本文選題：BRE　切入點(diǎn)：蛋白質(zhì)組學(xué)　出處：《汕頭大學(xué)》2007年碩士論文

【摘要】： 背景和目的： “Brain and Reproductive Organ-Expressed”(BRE)基因，首次發(fā)現(xiàn)于1995年。最初的研究發(fā)現(xiàn)BRE基因在小鼠的大腦、睪丸、卵巢中高表達(dá)，故命名之。隨后研究發(fā)現(xiàn)，BRE基因廣泛表達(dá)；其功能涉及到應(yīng)激反應(yīng)、凋亡、腫瘤生長(zhǎng)和類固醇激素合成等方面。目前，BRE基因具體作用機(jī)制尚不清楚。本研究首先利用RNAi技術(shù)，探討B(tài)RE基因在肝細(xì)胞系中的功能；進(jìn)一步以BRE轉(zhuǎn)基因小鼠肝臟為研究對(duì)象，利用蛋白質(zhì)組學(xué)技術(shù)探討B(tài)RE蛋白與其他蛋白質(zhì)之間的關(guān)系，旨在研究BRE蛋白在機(jī)體中的作用機(jī)制。 材料和方法： (1) RNAi抑制人正常肝細(xì)胞系-Chang細(xì)胞BRE基因表達(dá)，相差顯微鏡觀察活細(xì)胞形態(tài)、MTT法和BrdU摻入法檢測(cè)細(xì)胞增殖、流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡情況的改變。 (2)以BRE轉(zhuǎn)基因小鼠和其對(duì)照小鼠肝臟為對(duì)象，HE染色后測(cè)量肝細(xì)胞和細(xì)胞核的周長(zhǎng)及面積；利用蛋白質(zhì)組學(xué)方法研究BRE基因?qū)π∈蟾闻K蛋白質(zhì)譜的影響。 (3)以BRE轉(zhuǎn)基因小鼠以及BRE基因干擾后的Chang細(xì)胞為材料，RT-PCR方法從正反兩方面印證蛋白質(zhì)組學(xué)實(shí)驗(yàn)結(jié)果。 (4)實(shí)驗(yàn)數(shù)據(jù)用SPSS13.0統(tǒng)計(jì)軟件進(jìn)行分析處理。 結(jié)果： (1) BRE基因在BRE轉(zhuǎn)基因小鼠和ctl-siRNA處理的Chang細(xì)胞中高表達(dá)。 (2) BRE轉(zhuǎn)基因小鼠與其對(duì)照組小鼠肝細(xì)胞、RNAi處理前后Chang細(xì)胞的形態(tài)學(xué)分析顯示其形態(tài)沒有差別；RNAi處理后Chang細(xì)胞的MTT實(shí)驗(yàn)結(jié)果及RNAi處理后Chang細(xì)胞的BrdU實(shí)驗(yàn)結(jié)果顯示細(xì)胞在BRE基因干擾前后的增殖沒有差異；RNAi處理后Chang細(xì)胞凋亡減少。 (3)成功建立了BRE轉(zhuǎn)基因小鼠和其對(duì)照組小鼠的肝臟蛋白質(zhì)雙向凝膠電泳圖譜，多數(shù)蛋白集中于pH 5～7、分子量20～80KDa范圍。對(duì)照組和BRE轉(zhuǎn)基因組電泳圖譜的平均蛋白質(zhì)點(diǎn)數(shù)分別為804±154、798±144；對(duì)照組與實(shí)驗(yàn)組平均匹配率為89.75％。對(duì)比發(fā)現(xiàn)多個(gè)蛋白質(zhì)點(diǎn)表達(dá)有差異，經(jīng)數(shù)據(jù)庫查詢鑒定出15個(gè)差異蛋白質(zhì)。 (4) RT-PCR檢測(cè)HSP70-1、HSP70-2、Ubi-d4和G21在BRE轉(zhuǎn)基因小鼠肝臟和ctl-siRNA處理的Chang細(xì)胞中高表達(dá)，α-烯醇化酶在對(duì)照組小鼠肝臟組織和BRE-siRNA處理的Chang細(xì)胞中高表達(dá)。 結(jié)論： (1)高重復(fù)性的2-DE凝膠圖和MALD-TOF-MS結(jié)果表明蛋白質(zhì)組學(xué)結(jié)果是可靠的。 (2) BRE基因不能改變肝細(xì)胞的形態(tài)；BRE基因?qū)hang細(xì)胞的增殖沒有影響；BRE基因可以促進(jìn)Chang細(xì)胞凋亡。 (3) BRE基因可以上調(diào)HSP70-1和HSP70-2的表達(dá)，，或許通過HSP70在應(yīng)激反應(yīng)中發(fā)揮作用及促進(jìn)細(xì)胞凋亡。 (4) BRE基因可以調(diào)節(jié)α-烯醇化酶、Ubi-d4和G21的表達(dá)，可能在能量生成、腫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 鹵154798 鹵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 偽 -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 偽 -enolase Ubi-d4 and G21, which may play an important role in energy generation and tumorigenesis.
【學(xué)位授予單位】：汕頭大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R346

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