分別檢測兩株細(xì)胞的侵襲及遷移能力；同樣再以10uM濃度的乙酰膽堿刺激MKN-45及MGC-803細(xì)胞0 h、24 h及48 h,各提取全部mRNA和蛋白質(zhì),將mRNA逆轉(zhuǎn)錄成cDNA,實時定量PCR檢測EMT相關(guān)指標(biāo)(E-鈣黏蛋白、波形蛋白、纖連蛋白、MMP2及MMP9)的mRNA相對表達(dá)量,用Western blot的方法檢測上述EMT指標(biāo)的蛋白含量。我們也觀察了乙酰膽堿刺激前后兩株細(xì)胞的形態(tài)變化。3.M3型膽堿受體抑制劑對乙酰膽堿作用的影響在MKN-45及MGC-803胃癌細(xì)胞的培養(yǎng)基中加入M3型膽堿受體特異性抑制劑達(dá)非那新(10uM濃度),30 min后再加入10uM的乙酰膽堿,然后兩株細(xì)胞培養(yǎng)48 h。實驗一共分為四組：陰性對照、乙酰膽堿、達(dá)非那新、達(dá)非那新+乙酰膽堿。用帶有基質(zhì)膠的Transwell侵襲實驗及不帶基質(zhì)膠的Transwell遷移實驗分別檢測兩株細(xì)胞的侵襲及遷移能力。再按照上述分組培養(yǎng)MKN-45及MGC-803細(xì)胞株48 h,各提取兩株細(xì)胞的mRNA和全蛋白,mRNA逆轉(zhuǎn)錄成cDNA后,實時定量PCR檢測EMT相關(guān)指標(biāo)(E-鈣黏蛋白、波形蛋白、纖連蛋白、MMP2及MMP9)的mRNA相對表達(dá)量,用Western blot方法檢測上述EMT指標(biāo)的蛋白含量。4.乙酰膽堿的刺激對MACC1表達(dá)的影響生物信息學(xué)的方法分析M3型受體的編碼基因CHRM3的表達(dá)情況與磷酸化AMPK的相關(guān)性。我們考慮MACC1可能為乙酰膽堿下游信號,將10uM濃度的乙酰膽堿添加到MKN-45及MGC-803胃癌細(xì)胞的培養(yǎng)基,分別培養(yǎng)0h、24h及48 h,各提取全部mRNA和蛋白質(zhì),將mRNA逆轉(zhuǎn)錄成cDNA,實時定量PCR檢測MACC1的mRNA相對表達(dá)量,用Western blot的方法檢測MACC1的蛋白含量。然后將MKN-45及MGC-803胃癌細(xì)胞按照陰性對照、乙酰膽堿、達(dá)非那新、達(dá)非那新+乙酰膽堿四組培養(yǎng)48 h后,實時定量PCR及Western blot分別檢測MACC1的mRNA及蛋白含量。免疫熒光實驗檢測乙酰膽堿刺激后MACC1的核轉(zhuǎn)位情況。5.MACC1在乙酰膽堿促胃癌細(xì)胞侵襲／遷移及EMT中的重要性將MKN-45及MGC-803細(xì)胞轉(zhuǎn)染MACC1的沉默序列(siMACC1)或陰性對照序列(siCtrl),實時定量PCR及Western blot分別檢測mRNA及蛋白水平的沉默效果。然后在轉(zhuǎn)染有siMACC1或siCtrl的細(xì)胞中加入10uM乙酰膽堿進(jìn)行培養(yǎng),共分成四組：siCtrl、siMACC1、siCtrl+乙酰膽堿、siMACC1+乙酰膽堿。用帶有基質(zhì)膠的Transwell侵襲實驗及不帶基質(zhì)膠的Transwell遷移實驗分別檢測兩株細(xì)胞的侵襲及遷移能力。實時定量PCR及Western blot分別在mRNA及蛋白水平檢測EMT相關(guān)指標(biāo)(E-鈣黏蛋白、波形蛋白、纖連蛋白、MMP2及MMP9)的變化。6.驗證AMPK是乙酰膽堿/M3型膽堿受體與MACC1的中間信號將MKN-45及MGC-803胃癌細(xì)胞按照陰性對照、乙酰膽堿、達(dá)非那新、達(dá)非那新+乙酰膽堿四組培養(yǎng)48 h后,提取全蛋白,Western blot檢測p-AMPK的蛋白含量。在兩株細(xì)胞的培養(yǎng)基中加入8uM濃度的AMPK抑制劑Dorsomorphin,30 min后再加入10uM的乙酰膽堿,一共分為四組：陰性對照、乙酰膽堿、Dorsomorphin、達(dá)非那新+Dorsomorphin,培養(yǎng)48 h后,Western blot檢測p-AMPK及MACC1的蛋白含量。7.統(tǒng)計方法所有的統(tǒng)計學(xué)分析都是用SPSS 20.0完成的,本文數(shù)據(jù)以平均值±標(biāo)準(zhǔn)誤的形式表示,用one-way ANOVA的方法來分析組間的差異,雙邊的P值小于0.05被認(rèn)為有統(tǒng)計學(xué)差異。研究結(jié)果：1.篩選M3型膽堿受體高表達(dá)的胃癌細(xì)胞株實時定量PCR檢測各胃癌細(xì)胞株M3型膽堿受體的mRNA表達(dá),發(fā)現(xiàn)MKN-45及MGC-803兩株細(xì)胞CHRM3的mRNA表達(dá)最高,因此選擇這兩株細(xì)胞進(jìn)行后續(xù)實驗。2.乙酰膽堿刺激胃癌細(xì)胞侵襲／遷移并誘導(dǎo)EMT在Transwell侵襲及遷移實驗中,在48 h內(nèi),乙酰膽堿逐漸增加了MKN-45及MGC-803胃癌細(xì)胞的侵襲及遷移數(shù)量,并且從實時定量PCR的結(jié)果可以看到,隨著刺激時間增加,乙酰膽堿能逐漸降低兩株細(xì)胞E-鈣黏蛋白的mRNA表達(dá),并增加了波形蛋白、纖連蛋白、MMP2及MMP9的mRNA表達(dá)。Western blot檢測的蛋白水平也得到了與mRNA相同的趨勢,說明48 h內(nèi)乙酰膽堿逐漸誘導(dǎo)了胃癌細(xì)胞EMT。但是,乙酰膽堿的刺激未改變兩株胃癌細(xì)胞的形態(tài)。3.M3型膽堿受體介導(dǎo)乙酰膽堿促胃癌侵襲／遷移及EMT在Transwell侵襲及遷移實驗中,乙酰膽堿顯著增加了MKN-45及MGC-803胃癌細(xì)胞的侵襲及遷移數(shù)量,而加入M3型膽堿受體抑制劑達(dá)非那新以后,乙酰膽堿對兩株細(xì)胞的侵襲及遷移數(shù)量的改變均受到抑制。實時定量PCR及westernblot的結(jié)果顯示,乙酰膽堿所誘導(dǎo)的E-鈣黏蛋白表達(dá)下降,波形蛋白、纖連蛋白、MMP2及MMP9表達(dá)的上升,在達(dá)非那新存在的情況下都被逆轉(zhuǎn)。并且,單獨的達(dá)非那新也表現(xiàn)出抑制侵襲／遷移及EMT的能力。4.乙酰膽堿刺激MACC1表達(dá)上調(diào)通過對TCGA提取的胃腺癌相關(guān)數(shù)據(jù)分析得到,AMPK蘇氨酸172位點磷酸化水平與CHRM3的mRNA表達(dá)呈正相關(guān)。結(jié)合我們實驗室前期研究結(jié)論：磷酸化的AMPK可以上調(diào)MACC1,我們接下來驗證了乙酰膽堿與MACC1的聯(lián)系。我們發(fā)現(xiàn),48 h內(nèi)乙酰膽堿逐漸增加了MACC1的mRNA及蛋白表達(dá),并且達(dá)非那新能逆轉(zhuǎn)這一作用。但是乙酰膽堿對于MACC1的核轉(zhuǎn)位沒有明顯改變。5.MACC1在乙酰膽堿促胃癌細(xì)胞侵襲／遷移及EMT中發(fā)揮了重要作用我們將MKN-45及MGC-803細(xì)胞轉(zhuǎn)染了MACC1的沉默序列后,MACC1的mRNA及蛋白水平的表達(dá)都明顯下降,說明沉默效果較好。MACC1的表達(dá)被沉默后,乙酰膽堿促胃癌細(xì)胞侵襲／遷移的作用都被抑制了。且不論從mRNA還是蛋白水平,乙酰膽堿降低E-鈣黏蛋白,增加波形蛋白、纖連蛋白、MMP2及MMP9的作用也在沉默MACC1后逆轉(zhuǎn)了。6.乙酰膽堿/M3型膽堿受體通過AMPK途徑上調(diào)MACC1Western blot的結(jié)果顯示,乙酰膽堿促進(jìn)了p-AMPK的蛋白水平,并且這一作用可以被M3型膽堿受體抑制劑所逆轉(zhuǎn)。另外,當(dāng)使用Dorsomorphin抑制了AMPK活性后,乙酰膽堿促進(jìn)MACC1表達(dá)的作用就被抑制了,說明AMPK是乙酰膽堿/M3型膽堿受體與MACC1的中間信號。研究結(jié)論：乙酰膽堿通過M3型膽堿受體/AMPK/MACC1信號通路促進(jìn)了胃癌細(xì)胞侵襲／遷移并誘導(dǎo)了EMT。
