本文選題：GNMT + 前列腺癌細(xì)胞系　； 參考：《復(fù)旦大學(xué)》2014年博士論文

【摘要】：前列腺癌的發(fā)病率在世界范圍內(nèi)及我國均呈逐年上升趨勢,眾多的分子生物學(xué)及遺傳學(xué)機制主導(dǎo)著這一疾病的進程。Sreekumar等通過代謝組學(xué)的研究方法,發(fā)現(xiàn)肌氨酸與前列腺癌的發(fā)生及進展密切相關(guān)。它在前列腺癌尤其是侵襲性前列腺癌組織中的濃度明顯高于良性前列腺增生組織中的濃度,并且進一步的研究發(fā)現(xiàn)在良性前列腺增生細(xì)胞系中加入肌氨酸后可以使細(xì)胞具有侵襲性。組織細(xì)胞中的肌氨酸濃度受到甘氨酸N-甲基轉(zhuǎn)移酶(GNMT)的調(diào)節(jié)而發(fā)生變化。最新研究發(fā)現(xiàn),前列腺癌細(xì)胞比正常前列腺上皮細(xì)胞表達更多的IGNMT,并且敲除GNMT基因可以抑制前列腺癌細(xì)胞增殖,并誘導(dǎo)其凋亡。因此,調(diào)節(jié)GNMT的表達或功能,有望為前列腺癌的發(fā)生及進展機制和治療方法的研究提供一種全新的思路。GNMT作為一種具有多種功能的蛋白質(zhì),其甲基轉(zhuǎn)移酶的活性可以被其催化產(chǎn)物S-腺苷高半胱氨酸(SAH)所抑制,這種作用在體外及體內(nèi)均可觀察到,并且SAH還可以在轉(zhuǎn)錄水平上抑制GNMT的表達。維甲酸(retinoic acid, RA)可以顯著增加小鼠體內(nèi)GNMT的活性,并可在轉(zhuǎn)錄水平上促進其表達。因此,我們可以嘗試?yán)肧AH和RA來實現(xiàn)體外和體內(nèi)對GNMT表達及活性的調(diào)控,從而進行進一步的研究。目前尚無研究證實,外源性調(diào)控GNMT是否能夠改變前列腺癌細(xì)胞的生物學(xué)特性,而這正是我們這一研究試圖回答的問題。我們旨在通過對GNMT的體外及體內(nèi)調(diào)控,觀察其對人前列腺癌細(xì)胞系LNCap和PC3的分化、凋亡、增殖、侵襲性以及成瘤能力的影響,從而對前列腺癌的發(fā)生機制及治療途徑進行新的探索。第一部分 GNMT在人前列腺癌細(xì)胞系LNCap和PC3中的表達目的：研究不同侵襲性的人前列腺癌細(xì)胞系LNCap和PC3中GNMT的表達差異,分析GNMT與前列腺癌細(xì)胞侵襲性的關(guān)系。方法：RT-PCR檢測LNCap和PC3細(xì)胞內(nèi)GNMTmRNA的表達,Western Blot檢測兩種細(xì)胞內(nèi)GNMT蛋白的表達。結(jié)果：LNCap細(xì)胞中GNMTmRNA的相對表達量為(1.000±0.331),PC3細(xì)胞中GNMT mRNA的相對表達量為(2.579±0.863),二者的差異有統(tǒng)計學(xué)意義,p0.05。LNCap細(xì)胞中GNMT蛋白的相對表達量為(0.341±0.072),PC3細(xì)胞中3NMT蛋白的相對表達量為(0.841±0.116),二者的差異有顯著統(tǒng)計學(xué)意義p0.01。結(jié)論：GNMT的表達量和前列腺癌細(xì)胞系的侵襲性相關(guān),侵襲性較高的PC3細(xì)胞中表達較高,侵襲性較低的LNCap細(xì)胞中表達較低。第二部分 GNMT的體外調(diào)控對人前列腺癌細(xì)胞系LNCap和PC3生物學(xué)特性的影響目的：研究GNMT的體外調(diào)控對人前列腺癌細(xì)胞系LNCap和PC3的分化、凋亡、增殖和侵襲性等各項生物學(xué)特性的影響。方法：細(xì)胞培養(yǎng)液中分別加入GNMT (10μg/ml)和SAH (10ng/ml)來上調(diào)和下調(diào)LNCap和PC3細(xì)胞內(nèi)GNMT的活性。在12h、24h、36h和48h這4個時間點,光鏡下觀察細(xì)胞分化,TUNEL和流式細(xì)胞法檢測細(xì)胞凋亡,MTT法檢測細(xì)胞增殖,Transwell實驗檢測細(xì)胞侵襲性。結(jié)果：①光鏡觀察第48h,GNMT和SAH處理過的LNCap和PC3細(xì)胞形態(tài)在200倍光鏡下觀察,均沒有發(fā)生明顯變化,延長培養(yǎng)時間至5d后結(jié)果仍是一樣。②TUNEL兩種細(xì)胞中凋亡率,GNMT組均低于對照組,SAH組均高于對照組。其差異在LNCap細(xì)胞中GNMT組和對照組之間,第48h無統(tǒng)計學(xué)意義,p0.05,第12h、24h和36h有統(tǒng)計學(xué)意義,p0.05; SAH組和對照組之間,4個時間點均有統(tǒng)計學(xué)意義,p0.05。在PC3細(xì)胞中GNMT組和對照組之間,第12h和48h無統(tǒng)計學(xué)意義,p0.05,第24h和36h有統(tǒng)計學(xué)意義,p0.05; SAH組和對照組之間,第12h無統(tǒng)計學(xué)意義,p0.05,第24h、36h和48h有統(tǒng)計學(xué)意義,p0.05。③流式細(xì)胞兩種細(xì)胞中凋亡率,GNMT組均低于對照組,SAH組均高于對照組。其差異在LNCap細(xì)胞中3組之間兩兩比較,4個時間點均有顯著統(tǒng)計學(xué)意義,p0.01。在PC3細(xì)胞中GNMT組和對照組之間,第48h無統(tǒng)計學(xué)意義,p0.05,第12h、24h和36h有統(tǒng)計學(xué)意義,p0.05; SAH組和對照組之間,4個時間點均有顯著統(tǒng)計學(xué)意義,p0.01。 ④MTT兩種細(xì)胞中吸光度值,GNMT組均高于對照組,SAH組均低于對照組。其差異只在PC3細(xì)胞中的48h時間點SAH組與對照組之間有統(tǒng)計學(xué)意義,p0.05。⑤Transwell兩種細(xì)胞中穿膜細(xì)胞數(shù),GNMT組均高于對照組,SAH組均低于對照組。其差異在兩種細(xì)胞中均在36h和48h時間點才有統(tǒng)計學(xué)意義,p0.05。結(jié)論：在細(xì)胞培養(yǎng)液中分別加入GNMT和SAH,可以實現(xiàn)對GNMT的體外調(diào)控,可以改變前列腺癌細(xì)胞系LNCap和PC3除細(xì)胞分化以外的各項生物學(xué)特性。GNMT活性上調(diào)表現(xiàn)出的作用：可以抑制LNCap和PC3細(xì)胞凋亡,并且程度與細(xì)胞的雄激素依賴特性無關(guān)；對LNCap和PC3細(xì)胞的增殖沒有明顯影響；可以增加前列腺癌細(xì)胞,尤其是PC3細(xì)胞的侵襲性。GNMT活性下調(diào)表現(xiàn)出的作用：可以促進LNCap和PC3細(xì)胞凋亡,并且程度與細(xì)胞的雄激素依賴特性無關(guān)；可以抑制PC3細(xì)胞增殖,但是不能抑制LNCap細(xì)胞增殖；可以減弱LNCap和PC3細(xì)胞的侵襲性,并且程度與細(xì)胞的雄激素依賴特性無關(guān)。