MACC1介导乙酰胆碱促人胃癌细胞侵袭及迁移的研究

发布时间：2018-05-30 11:31

本文选题：乙酰胆碱 + MACC1　；参考：《南方医科大学》2016年硕士论文

【摘要】：研究背景：胃癌是世界上常见的恶性肿瘤之一,在全球癌症总发病率中排第五位,在癌症死亡原因中排第三位,其中以中国的发病率最高。胃癌发病率在我国男性中排第二位,在女性中排第三位,虽然近年来我国胃癌的发病率有下降趋势,但是发病人数依然较多,2015年我国估计有67.91万人诊断出胃癌,有49.8万人死于胃癌。早期胃癌的治疗主要以手术为主,但是绝大多数胃癌往往到了晚期才被确诊,晚期胃癌只能采取放化疗、靶向药物等治疗方法,疗效往往不理想。晚期胃癌往往伴随广泛浸润或远处转移,因此研究胃癌转移机制有助于进一步理解晚期胃癌的恶性生物学行为及发现新的治疗新策略。肿瘤转移是指肿瘤细胞脱离原发病灶在体内播散,并在其他部位定植生长形成继发肿瘤病灶。上皮间质转化(epithelial-mesenchymal transition, EMT)被认为是恶性肿瘤转移的起始步骤。在EMT中,非运功的、有极性的、细胞连接紧密的上皮来源肿瘤细胞,转化成为非极性的、运动的、侵袭性的间质性细胞。发生EMT的肿瘤细胞,其分子标志发生了显著的变化,例如,上皮细胞-细胞粘附分子E-钙黏蛋白表达下降,而间质细胞的一些表型的表达则会上调,如波形蛋白、纤连蛋白。另外,肿瘤细胞也可改造细胞外基质(extracellular matrix, ECM)促进侵袭和转移,其中各种基质金属蛋白酶(matrix metalloproteinase, MMP)的分泌发挥了重要作用。肿瘤微环境在肿瘤发生、发展及转移中的重要作用近年来受到了高度的重视。神经是肿瘤微环境的重要组成部分,虽然已经有研究证实肿瘤可以沿神经生长,然后通过神经途径播散,即嗜神经侵袭(perineural invasion, PNI),但是神经在肿瘤发生发展中所发挥的主动性作用却很少受到关注。早在19世纪40年代,就已经有研究探索了去除肿瘤神经支配所带来的效果,但是并未引起研究者重视神经对肿瘤的作用。2013年,Magnon等人发表的研究使研究者们重新关注到神经对于肿瘤的作用。他们揭示了自主神经系统对小鼠前列腺癌发生发展至关重要,其中,去除交感神经系统(sympathetic nervous system, SNS)主要在肿瘤发生的早期抑制生长,而去除副交感神经系统(parasympathetic nervous system, PNS)主要在肿瘤发展的后期抑制转移。2014年Zhao等人发表的另一篇文献报道,在小鼠胃癌模型中,手术或药物去除胃神经支配(即迷走神经切断术或局部注射神经毒性药物)能显著降低胃癌发生概率及胃癌进展速度。迷走神经末梢可以合成和释放乙酰胆碱,乙酰胆碱及其M3型胆碱受体已被报道参与了肺癌、肝癌、结肠癌及前列腺癌等的生长与转移,但是其对胃癌转移的作用及机制尚不明确。结肠转移相关基因-1(metastasis-associated in colon cancer-1, MACC1)是2008年在结肠癌中首先发现的一个促癌基因。Stein等人发现MACC1在肠结癌组织中的表达要高于正常结肠粘膜上皮,且是结肠癌病人的一个独立预后因素。2013年我们实验团队证实了MACC1在胃癌组织中高表达并与胃癌病人分期及预后相关。MACC1促进肝细胞生长因子受体c-Met信号并诱导胃癌细胞EMT、促进MMP2及MMP9的表达,从而促进胃癌生长、侵袭及转移。并且在糖剥夺情况下,胃癌细胞可通过磷酸化的腺苷酸活化蛋白激酶(AMP-activated protein kinase, AMPK)上调MACC1, MAAC1通过瓦伯格效应抵抗胃癌细胞代谢应激所带来的损伤,从而保持胃癌细胞的存活。我们通过生物信息学的方法发现胃癌中磷酸化的AMPK水平与M3型胆碱受体的编码基因CHRM3 (M3型胆碱受体的编码基因)的表达呈正相关。我们推测,乙酰胆碱作用于M3型胆碱受体后,可能通过磷酸化的AMPK促进MACC1的表达,并且MACC1在乙酰胆碱促胃癌转移过程中发挥了介导作用。