[Abstract]:Background: gastric cancer is one of the most common malignant tumors in the world. It ranks fifth in the total incidence of global cancer and third in the cause of cancer death. Among them, the incidence of cancer is the highest in China. The incidence of gastric cancer is second in the male and third in women. Although the incidence of gastric cancer in China has declined in recent years, the incidence of gastric cancer has declined. However, there are still more people in the disease. In 2015, 679 thousand and 100 people were estimated to have diagnosed gastric cancer and 498 thousand people died of gastric cancer. Early gastric cancer was mainly treated with surgery, but most of the gastric cancer was diagnosed at the late stage. Advanced gastric cancer can only be treated by radiotherapy and chemotherapy and targeted drugs, and the curative effect is often not ideal. Gastric cancer is often accompanied by extensive infiltration or distant metastasis. Therefore, the study of the mechanism of metastasis of gastric cancer is helpful to further understand the malignant biological behavior of advanced gastric cancer and the discovery of a new therapeutic strategy. Epithelial-mesenchymal transition (EMT) is considered to be the starting step of metastasis of malignant tumor. In EMT, nonoperational, polar, tightly connected epithelial cells are derived from tumor cells, transforming into non polar, moving, invasive interstitial cells. The tumor cells of EMT have a significant change in molecular markers. For example, the expression of epithelial cell adhesion molecule E- calcium mucin is decreased, while some of the expression of mesenchymal cells can be up-regulated, such as vimentin and fibronectin. In addition, tumor cells can also transform extracellular matrix (ECM) to promote invasion and metastasis, including various matrix metalloproteinases (matrix metalloprotei). The secretion of nase, MMP, plays an important role. The important role of the tumor microenvironment in the occurrence, development and metastasis of tumor has been highly valued in recent years. Nerve is an important part of the tumor microenvironment. Although studies have shown that the tumor can grow along the nerve and then spread through the neural pathway, that is, the nerve invasion (perineura L invasion, PNI), but the active role of nerve in the development of tumor is rarely concerned. As early as 1840s, the effects of removing tumor nerve innervation had been explored, but researchers did not pay attention to the effect of nerve to tumor for.2013 years, and the research published by Magnon et al. They reconcerned the role of the nerve in the tumor. They revealed that the autonomic nervous system was essential for the development of prostate cancer in mice, in which the sympathetic nervous system (SNS) was mainly in the early inhibition of growth of the tumor, and the parasympathetic nervous system (parasympathetic nervous system, P) was removed. NS) another literature report, published by Zhao et al..2014, was published mainly in the late metastasis of tumor development. In the mouse model of gastric cancer, surgical or drug removal of gastric innervation (vagotomy or local injection of neurotoxic drugs) can significantly reduce the incidence of gastric cancer and the