本文選題：DNA甲基化轉移酶1 + 激活素A��； 參考：《吉林大學》2017年博士論文

【摘要】：研究背景激活素A屬于TGF-β超家族成員,其受體屬于絲氨酸/蘇氨酸激酶(Ser/Thr)型受體,分為I型受體及II型受體,激活素與II型結合,再招募I型受體,進而通過細胞內(nèi)Smads信號蛋白,或非Smads依賴的MAPK通路、Rho GTPase通路和PI3K/Akt通路傳導信號至細胞核,激活靶基因轉錄。有關研究顯示,激活素A的表達與腫瘤的發(fā)生發(fā)展密切相關,參與多種腫瘤細胞增殖及凋亡調(diào)控。如大腸癌及骨肉瘤等患者外周血激活素A水平升高,大腸癌及卵巢癌等組織高表達激活素A,激活素A可以誘導肺腺癌細胞凋亡、促進前列腺癌細胞轉移,激活素A表達異常還與腫瘤惡液質(zhì)形成有關等。DNA甲基化是由DNA甲基化轉移酶DNMTs(DNA methyltransferases,Dnmts)催化并維持的,DNMTs家族包括DNMT1、DNMT2、DNMT3a、DNMT3b及DNMT3L。DNMTs是調(diào)控表觀遺傳學DNA甲基化的重要分子,DNMT1主要起維持性甲基化酶作用。以往的研究顯示,DNMTs在腫瘤細胞中高表達,DNMTs的表達還能影響胚胎發(fā)育過程,推測細胞核潛能越高與DNMTs的調(diào)控關系越密切。激活素也具有調(diào)控中胚層細胞發(fā)育及腫瘤細胞增殖的作用,但是,激活素A能否通過調(diào)控DNMTs表達,影響腫瘤細胞增殖及凋亡,仍不清楚。因此,本研究通過預實驗,選擇對激活素A敏感的小鼠骨髓瘤細胞系NS-1細胞作為靶細胞,探討激活素A調(diào)控NS-1細胞增殖及凋亡與DNMT1表達的關系。課題分為四方面進行論述,第一部分分析了激活素A對多種腫瘤細胞的生物學作用,探討激活素A作用與DNMT1關系。第二部分闡述了激活素A調(diào)控小鼠骨髓瘤細胞NS-1增殖及凋亡作用及其信號機制,分析激活素信號蛋白Smad3 Knock-down及過表達對DNMT1的影響。第三部分通過DNMT1Knock-down,進一步探討改變DNMT1表達對激活素A抑制NS-1細胞增殖及誘導凋亡的影響及其機制。第四部分建立NS-1荷瘤鼠模型,評價激活素A及DNMT1 Knock-down對小鼠體內(nèi)NS-1細胞成瘤的影響。研究方法RT-PCR及Western blotting檢測DNMT1在NS-1細胞的表達。MTT法、CFSE染色、Hochest33342染色及Annexin V-7AAD評價NS-1腫瘤細胞活力及凋亡。構建pLVX-U6/Puro-DNMT1 sh RNA表達質(zhì)粒,轉染NS-1細胞評價敲低DNMT1基因?qū)毎鲋臣暗蛲龅挠绊憽＝S-1細胞荷瘤鼠模型,HE染色、免疫組織化學染色、Tunel法評價DNMT1基因Knock-down對NS-1細胞成瘤的影響。研究結果第一部分激活素A對多種腫瘤細胞活性及DNMT1表達的影響DNMT1在腫瘤細胞高表達,而在正常組織來源細胞低表達。Giemsa染色法顯示激活素A作用于SW480、Hela、A549、NS-1細胞后發(fā)生明顯的細胞形態(tài)學改變,提示激活素A可以影響NS-1細胞形態(tài)變化。且在低濃度5ng/ml時激活素A即可影響NS-1細胞形態(tài)變化。同時可見激活素A可以顯著抑制骨髓瘤細胞系NS-1細胞DNMT1表達。因此,實驗采用NS-1細胞作為激活素A敏感細胞,進行后續(xù)研究。第二部分激活素A誘導小鼠骨髓瘤細胞系NS-1凋亡為了確定激活素A對NS-1細胞的作用,實驗首先采用MTT法檢測NS-1細胞活力,結果顯示激活素A可以抑制NS-1細胞活力。CFSE染色進一步顯示,激活素A抑制了NS-1細胞增殖。Hocheast染色和Annexin V-7AAD結果顯示激活素A可以促進NS-1細胞凋亡。Western blotting結果顯示激活素A上調(diào)NS-1細胞Bax、Cyc C及Caspase3表達,而抑制Bcl2及DNMT1表達,提示激活素A通過線粒體凋亡途徑誘導NS-1細胞凋亡,其作用可能與下調(diào)DNMT1表達有關。進一步研究顯示,激活素A可以促進NS-1細胞表達激活素信號蛋白Smad3,但過表達和Knock-down Smad3不能影響NS-1細胞DNMT1表達,提示激活素A可能通過Smad3信號非依賴途徑調(diào)控DNMT1表達。第三部分DNMT1 Knock-down對NS-1細胞的活力及凋亡的影響Western blotting結果顯示構建的pLVX-DNMT1干涉質(zhì)粒轉染NS-1細胞成功敲低了DNMT1表達,同時上調(diào)NS-1細胞Cyc C及Caspase3表達及Bax/Bcl2比值。MTT結果顯示NS-1細胞轉染0.5μg pLVX-DNMT1質(zhì)粒24h后,與pLVX對照組質(zhì)粒轉染NS-1細胞組比較細胞活力明顯下降;添加5ng/ml激活素A組,較激活素A處理的pLVX對照組質(zhì)粒轉染NS-1細胞組細胞活力進一步下降。Annexin V-7AAD結果顯示轉染pLVX-DNMT1質(zhì)�？梢哉T導NS-1細胞發(fā)生凋亡。第四部分DNMT1 Knock-down對小鼠NS-1細胞成瘤的影響為了進一步確定DNMT1 Knock-down對激活素A誘導NS-1細胞凋亡的影響,實驗首先采用不同濃度的NS-1細胞接種Balb/c小鼠背部,觀察成瘤情況。結果顯示2.0x106 NS-1細胞/0.1ml接種小鼠背部皮下,成功構建了NS-1細胞荷瘤鼠模型。進一步分析DNMT1 Knock-down對小鼠NS-1細胞體內(nèi)成瘤影響。實驗采用預轉染pLVX-NC空質(zhì)粒、轉染pLVX-DNMT1 sh RNA表達質(zhì)粒及未轉染質(zhì)粒的野生型NS-1接種Balb/c小鼠背部,體內(nèi)測量腫瘤體積結果顯示,與pLVX-NC組相比,pLVX-DNMT1組的NS-1細胞成瘤能力明顯減弱,且體積增長減慢。提示DNMT1 Knock-down可以抑制NS-1細胞體內(nèi)成瘤能力。待腫瘤生長到第18天處死Balb/c小鼠取腫瘤,結果顯示與pLVX-NC組相比,pLVX-DNMT1組的NS-1細胞腫瘤體積明顯降低,具有統(tǒng)計學差異。提取腫瘤組織總蛋白Westernblotting結果顯示pLVX-DNMT1組成功敲底了DNMT1的表達,且HE染色結果和免疫組織化學染色結果顯示與pLVX-NC組相比,pLVX-DNMT1組細胞核漿比例明顯增加、排列紊亂、局部出現(xiàn)膿腫和壞死,提示DNMT1 Knock-down可以誘導NS-1細胞瘤發(fā)生凋亡,可能具有骨髓瘤治療價值。Tunnel染色顯示與激活素A處理的pLVX-NC組相比,激活素A處理的DNMT1 Knock-down的NS-1細胞腫瘤組織存在更多的染色陽性細胞,提示DNMT1 Knock-down促進了激活素A誘導NS-1細胞凋亡作用。綜上所述,DNMT1是DNA甲基化表觀遺傳學調(diào)控的重要分子蛋白,降低NS-1細胞DNMT1的表達水平,可以通過線粒體凋亡途徑抑制NS-1細胞增殖、促進NS-1細胞凋亡。激活素A可以調(diào)控腫瘤細胞DNMT1表達,對治療DNA甲基化修飾異常所引發(fā)的腫瘤疾病具有重要的意義。