因此本研究旨在探索乙酰胆碱对人胃癌细胞侵袭／迁移及EMT的影响,阐明M3型胆碱受体/AMPK/MACC1信号通路及其在人胃癌细胞侵袭／迁移及EMT中的作用。研究假设：1.乙酰胆碱促进人胃癌细胞侵袭／迁移及EMT；2.M3型胆碱受体介导乙酰胆碱促人胃癌细胞侵袭／迁移及EMT的作用；3.乙酰胆碱作用于M3型胆碱受体后,通过AMPK上调MACC1的表达,且MACC1在乙酰胆碱促人胃癌细胞侵袭／迁移及EMT中发挥重要的中间信号作用。研究内容：1.筛选M3型胆碱受体高表达的胃癌细胞株分别提取BGC-803、BGC-823、MGC-803、MKN-28、MKN-45、SGC-7901人胃癌细胞的RNA,逆转录成cDNA后,实时定量PCR检测各细胞株M3型胆碱受体的mRNA表达情况,并选取表达量最高的两株细胞进行后续实验。2.乙酰胆碱对胃癌细胞侵袭／迁移及EMT的影响将10uM浓度的乙酰胆碱添加到MKN-45及MGC-803胃癌细胞的培养基,分别培养0h、24h及48 h,用带有基质胶的Transwell侵袭实验及不带基质胶的Transwell迁移实验分别检测两株细胞的侵袭及迁移能力；同样再以10uM浓度的乙酰胆碱刺激MKN-45及MGC-803细胞0 h、24 h及48 h,各提取全部mRNA和蛋白质,将mRNA逆转录成cDNA,实时定量PCR检测EMT相关指标(E-钙黏蛋白、波形蛋白、纤连蛋白、MMP2及MMP9)的mRNA相对表达量,用Western blot的方法检测上述EMT指标的蛋白含量。我们也观察了乙酰胆碱刺激前后两株细胞的形态变化。3.M3型胆碱受体抑制剂对乙酰胆碱作用的影响在MKN-45及MGC-803胃癌细胞的培养基中加入M3型胆碱受体特异性抑制剂达非那新(10uM浓度),30 min后再加入10uM的乙酰胆碱,然后两株细胞培养48 h。实验一共分为四组：阴性对照、乙酰胆碱、达非那新、达非那新+乙酰胆碱。用带有基质胶的Transwell侵袭实验及不带基质胶的Transwell迁移实验分别检测两株细胞的侵袭及迁移能力。再按照上述分组培养MKN-45及MGC-803细胞株48 h,各提取两株细胞的mRNA和全蛋白,mRNA逆转录成cDNA后,实时定量PCR检测EMT相关指标(E-钙黏蛋白、波形蛋白、纤连蛋白、MMP2及MMP9)的mRNA相对表达量,用Western blot方法检测上述EMT指标的蛋白含量。4.乙酰胆碱的刺激对MACC1表达的影响生物信息学的方法分析M3型受体的编码基因CHRM3的表达情况与磷酸化AMPK的相关性。我们考虑MACC1可能为乙酰胆碱下游信号,将10uM浓度的乙酰胆碱添加到MKN-45及MGC-803胃癌细胞的培养基,分别培养0h、24h及48 h,各提取全部mRNA和蛋白质,将mRNA逆转录成cDNA,实时定量PCR检测MACC1的mRNA相对表达量,用Western blot的方法检测MACC1的蛋白含量。然后将MKN-45及MGC-803胃癌细胞按照阴性对照、乙酰胆碱、达非那新、达非那新+乙酰胆碱四组培养48 h后,实时定量PCR及Western blot分别检测MACC1的mRNA及蛋白含量。免疫荧光实验检测乙酰胆碱刺激后MACC1的核转位情况。5.MACC1在乙酰胆碱促胃癌细胞侵袭／迁移及EMT中的重要性将MKN-45及MGC-803细胞转染MACC1的沉默序列(siMACC1)或阴性对照序列(siCtrl),实时定量PCR及Western blot分别检测mRNA及蛋白水平的沉默效果。然后在转染有siMACC1或siCtrl的细胞中加入10uM乙酰胆碱进行培养,共分成四组：siCtrl、siMACC1、siCtrl+乙酰胆碱、siMACC1+乙酰胆碱。用带有基质胶的Transwell侵袭实验及不带基质胶的Transwell迁移实验分别检测两株细胞的侵袭及迁移能力。实时定量PCR及Western blot分别在mRNA及蛋白水平检测EMT相关指标(E-钙黏蛋白、波形蛋白、纤连蛋白、MMP2及MMP9)的变化。6.