第三部分 GNMT的體內(nèi)調(diào)控對人前列腺癌細(xì)胞系PC3成瘤作用的影響目的：研究GNMT的體內(nèi)調(diào)控對PC3細(xì)胞在裸鼠體內(nèi)成瘤能力的影響。方法：將人前列腺癌細(xì)胞系PC3注入裸鼠皮下成瘤。隨機分為3組,2個實驗組每天分別喂食順式維甲酸(CRA,30μmol/kg)口S-腺苷高半胱氨酸(SAH, 10g/kg),對照組正常飲食,連續(xù)6周,每周測量腫瘤的大小,繪制腫瘤生長曲線,處死后免疫組化檢測瘤體內(nèi)GNMT的表達。結(jié)果：6周后,腫瘤體積CRA組比對照組明顯增大,差異有統(tǒng)計學(xué)意義,p0.05；SAH組比對照組明顯縮小,差異有統(tǒng)計學(xué)意義,p0.05。瘤體內(nèi)GNMT表達CRA組比對照組明顯增加,差異有統(tǒng)計學(xué)意義(陽性細(xì)胞率差異,p0.01；累積光密度差異,p0.05)；SAH組比對照組明顯減少,差異有統(tǒng)計學(xué)意義(陽性細(xì)胞率差異,p0.01；累積光密度差異,p0.05)。結(jié)論：通過飲食攝入CRA和SAH可以改變荷瘤裸鼠PC3細(xì)胞所致人前列腺癌腫瘤的生長。這一影響可能是通過調(diào)控腫瘤內(nèi)GNMT的活性和表達實現(xiàn)的,攝入CRA可以上調(diào)GNMT從而促進腫瘤生長；攝入SAH可以下調(diào)GNMT從而抑制腫瘤生長。
[Abstract]:The incidence of prostate cancer is increasing worldwide and in our country. Many molecular biological and genetic mechanisms dominate the process of the disease, such as.Sreekumar, and so on. By metabonomics, it is found that muscle ammonia is closely related to the development and progression of prostate cancer. It is especially an aggressive prostatic cancer in prostate cancer. The concentration in the adenocarcinoma tissue is significantly higher than that in the benign prostatic hyperplasia tissue, and further studies have found that the addition of mylysine to the benign prostatic hyperplasia cell line can make the cells invasive. The concentration of creatine in the tissue cells is changed by the regulation of glycine N- methyltransferase (GNMT). The latest study It is found that prostate cancer cells express more IGNMT than normal prostatic epithelial cells, and knockout GNMT gene can inhibit the proliferation of prostate cancer cells and induce apoptosis. Therefore, the regulation of GNMT expression or function may provide a new idea for the study of the pathogenesis and progression of prostate cancer and the treatment methods,.GNMT as a new way of thinking. A protein with multiple functions, the activity of its methyltransferase can be inhibited by its catalytic product S- adenosine homocysteine (SAH), which can be observed in vitro and in vivo, and SAH can also inhibit the expression of GNMT at transcriptional level. Retinoic acid (RA) can significantly increase the activity of GNMT in mice. Therefore, we can try to use SAH and RA to regulate the expression and activity of GNMT in vitro and in vivo, so that we can do further research. There is no research on whether exogenous GNMT can change the biological characteristics of prostate cancer cells, and this is our study. To answer the question, we aim to observe the effects of GNMT on the differentiation, apoptosis, proliferation, invasiveness and tumorigenicity of human prostate cancer cell lines, LNCap and PC3 in vitro and in vivo, and to explore the mechanism and treatment of prostate cancer. The first part of GNMT is in the human prostate cancer cell line LNCap and in the first part of the human prostate cancer cell line LNCap PC3 expression objective: To study the difference of GNMT expression in LNCap and PC3 of different invasive human prostate cancer cell lines and to analyze the relationship between GNMT and the invasiveness of prostate cancer cells. Methods: RT-PCR was used to detect