[Abstract]:Background activin A belongs to the member of the TGF- beta superfamily, whose receptor belongs to the serine / threonine kinase (Ser/Thr) receptor, which is divided into I type and II receptor. Activin is combined with II type, and then recruitment of I type receptor, and then through intracellular Smads signal protein, or MAPK pathway of non Smads dependent Lai, Rho GTPase pathway and transduction pathway. The study shows that the expression of activin A is closely related to the occurrence and development of tumor, and participates in the regulation of proliferation and apoptosis of various tumor cells. The levels of peripheral blood activin A in patients with colorectal cancer and osteosarcoma, such as large intestine and ovarian cancer, are high expression of activin A, and activin A can induce lung gland. The apoptosis of cancer cells, the promotion of the metastasis of prostate cancer cells, the abnormal expression of activin A and the formation of.DNA methylation related to the formation of malignant tumor fluid are catalyzed and maintained by DNA methyltransferase DNMTs (DNA methyltransferases, Dnmts). The DNMTs family consists of DNMT1, DNMT2, DNMT3a, DNMT3b, and regulating the weight of epigenetic methylation. DNMT1 mainly acts as a maintainable methylation enzyme. Previous studies have shown that DNMTs is highly expressed in tumor cells, and the expression of DNMTs can also affect the process of embryonic development. It is presumed that the higher the cell nuclear potential is, the closer the relationship between the regulation of DNMTs and the regulation of mesoderm cell development and tumor cell proliferation. Whether or not A can affect the proliferation and apoptosis of tumor cells by regulating the expression of DNMTs and affecting the proliferation and apoptosis of tumor cells is still unclear. Therefore, this study selects the NS-1 cell line of murine myeloma cell line sensitive to activin A as a target cell, and discusses the relationship between activin A regulation of NS-1 cell proliferation and apoptosis and the expression of DNMT1. The subject is divided into four aspects. The first part analyses the biological effect of activin A on a variety of tumor cells, and discusses the relationship between activin A and DNMT1. Second the effect of activin A on the proliferation and apoptosis of murine myeloma cells and its signal mechanism and the effect of Smad3 Knock-down on activin signal protein and the effect of overexpression on DNMT1 are discussed. The third part is connected with the activity of activin protein. DNMT1Knock-down, further explore the effect and mechanism of DNMT1 expression on activin A inhibition of NS-1 cell proliferation and induction of apoptosis. Fourth the model of NS-1 bearing mice was established to evaluate the effect of activin A and DNMT1 Knock-down on the tumor formation of NS-1 cells in