本文選題：前列腺癌細(xì)胞 + RTN4��； 參考：《福建醫(yī)科大學(xué)》2016年碩士論文

【摘要】：目的:前列腺癌是泌尿生殖系統(tǒng)的主要惡性腫瘤之一。在歐美前列腺癌是男性最易發(fā)生的腫瘤,其死亡率僅次于肺癌。在我國(guó)前列腺癌的檢出率和發(fā)病率呈逐年上升趨勢(shì)。在前期的研究中發(fā)現(xiàn),RTN4的表達(dá)在前列腺癌與正常前列腺組織之間存在差異。但是,RTN4在前列腺癌細(xì)胞中的表達(dá)及其作用目前尚無(wú)研究。本研究通過(guò)體外實(shí)驗(yàn)探討漿膜蛋白(Reticulon-4,RTN4)基因?qū)η傲邢侔┘?xì)胞增殖和衰老的影響。方法:(1)采用實(shí)時(shí)熒光定量PCR和Western blotting技術(shù)檢測(cè)RTN4在前列腺癌細(xì)胞株P(guān)C3,DU145和LNCaP中mRNA和蛋白的表達(dá)。(2)通過(guò)重組質(zhì)粒,瞬時(shí)轉(zhuǎn)染,將攜帶RTN4 shRNA和shCon的慢病毒載體轉(zhuǎn)染前列腺癌細(xì)胞,建立穩(wěn)定沉默表達(dá)RTN4的細(xì)胞株和對(duì)照組細(xì)胞株,分別用實(shí)時(shí)熒光定量PCR技術(shù)檢測(cè)前列腺癌細(xì)胞中RTN4 mRNA的表達(dá)。采用MTT法、平板克隆形成實(shí)驗(yàn)、FCM法、PI法和β-半乳糖苷酶(senescence-associated betaga1actosidase,SA-β-Ga1)染色等方法檢測(cè)細(xì)胞的增殖、細(xì)胞周期和細(xì)胞衰老情況。結(jié)果:采用實(shí)時(shí)熒光定量PCR和Western blotting技術(shù)檢測(cè)RTN4在前列腺癌細(xì)胞株P(guān)C3,DU145和LNCaP中的表達(dá)情況。結(jié)果顯示:RTN4在前列腺癌細(xì)胞中均可表達(dá)。穩(wěn)定沉默表RTN4的細(xì)胞株構(gòu)建成功后,采用實(shí)時(shí)熒光定量PCR技術(shù)檢測(cè)RTN4在前列腺癌細(xì)胞中表達(dá)情況。結(jié)果顯示:與scrambled組和空白對(duì)照組相比,實(shí)驗(yàn)組水平明顯降低(P0.01)。采用MTT法和FCM法檢測(cè)細(xì)胞增殖,結(jié)果顯示:與scrambled組和空白對(duì)照組相比,轉(zhuǎn)染后LV-shRTN4組細(xì)胞的增殖力受到抑制,細(xì)胞數(shù)明顯減少,克隆形成能力下降(P0.01)。采用PI法檢測(cè)細(xì)胞周期變化,結(jié)果顯示:與scrambled組和空白對(duì)照組相比,轉(zhuǎn)染后LV-shRTN4組細(xì)胞所含DNA在S期的比例明顯增加(P0.01)。采用β-半乳糖苷酶染色法檢測(cè)細(xì)胞衰老變化,結(jié)果顯示:與scrambled組和空白對(duì)照組相比,轉(zhuǎn)染后LV-shRTN4組衰老細(xì)胞所占百分?jǐn)?shù)明顯增多(P0.01)。結(jié)論:(1)RTN4 mRNA在前列腺癌細(xì)胞株P(guān)C3,DU145和LNCaP均有表達(dá),提示RTN4可能在前列腺癌的發(fā)生發(fā)展中發(fā)揮作用。(2)沉默表達(dá)PC3細(xì)胞中RTN4基因,細(xì)胞生長(zhǎng)受到抑制,可抑制人前列腺癌細(xì)胞的增殖力、加速前列腺癌細(xì)胞衰老。進(jìn)一步證實(shí)了RTN4可能在PC3發(fā)揮作用,RTN4可能成為治療前列腺癌的新靶點(diǎn)。
[Abstract]:Objective: prostate cancer is one of the major malignant tumors in the genitourinary system. Prostate cancer is the most common cancer in men in Europe and America, and its mortality is second only to lung cancer. The detection rate and incidence of prostate cancer in China are increasing year by year. In previous studies, the expression of RTN4 was found to be different between prostate cancer and normal prostate tissues. However, the expression and role of RTN4 in prostate cancer cells have not been studied. This study was designed to investigate the effects of serosal protein Reticulon-4 (RTN4) gene on the proliferation and senescence of prostate cancer cells in vitro. Methods the expression of mRNA and protein in prostate cancer cell line PC3DU145 and LNCaP was detected by real-time fluorescence quantitative PCR and Western blotting techniques. The lentivirus vector carrying RTN4 shRNA and shCon was transfected into prostate cancer cells by transient transfection with recombinant plasmid. The expression of RTN4 mRNA in prostate cancer cells was detected by real-time fluorescence quantitative PCR. The proliferation, cell cycle and cell senescence of cells were detected by MTT, FCM and 尾 -galactosidase associated betaga1 actosidase SA- 尾 -Ga1 staining. Results: the expression of RTN4 in prostate cancer cell line PC3DU145 and LNCaP was detected by real-time fluorescence quantitative PCR and Western blotting. The results showed that the expression of 10% RTN4 in prostate cancer cells could be detected. The expression of RTN4 in prostate cancer cells was detected by real-time fluorescence quantitative PCR after the successful construction of the cell line of stable silencing table RTN4. The results showed that compared with the scrambled group and the blank control group, the level of the experimental group was significantly lower than that of the control group. MTT and FCM methods were used to detect cell proliferation. The results showed that compared with scrambled group and blank control group, the proliferation of transfected LV-shRTN4 cells was inhibited, the number of cells was significantly decreased, and the ability of clone formation was decreased (P0.01). Pi assay was used to detect the cell cycle changes. The results showed that compared with scrambled group and blank control group, the proportion of DNA in S phase of transfected LV-shRTN4 group was significantly increased (P 0.01). 尾 -galactosidase staining was used to detect the changes of cell senescence. The results showed that compared with scrambled group and blank control group, the percentage of senescent cells in LV-shRTN4 group after transfection was significantly higher than that in LV-shRTN4 group. Conclusion the expression of RTN4 mRNA in prostate cancer cell line PC3nDU145 and LNCaP suggests that RTN4 may play a role in the pathogenesis and development of prostate cancer.) RTN4 silenced the expression of RTN4 gene in PC3 cells. The cell growth was inhibited and the proliferation of human prostate cancer cells was inhibited. Accelerate the aging of prostate cancer cells. It is further confirmed that RTN4 may play a role in PC3 and RTN4 may be a new target for prostate cancer treatment.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R737.25

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