speed of gastric cancer. The vagus nerve ends can be synthesized. And the release of acetylcholine, acetylcholine and its M3 type choline receptors have been reported to be involved in the growth and metastasis of lung cancer, liver cancer, colon cancer and prostate cancer, but its role and mechanism for the metastasis of gastric cancer is not clear. The colon metastasis related gene -1 (metastasis-associated in colon cancer-1, MACC1) is the first in colon cancer in 2008. A oncogene.Stein and others found that the expression of MACC1 in colon cancer tissue is higher than normal colonic mucosa epithelium, and it is an independent prognostic factor of colon cancer patients. Our experimental team confirmed the high expression of MACC1 in gastric cancer tissue and related to the stage and prognosis of gastric cancer patients with.MACC1 to promote the growth of hepatocyte. The subreceptor c-Met signal and the induction of gastric cancer cell EMT promote the expression of MMP2 and MMP9, thus promoting the growth, invasion and metastasis of gastric cancer. And in the case of sugar deprivation, gastric cancer cells can regulate MACC1 through the phosphorylated adenylate activated protein kinase (AMP-activated protein kinase, AMPK), and MAAC1 can resist the gastric cancer cell generation through the effect of the flower bud effect. We have found that the AMPK level of phosphorylation in gastric cancer is positively related to the expression of the M3 cholinergic receptor encoding gene CHRM3 (the encoding gene of the M3 cholinergic receptor) in gastric cancer by bioinformatics. We speculate that acetylcholine may be used after the M3 type cholinergic receptor, which may pass through the M3 type cholinergic receptor. The phosphorylated AMPK promotes the expression of MACC1, and MACC1 plays a mediating role in the process of acetylcholine promoting gastric cancer metastasis. Therefore, this study aims to explore the effect of acetylcholine on the invasion / migration and EMT of human gastric cancer cells, and to elucidate the /AMPK/MACC1 signaling pathway of the M3 cholinergic receptor and its role in the invasion / migration of human gastric cancer cells and in EMT. Use. Research hypothesis: 1. acetylcholine promotes invasion / migration of human gastric cancer cells and EMT; 2.M3 type choline receptors mediate the invasion / migration of human gastric cancer cells and the role of EMT; 3. acetylcholine acts on the type M3 choline receptor, up regulation of MACC1 expression through AMPK, and MACC1 in acetylcholine promoting invasion / migration of human gastric cancer cells And EMT plays an important intermediate signal role. Research content: 1. screening the M3 cholinergic receptor high expression gastric cancer cell lines to extract RNA of BGC-803, BGC-823, MGC-803, MKN-28, MKN-45, SGC-7901 human gastric cancer cells. After reverse transcription to cDNA, the expression of the M3 type cholinergic receptor of each cell line was detected in real time, and the expression amount was selected. The highest two cells were tested for the effect of acetylcholine on the invasion / migration of gastric cancer cells and the effect of EMT on the invasion / migration of gastric cancer cells. The 10uM concentration of acetylcholine was added to the culture medium of MKN-45 and MGC-803 gastric cancer cells, and 0h, 24h and 48 h were cultured respectively. The experiment of Transwell invasion with