mice. The expression of.MTT, CFSE staining, Hochest33342 staining and Annexin V-7AAD were used to evaluate the viability and apoptosis of NS-1 tumor cells. PLVX-U6/Puro-DNMT1 sh RNA expression plasmid was constructed, and NS-1 cells were transfected to evaluate the effect of low DNMT1 gene on cell proliferation and apoptosis. The effect of NMT1 gene Knock-down on the tumor formation of NS-1 cells. The first part of the study was the effect of activin A on the activity of various tumor cells and the expression of DNMT1, DNMT1 was highly expressed in the tumor cells, and the expression of activin A in the normal tissue derived cells showed that the activin A acted upon SW480, Hela, A549, and NS-1 cells. It is suggested that activin A can affect the morphological changes of NS-1 cells, and activin A can affect the morphological changes of NS-1 cells at low concentration of 5ng/ml. At the same time, activin A can significantly inhibit the expression of DNMT1 in myeloma cell line NS-1 cells. Therefore, the experiment uses NS-1 cells as activin A sensitive cells for follow-up study. Second parts Activin A induced the apoptosis of mouse myeloma cell line NS-1 in order to determine the effect of activin A on NS-1 cells. First, the MTT assay was used to detect the activity of NS-1 cells. The results showed that activin A can inhibit the.CFSE staining of NS-1 cell viability further. Activin A inhibits NS-1 cell proliferation.Hocheast staining and the result of NS-1. Activin A can promote NS-1 cell apoptosis,.Western blotting results show that activin A up-regulated NS-1 cells Bax, Cyc C and Caspase3 expression, and inhibits Bcl2 and DNMT1 expression, suggesting that activin may induce apoptosis through mitochondrial apoptosis pathway, and its role may be related to down regulation. The expression of activin signal protein Smad3 was expressed in NS-1 cells, but overexpression and Knock-down Smad3 did not affect the DNMT1 expression in NS-1 cells, suggesting that activin A may regulate DNMT1 expression through non dependent pathway of Smad3 signal. Third part DNMT1 Knock-down on the vitality and apoptosis of NS-1 cells The transfection of NS-1 cells with the interference plasmid successfully knocked down the expression of DNMT1, at the same time, the expression of Cyc C and Caspase3 in NS-1 cells and the Bax/Bcl2 ratio.MTT results showed that the NS-1 cells transfected to 0.5 mu g pLVX-DNMT1 plasmid 24h, compared with the control group plasmids transfected to the cell group, the cell viability was significantly decreased. LVX control group plasmid transfected NS-1 cell cell viability further decreased.Annexin V-7AAD results showed that transfection of pLVX-DNMT1 plasmids could induce apoptosis of NS-1 cells. Fourth the effect of DNMT1 Knock-down on the tumor formation of mice NS-1 cells in order to further determine the effect of DNMT1 Knock-down on the apoptosis of activin A induced cells. First of all, NS-1 cells with different concentrations were inoculated on the back of Balb/c