【摘要】：目的:探討蛋白精氨酸甲基轉(zhuǎn)移酶2(protein arginine methyltransferase 2,PRMT2)高表達(dá)對(duì)甲狀腺癌TPC-1細(xì)胞增殖的影響,并初步探討其調(diào)控細(xì)胞周期蛋白D1(Cyclin D1,CCND1)的分子機(jī)制。方法:將已構(gòu)建好的PRMT2慢病毒載體轉(zhuǎn)染TPC-1細(xì)胞,構(gòu)建TPC-1-PRMT2穩(wěn)定細(xì)胞株,采用蛋白免疫印跡(Western Blot)鑒定甲狀腺癌TPC-1-PRMT2穩(wěn)定細(xì)胞株;設(shè)立實(shí)驗(yàn)組即四環(huán)素誘導(dǎo)組(+Dox),陰性對(duì)照組即未加四環(huán)素誘導(dǎo)組(-Dox)。以上述兩組細(xì)胞為實(shí)驗(yàn)對(duì)象,分別采用MTT檢測(cè)方法、克隆形成實(shí)驗(yàn)檢測(cè)各組細(xì)胞的增殖及克隆形成能力;Western Blot檢測(cè)甲狀腺癌中高表達(dá)PRMT2后對(duì)p-Akt、p-GSK-3β、CCND1蛋白表達(dá)的影響,并對(duì)其影響甲狀腺癌細(xì)胞增殖的分子機(jī)制進(jìn)行初步探討。免疫共沉淀實(shí)驗(yàn)判斷在普通甲狀腺癌TPC-1細(xì)胞中,PRMT2與TRβ的相互作用。結(jié)果:1.轉(zhuǎn)染TPC-1細(xì)胞后獲得TPC-1-PRMT2穩(wěn)定細(xì)胞株,經(jīng)四環(huán)素誘導(dǎo)后Western Blot檢測(cè)到PRMT2-3Flag蛋白的表達(dá);2.MTT檢測(cè)及克隆形成實(shí)驗(yàn)表明,PRMT2高表達(dá)后可降低TPC-1細(xì)胞的增殖能力;3.Western Blot檢測(cè)提示,PRMT2高表達(dá)后p-Akt、p-GSK-3β、CCND1的表達(dá)均減少。而p-ERK蛋白的表達(dá)未見(jiàn)明顯減少。且不影響TRβ的表達(dá)。4.免疫共沉淀實(shí)驗(yàn)表明,在普通TPC-1細(xì)胞內(nèi),PRMT2與TRβ存在相互作用。結(jié)論:1.PRMT2高表達(dá)后抑制甲狀腺癌TPC-1細(xì)胞的增殖。2.PRMT2高表達(dá)后抑制甲狀腺癌TPC-1細(xì)胞的增殖,可能通過(guò)調(diào)控Akt/GSK 3β信號(hào)通路,下調(diào)細(xì)胞周期蛋白CCND1表達(dá)水平有關(guān);3.在普通TPC-1細(xì)胞內(nèi),PRMT2能和TRβ發(fā)生相互作用。
[Abstract]:Aim: to investigate the effect of the overexpression of arginine methyltransferase 2 (protein arginine methyltransferase 2 (PRMT2) on the proliferation of thyroid carcinoma TPC-1 cells and the molecular mechanism of its regulation of cyclin D1 (Cyclin D1). Methods: the constructed PRMT2 lentivirus vector was transfected into TPC-1 cells to construct TPC-1-PRMT2 stable cell line. Western blot (Western Blot) was used to identify TPC-1-PRMT2 stable cell line of thyroid carcinoma. Establish experimental group, tetracycline induced group, (Dox), negative control group, that is, no tetracycline induced group (- Dox).) The above two groups of cells were used as experimental objects. The proliferation and clone forming ability of each group were detected by MTT assay and clone formation assay respectively. The effects of high expression of PRMT2 on the expression of p-Akttnp-GSK-3 尾 and CCND1 protein in thyroid carcinoma were detected by Western Blot, and the molecular mechanism of its influence on the proliferation of thyroid cancer cells was discussed. The interaction of PRMT2 and TR 尾 in normal thyroid carcinoma TPC-1 cells was determined by immunoprecipitation assay. The result is 1: 1. After transfection of TPC-1 cells, TPC-1-PRMT2 stable cell lines were obtained, and the expression of PRMT2-3Flag protein was detected by Western Blot induced by tetracycline, 2.MTT detection and clone formation assay showed that high expression of PRMT2 could reduce the proliferation ability of TPC-1 cells. The results of 3.Western Blot showed that the expression of p-Aktbumin p-GSK-3 尾 and CCND1 decreased after high expression of PRMT2. However, the expression of p-ERK protein was not significantly decreased. The expression of TR 尾 was not affected. 4. Immunoprecipitation assay showed that PRMT2 interacted with TR 尾 in normal TPC-1 cells. Conclusion: the high expression of 1.PRMT2 inhibits the proliferation of TPC-1 cells in thyroid carcinoma, and the high expression of 2.PRMT2 inhibits the proliferation of TPC-1 cells of thyroid carcinoma, which may be through the regulation of Akt/GSK 3 尾 signaling pathway. The down-regulation of cyclin CCND1 expression was related to the expression of cyclin CCND1. 3. In normal TPC-1 cells, PRMT2 can interact with TR 尾.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R736.1

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