慢病毒介导的RTN4基因敲除对前列腺癌细胞增殖和衰老的影响

发布时间：2018-05-15 21:37

本文选题：前列腺癌细胞 + RTN4　；参考：《福建医科大学》2016年硕士论文

【摘要】：目的:前列腺癌是泌尿生殖系统的主要恶性肿瘤之一。在欧美前列腺癌是男性最易发生的肿瘤,其死亡率仅次于肺癌。在我国前列腺癌的检出率和发病率呈逐年上升趋势。在前期的研究中发现,RTN4的表达在前列腺癌与正常前列腺组织之间存在差异。但是,RTN4在前列腺癌细胞中的表达及其作用目前尚无研究。本研究通过体外实验探讨浆膜蛋白(Reticulon-4,RTN4)基因对前列腺癌细胞增殖和衰老的影响。方法:(1)采用实时荧光定量PCR和Western blotting技术检测RTN4在前列腺癌细胞株PC3,DU145和LNCaP中mRNA和蛋白的表达。(2)通过重组质粒,瞬时转染,将携带RTN4 shRNA和shCon的慢病毒载体转染前列腺癌细胞,建立稳定沉默表达RTN4的细胞株和对照组细胞株,分别用实时荧光定量PCR技术检测前列腺癌细胞中RTN4 mRNA的表达。采用MTT法、平板克隆形成实验、FCM法、PI法和β-半乳糖苷酶(senescence-associated betaga1actosidase,SA-β-Ga1)染色等方法检测细胞的增殖、细胞周期和细胞衰老情况。结果:采用实时荧光定量PCR和Western blotting技术检测RTN4在前列腺癌细胞株PC3,DU145和LNCaP中的表达情况。结果显示:RTN4在前列腺癌细胞中均可表达。稳定沉默表RTN4的细胞株构建成功后,采用实时荧光定量PCR技术检测RTN4在前列腺癌细胞中表达情况。结果显示:与scrambled组和空白对照组相比,实验组水平明显降低(P0.01)。采用MTT法和FCM法检测细胞增殖,结果显示:与scrambled组和空白对照组相比,转染后LV-shRTN4组细胞的增殖力受到抑制,细胞数明显减少,克隆形成能力下降(P0.01)。采用PI法检测细胞周期变化,结果显示:与scrambled组和空白对照组相比,转染后LV-shRTN4组细胞所含DNA在S期的比例明显增加(P0.01)。采用β-半乳糖苷酶染色法检测细胞衰老变化,结果显示:与scrambled组和空白对照组相比,转染后LV-shRTN4组衰老细胞所占百分数明显增多(P0.01)。结论:(1)RTN4 mRNA在前列腺癌细胞株PC3,DU145和LNCaP均有表达,提示RTN4可能在前列腺癌的发生发展中发挥作用。(2)沉默表达PC3细胞中RTN4基因,细胞生长受到抑制,可抑制人前列腺癌细胞的增殖力、加速前列腺癌细胞衰老。进一步证实了RTN4可能在PC3发挥作用,RTN4可能成为治疗前列腺癌的新靶点。
[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.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.25

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