matrix glue and the Transwell migration test without matrix glue were detected respectively. The invasiveness and migration ability of two cells, as well as 10uM concentration of acetylcholine stimulated MKN-45 and MGC-803 cells 0 h, 24 h and 48 h, each extracted all mRNA and protein, reverse transcriptase mRNA to cDNA, and real-time quantitative PCR detection of the relative expression of EMT related indexes (E- calcium mucin, vimentin, fibronins, etc.) Blot method was used to detect the protein content of the above EMT index. We also observed the morphological changes of two cells before and after acetylcholine stimulation. The effect of.3.M3 type cholinergic receptor inhibitor on acetylcholine was added to the culture medium of MKN-45 and MGC-803 gastric cancer cells with M3 cholinergic receptor specific inhibitor, Da NACHIN (10uM concentration), and 30 min Then the acetylcholine was added to 10uM, then the two cell culture 48 h. experiments were divided into four groups: negative control, acetylcholine, Da afien, nafna New + acetylcholine. The invasion and migration ability of two cells were detected by Transwell invasion experiment with matrix glue and Transwell migration test without matrix glue. MKN-45 and MGC-803 cell line 48 h were cultured. MRNA and whole protein were extracted from each cell. After mRNA was reverse transcribed to cDNA, the relative expression of EMT related indexes (E- calcium mucin, vimentin, fibronectin, MMP2 and MMP9) was detected by real-time quantitative PCR. The effect of stimulation on the expression of MACC1 in bioinformatics analysis of the expression of the encoding gene CHRM3 of M3 type receptor and the correlation of phosphorylated AMPK. We consider that MACC1 may be a downstream signal of acetylcholine, adding 10uM concentration of acetylcholine to the medium of MKN-45 and MGC-803 gastric cancer cells, respectively, to develop 0h, 24h and 48 h, each extraction. All mRNA and protein, mRNA reverse transcriptase cDNA, real-time quantitative PCR detection of MACC1 mRNA relative expression, Western blot method to detect the protein content of MACC1. Then MKN-45 and MGC-803 gastric cancer cells according to negative control, acetylcholine, Da afinxin, the new + acetylcholine four groups after 48 h, real-time quantitative and quantitative The mRNA and protein content of MACC1 were detected by blot respectively. Immunofluorescence test detected the nuclear translocation of MACC1 after acetylcholine stimulation.5.MACC1 was important in the invasion / migration of gastric cancer cells by acetylcholine and EMT, MKN-45 and MGC-803 cells were transfected to MACC1 silent sequence (siMACC1) or negative control sequence (siCtrl). Ern blot detected the silencing effect of mRNA and protein level respectively. Then, 10uM acetyl choline was added to cells transfected with siMACC1 or siCtrl to be cultured and divided into four groups: siCtrl, siMACC1, siCtrl+ acetyl choline, siMACC1+ acetyl choline. The Transwell invasion experiment with matrix glue and Transwell migration experiment without matrix glue Do not detect the invasion and migration of two cells. Real-time quantitative PCR and Western blot test the changes of EMT related indexes (E- calcium mucin, vimentin, fibronectin, MMP2 and MMP9) at mRNA and protein levels, respectively. AMPK is the intermediate signal of acetylcholine /M3 cholinergic receptor and MACC1. Sex control, acetylcholine, Da afen, four