the expression of GNMTmRNA in LNCap and PC3 cells. Western Blot detected the expression of two kinds of intracellular GNMT proteins. The relative expression amount was (1 + 0.331), the relative expression of GNMT mRNA in PC3 cells was (2.579 + 0.863), the difference between the two was statistically significant, the relative expression of GNMT protein in p0.05.LNCap cells was (0.341 + 0.072), and the relative expression of 3NMT protein in PC3 cells was (0.841 + 0.116), and the differences of two were statistically significant p0.01. knots. The expression of GNMT is associated with the invasiveness of the prostate cancer cell lines, high expression of PC3 cells with higher invasiveness and low expression in the less invasive LNCap cells. The effect of the second part of GNMT on the biological characteristics of LNCap and PC3 in human prostate cancer cell lines: the study of GNMT in vitro regulation of human prostate cancer The effects of the differentiation, apoptosis, proliferation and invasiveness of cell lines LNCap and PC3. Methods: GNMT (10 mu g/ml) and SAH (10ng/ml) were added to the cell culture solution to up and down the activity of GNMT in LNCap and PC3 cells. At the 4 time points of 12h, 24h, 36h, and PC3, the cell differentiation was observed under light microscope, and the flow cytometry was observed. Detection of cell apoptosis, MTT assay to detect cell proliferation and Transwell test to detect cell invasiveness. Results: (1) the morphology of LNCap and PC3 cells treated by 48h, GNMT and SAH was observed under 200 times of light microscope, and no obvious changes were observed, and the same results were found after prolonging the incubation time to 5D. (2) the apoptotic rate in the TUNEL two cells and the GNMT group were all The difference between group SAH and control group was higher than that of control group. The difference between group GNMT and control group in LNCap cells was not statistically significant, P0.05, 12h, 24h and 36h were statistically significant, P0.05, between the SAH group and the control group, the 4 time points were statistically significant, p0.05. was between the PC3 cells and the control group, and there was no statistical difference. Significance, P0.05, 24h and 36h were statistically significant, P0.05, SAH group and control group, 12h had no statistical significance, P0.05, 24h, 36h and 48h were statistically significant, p0.05. 3 cell apoptosis rate in the two cells, GNMT groups were higher than those in the control group. The difference between the 3 groups was 22 and 4 times. There was significant statistical significance. There was no statistical significance in p0.01. between group GNMT and control group in PC3 cells. P0.05, 12h, 24h and 36h were statistically significant, and there were significant statistical significance in the 4 time points between the SAH group and the control group. The absorbance value of the p0.01. MTT two cells was higher than that of the control group, and the groups were all lower than those in the control group. In the control group, the difference was only statistically significant