肝细胞中BRE基因功能的初步研究

发布时间：2018-03-27 05:32

本文选题：BRE　切入点：蛋白质组学　出处：《汕头大学》2007年硕士论文

【摘要】： 背景和目的： “Brain and Reproductive Organ-Expressed”(BRE)基因，首次发现于1995年。最初的研究发现BRE基因在小鼠的大脑、睾丸、卵巢中高表达，故命名之。随后研究发现，BRE基因广泛表达；其功能涉及到应激反应、凋亡、肿瘤生长和类固醇激素合成等方面。目前，BRE基因具体作用机制尚不清楚。本研究首先利用RNAi技术，探讨BRE基因在肝细胞系中的功能；进一步以BRE转基因小鼠肝脏为研究对象，利用蛋白质组学技术探讨BRE蛋白与其他蛋白质之间的关系，旨在研究BRE蛋白在机体中的作用机制。材料和方法： (1) RNAi抑制人正常肝细胞系-Chang细胞BRE基因表达，相差显微镜观察活细胞形态、MTT法和BrdU掺入法检测细胞增殖、流式细胞术检测细胞凋亡情况的改变。 (2)以BRE转基因小鼠和其对照小鼠肝脏为对象，HE染色后测量肝细胞和细胞核的周长及面积；利用蛋白质组学方法研究BRE基因对小鼠肝脏蛋白质谱的影响。 (3)以BRE转基因小鼠以及BRE基因干扰后的Chang细胞为材料，RT-PCR方法从正反两方面印证蛋白质组学实验结果。 (4)实验数据用SPSS13.0统计软件进行分析处理。结果： (1) BRE基因在BRE转基因小鼠和ctl-siRNA处理的Chang细胞中高表达。 (2) BRE转基因小鼠与其对照组小鼠肝细胞、RNAi处理前后Chang细胞的形态学分析显示其形态没有差别；RNAi处理后Chang细胞的MTT实验结果及RNAi处理后Chang细胞的BrdU实验结果显示细胞在BRE基因干扰前后的增殖没有差异；RNAi处理后Chang细胞凋亡减少。 (3)成功建立了BRE转基因小鼠和其对照组小鼠的肝脏蛋白质双向凝胶电泳图谱，多数蛋白集中于pH 5～7、分子量20～80KDa范围。对照组和BRE转基因组电泳图谱的平均蛋白质点数分别为804±154、798±144；对照组与实验组平均匹配率为89.75％。对比发现多个蛋白质点表达有差异，经数据库查询鉴定出15个差异蛋白质。 (4) RT-PCR检测HSP70-1、HSP70-2、Ubi-d4和G21在BRE转基因小鼠肝脏和ctl-siRNA处理的Chang细胞中高表达，α-烯醇化酶在对照组小鼠肝脏组织和BRE-siRNA处理的Chang细胞中高表达。结论： (1)高重复性的2-DE凝胶图和MALD-TOF-MS结果表明蛋白质组学结果是可靠的。 (2) BRE基因不能改变肝细胞的形态；BRE基因对Chang细胞的增殖没有影响；BRE基因可以促进Chang细胞凋亡。 (3) BRE基因可以上调HSP70-1和HSP70-2的表达，，或许通过HSP70在应激反应中发挥作用及促进细胞凋亡。 (4) BRE基因可以调节α-烯醇化酶、Ubi-d4和G21的表达，可能在能量生成、肿瘤生成等方面中起作用。
[Abstract]:Background and purpose:. The "Brain and Reproductive Organ-Expressed" gene was first discovered in 1995.The initial study found that the BRE gene is highly expressed in the brain, testis and ovaries of mice, so it is named. Tumor growth and steroid hormone biosynthesis. At present, the specific mechanism of re gene is not clear. In this study, the function of BRE gene in liver cell line was studied by RNAi technique, and the liver of BRE transgenic mice was further studied. The relationship between BRE protein and other proteins was studied by proteomics in order to study the mechanism of BRE protein in organism. Materials and methods:. 1) RNAi inhibited the expression of BRE gene in human normal liver cell line -Chang. The morphology of living cells was observed by phase contrast microscopy and the proliferation of cells was detected by BrdU incorporation method. The apoptosis of the cells was detected by flow cytometry. (2) the liver of BRE transgenic mice and control mice were stained with HE to measure the perimeter and area of hepatocytes and nuclei, and the effect of BRE gene on the liver protein profile of mice was studied by proteomics. BRE transgenic mice and Chang cells interfered by BRE gene were used as materials to confirm the results of proteomics from both positive and negative aspects. 4) the experimental data are analyzed and processed by SPSS13.0 statistical software. Results:. BRE gene was overexpressed in BRE transgenic mice and Chang cells treated with ctl-siRNA. (2) morphological analysis of Chang cells in BRE transgenic mice and control mice before and after Chang treatment showed that there was no difference in morphology between Chang cells and Chang cells treated with RNAi. There was no difference in cell proliferation before and after interference of BRE gene. Apoptosis of Chang cells was decreased after treated with RNAi. A two-dimensional gel electrophoresis map of liver protein in BRE transgenic mice and control mice was successfully established. Most of the proteins were concentrated in pH 5 ~ 7 and molecular weight 20~80KDa range. The average number of protein in the electrophoretic map of control group and BRE transgenic group was 804 卤154798 卤144, respectively, and the average matching rate between control group and experimental group was 89.7575. The results showed that there were differences in the expression of multiple protein spots between the control group and the experimental group. Fifteen differential proteins were identified by database query. RT-PCR was used to detect the overexpression of HSP70-1HSP70-2Ubi-d4 and G21 in the liver of BRE transgenic mice and Chang cells treated with ctl-siRNA, and the overexpression of 伪 -enolase in the liver of control mice and Chang cells treated with BRE-siRNA. Conclusion:. The high reproducibility of 2-DE gel and MALD-TOF-MS showed that the proteomics results were reliable. (2) BRE gene could not change the morphology of hepatocytes and had no effect on the proliferation of Chang cells. It could promote the apoptosis of Chang cells. BRE gene can up-regulate the expression of HSP70-1 and HSP70-2, which may play a role in stress response and promote apoptosis through HSP70. (4) BRE gene can regulate the expression of 伪 -enolase Ubi-d4 and G21, which may play an important role in energy generation and tumorigenesis.
【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346

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