验证AMPK是乙酰胆碱/M3型胆碱受体与MACC1的中间信号将MKN-45及MGC-803胃癌细胞按照阴性对照、乙酰胆碱、达非那新、达非那新+乙酰胆碱四组培养48 h后,提取全蛋白,Western blot检测p-AMPK的蛋白含量。在两株细胞的培养基中加入8uM浓度的AMPK抑制剂Dorsomorphin,30 min后再加入10uM的乙酰胆碱,一共分为四组：阴性对照、乙酰胆碱、Dorsomorphin、达非那新+Dorsomorphin,培养48 h后,Western blot检测p-AMPK及MACC1的蛋白含量。7.统计方法所有的统计学分析都是用SPSS 20.0完成的,本文数据以平均值±标准误的形式表示,用one-way ANOVA的方法来分析组间的差异,双边的P值小于0.05被认为有统计学差异。研究结果：1.筛选M3型胆碱受体高表达的胃癌细胞株实时定量PCR检测各胃癌细胞株M3型胆碱受体的mRNA表达,发现MKN-45及MGC-803两株细胞CHRM3的mRNA表达最高,因此选择这两株细胞进行后续实验。2.乙酰胆碱刺激胃癌细胞侵袭／迁移并诱导EMT在Transwell侵袭及迁移实验中,在48 h内,乙酰胆碱逐渐增加了MKN-45及MGC-803胃癌细胞的侵袭及迁移数量,并且从实时定量PCR的结果可以看到,随着刺激时间增加,乙酰胆碱能逐渐降低两株细胞E-钙黏蛋白的mRNA表达,并增加了波形蛋白、纤连蛋白、MMP2及MMP9的mRNA表达。Western blot检测的蛋白水平也得到了与mRNA相同的趋势,说明48 h内乙酰胆碱逐渐诱导了胃癌细胞EMT。但是,乙酰胆碱的刺激未改变两株胃癌细胞的形态。3.M3型胆碱受体介导乙酰胆碱促胃癌侵袭／迁移及EMT在Transwell侵袭及迁移实验中,乙酰胆碱显著增加了MKN-45及MGC-803胃癌细胞的侵袭及迁移数量,而加入M3型胆碱受体抑制剂达非那新以后,乙酰胆碱对两株细胞的侵袭及迁移数量的改变均受到抑制。实时定量PCR及westernblot的结果显示,乙酰胆碱所诱导的E-钙黏蛋白表达下降,波形蛋白、纤连蛋白、MMP2及MMP9表达的上升,在达非那新存在的情况下都被逆转。并且,单独的达非那新也表现出抑制侵袭／迁移及EMT的能力。4.乙酰胆碱刺激MACC1表达上调通过对TCGA提取的胃腺癌相关数据分析得到,AMPK苏氨酸172位点磷酸化水平与CHRM3的mRNA表达呈正相关。结合我们实验室前期研究结论：磷酸化的AMPK可以上调MACC1,我们接下来验证了乙酰胆碱与MACC1的联系。我们发现,48 h内乙酰胆碱逐渐增加了MACC1的mRNA及蛋白表达,并且达非那新能逆转这一作用。但是乙酰胆碱对于MACC1的核转位没有明显改变。5.MACC1在乙酰胆碱促胃癌细胞侵袭／迁移及EMT中发挥了重要作用我们将MKN-45及MGC-803细胞转染了MACC1的沉默序列后,MACC1的mRNA及蛋白水平的表达都明显下降,说明沉默效果较好。MACC1的表达被沉默后,乙酰胆碱促胃癌细胞侵袭／迁移的作用都被抑制了。且不论从mRNA还是蛋白水平,乙酰胆碱降低E-钙黏蛋白,增加波形蛋白、纤连蛋白、MMP2及MMP9的作用也在沉默MACC1后逆转了。6.乙酰胆碱/M3型胆碱受体通过AMPK途径上调MACC1Western blot的结果显示,乙酰胆碱促进了p-AMPK的蛋白水平,并且这一作用可以被M3型胆碱受体抑制剂所逆转。另外,当使用Dorsomorphin抑制了AMPK活性后,乙酰胆碱促进MACC1表达的作用就被抑制了,说明AMPK是乙酰胆碱/M3型胆碱受体与MACC1的中间信号。研究结论：乙酰胆碱通过M3型胆碱受体/AMPK/MACC1信号通路促进了胃癌细胞侵袭／迁移并诱导了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.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.2

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