between the 48h time point SAH group and the control group in the PC3 cells. The number of membrane cells in the p0.05. Transwell two cells, the GNMT group were all higher than the control group, and the SAH group were all lower than the control group. The difference between the two cells was statistically significant at 36h and 48h time points, p0.05. conclusion: in cell culture The addition of GNMT and SAH, respectively, can be used to regulate and regulate GNMT in vitro, which can change the role of the biological properties of prostate cancer cell lines, LNCap and PC3, to inhibit the apoptosis of.GNMT and LNCap and PC3 cells, and the degree is not related to the androgen dependence of the cells; and LNCap and PC3 are fine. Cell proliferation has no obvious effect; it can increase the downregulation of invasive.GNMT activity of prostate cancer cells, especially PC3 cells: it can promote apoptosis of LNCap and PC3 cells, and is not related to the androgen dependence of cells; it can inhibit the proliferation of PC3 cells, but can not inhibit the proliferation of LNCap cells; can reduce the proliferation of cells; can reduce the proliferation of cells; The invasiveness of weak LNCap and PC3 cells and irrelevant to the androgen dependence of cells. Third the effect of part GNMT on human prostate cancer cell line PC3 tumorigenesis purpose: To study the effect of GNMT in vivo regulation of PC3 cells on the tumorigenicity of PC3 cells in nude mice. The mice were subcutaneously divided into 3 groups. The 2 experimental groups were fed with S- adenosine homocysteine (SAH, 10g/kg) at the mouth of CRA, 30 mu mol/kg. The control group had a normal diet for 6 weeks. The tumor size was measured every week and the tumor growth curve was plotted. After death, the expression of GNMT in the tumor was detected by immunohistochemistry. Results: 6 weeks after the tumor, the tumor body was detected. CRA group was significantly higher than the control group, the difference was statistically significant, P0.05, SAH group was significantly smaller than the control group, and the difference was statistically significant. The GNMT expression of CRA group in p0.05. tumor was significantly higher than the control group, the difference was statistically significant (the positive cell rate difference, P0.01; the cumulative light density difference, P0.05), and the SAH group was significantly less than the control group, and the difference was significantly lower than the control group. There is statistical significance (positive cell rate difference, P0.01; cumulative light density difference, P0.05). Conclusion: dietary intake of CRA and SAH can change the growth of human prostate cancer induced by PC3 cells in nude mice. This effect may be achieved by regulating the activity and expression of GNMT in the tumor. The intake of CRA can increase GNMT and thus promote the growth of the tumor. Tumor growth; ingestion of SAH can downregulate GNMT and inhibit tumor growth.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R737.25

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