mice to observe the tumor formation. The results showed that the 2.0x106 NS-1 cell /0.1ml was inoculated subcutaneously on the back of the mouse, and the tumor mouse model of NS-1 cells was successfully constructed. The effect of DNMT1 Knock-down on the tumor formation in the mice NS-1 cells was further analyzed. The experiment was carried out by pre transfection of pLVX-NC empty plasmid and transfection pLV. X-DNMT1 sh RNA expression plasmid and untransfected wild type NS-1 were inoculated on the back of Balb/c mice. The volume of tumor volume measured in vivo showed that the tumor formation ability of NS-1 cells in pLVX-DNMT1 group decreased significantly and slowed down compared with pLVX-NC group, suggesting that DNMT1 Knock-down could inhibit the tumorigenesis of NS-1 cells. 18 days after the death of Balb/c mice, the results showed that the volume of NS-1 cells in the group pLVX-DNMT1 was significantly lower than that in the pLVX-NC group, with a statistically significant difference. The result of the extraction of the total protein of the tumor tissue Westernblotting showed that the pLVX-DNMT1 group successfully knocked down the expression of DNMT1, and the results of HE staining and immunohistochemical staining showed pLVX-. Compared with group NC, the proportion of cell nuclear plasma in pLVX-DNMT1 group was obviously increased, arranged in disorder, local abscess and necrosis, suggesting that DNMT1 Knock-down could induce apoptosis of NS-1 cytoma, which might have the value of myeloma treatment with.Tunnel staining, compared with pLVX-NC group treated with activin A, DNMT1 Knock-down NS-1 cell swelling of activin A treatment. More staining positive cells exist in the tumor tissue, suggesting that DNMT1 Knock-down promotes the apoptosis of NS-1 cells induced by activin A. To sum up, DNMT1 is an important molecular protein of DNA methylation epigenetic regulation, which reduces the expression level of DNMT1 in NS-1 cells, and can inhibit the proliferation of NS-1 cells through mitochondrial apoptosis pathway and promote NS-1 cells. Apoptosis, activin A can regulate the expression of DNMT1 in tumor cells, which is of great significance in the treatment of tumor diseases caused by abnormal DNA methylation.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R730.2

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7 張宸豪;激活素受體相互作用蛋白5基因克隆及其在小鼠胰腺組織中的表達和作用研究[D];吉林大學;2010年

8 張學軍;激活素受體相互作用蛋白2在巨噬細胞中介導激活素信號傳導機制的研究[D];吉林大學;2004年

9 李占東;人激活素受體結合蛋白2的克隆、鑒定及其與乳腺癌相關性的研究[D];東北師范大學;2009年

10 李俊;激活素A對人早孕滋養(yǎng)層細胞凋亡的調(diào)節(jié)及與妊娠期高血壓疾病關系研究[D];中國醫(yī)科大學;2005年

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2 李繼茹;激活素A和脂多糖對小鼠腹腔巨噬細胞的活化作用及其機制研究[D];吉林大學;2013年

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6 徐妍婷;胎盤中激活素受體的表達及與胎兒生長受限的關系[D];蘇州大學;2014年

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9 宋春雷;草魚激活素Ⅰ型和Ⅱ型受體的cDNA克隆、結構分析及其在垂體細胞中的表達調(diào)控的研究[D];電子科技大學;2009年

10 常穎;激活素A促進人晶狀體上皮細胞增殖作用的研究[D];吉林大學;2006年

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