groups of new + acetylcholine culture 48 h, extract whole protein, Western blot to detect the protein content of p-AMPK. Add 8uM concentration AMPK inhibitor Dorsomorphin in the medium of two cells, and then add 10uM acetyl choline after 30 min, and divide into four groups: negative control, acetylcholine, Dor. Somorphin, Da +Dorsomorphin, after training 48 h, Western blot tests the protein content of p-AMPK and MACC1 in.7. statistics, all statistical analysis is performed with SPSS 20. This data is expressed in the form of mean value of standard error, using one-way ANOVA method to analyze the differences between groups, bilateral P values less than 0.05 are considered to be The results were statistically significant: 1. the mRNA expression of M3 type cholinergic receptor of gastric cancer cell lines was detected by screening the M3 cholinergic receptor high expression of gastric cancer cell line in real time. It was found that the mRNA expression of CHRM3 in MKN-45 and MGC-803 two cells was the highest. Therefore, the two cells were selected for the sequel experiment of.2. acetylcholine to stimulate the invasion of gastric cancer cells. / migration and induction of EMT in Transwell invasion and migration experiments, in 48 h, acetylcholine gradually increased the invasion and migration of MKN-45 and MGC-803 gastric cancer cells, and from the real-time quantitative PCR results, it can be seen that acetylcholine gradually reduced the mRNA expression of the two cell E- calcium mucin as the time of stimulation increased and increased. The protein levels of vimentin, fibronectin, MMP2 and MMP9 mRNA expression.Western blot also obtained the same trend as mRNA, indicating that acetylcholine gradually induced gastric cancer cell EMT. in 48 h, but acetylcholine stimulation did not alter the morphology of the two gastric cancer cells to mediate the invasion / migration of acetylcholine promoting gastric cancer. Acetylcholine significantly increased the invasion and migration of MKN-45 and MGC-803 gastric cancer cells in the Transwell invasion and migration experiments with EMT. After the addition of M3 type cholinergic receptor inhibitor to nachnat, the changes of the invasion and migration of acetylcholine to two cells were inhibited. The results of real-time quantitative PCR and Westernblot showed a significant result. The expression of E- calcium mucin induced by acetylcholine decreased, and the expression of vimentin, fibronectin, MMP2 and MMP9 was upside down in the new presence of dionna, and the individual Da van new also showed the ability to inhibit invasion / migration and EMT by.4. acetylcholine stimulation of MACC1 expression up-regulated through TCGA extraction of gastric adenocarcinoma Related data analysis showed that the phosphorylation level of AMPK threonine 172 site was positively correlated with the mRNA expression of CHRM3. Combined with our previous laboratory study conclusion that phosphorylated AMPK can up regulate MACC1, we later verified the relationship between acetylcholine and MACC1. We found that 48 h acetylcholine gradually increased the mRNA and protein table of MACC1. Da, and Da Fei Xin can reverse this effect. However, acetylcholine has no significant change in the nuclear translocation of MACC1,.5.MACC1 is promoted by acetylcholine.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R735.2

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