NF-κB通路論文：CARD結(jié)構(gòu)域?qū)ωiBCL 10表達(dá)及細(xì)胞周期的影響

1、. NF-B通路論文：CARD結(jié)構(gòu)域?qū)ωiBCL 10表達(dá)及細(xì)胞周期的影響【中文摘要】在細(xì)胞的信號轉(zhuǎn)導(dǎo)過程中,NF-B通路可以激活或者抑制核內(nèi)基因的表達(dá),從而將外界的刺激轉(zhuǎn)化成細(xì)胞的信號。在免疫系統(tǒng)中,NF-B在控制抗原受體信號轉(zhuǎn)導(dǎo)通路、調(diào)控誘導(dǎo)核轉(zhuǎn)錄因子中同樣起關(guān)鍵作用。BCL 10是參與NF-B通路的一個重要功能基因。BCL 10正常的表達(dá)水平可以促進(jìn)免疫因子的產(chǎn)生,增強(qiáng)機(jī)體的天然免疫和抵御感染的能力。反之,BCL 10的異常表達(dá),比如過表達(dá)、過度磷酸化和核內(nèi)表達(dá)會導(dǎo)致一系列細(xì)胞因子的非正常表達(dá),從而影響正常細(xì)胞的生理功能,甚至?xí)?dǎo)致淋巴瘤等多種疾病的發(fā)生。已知BCL10的CARD結(jié)構(gòu)域、磷

2、酸化位點(diǎn)以及C末端序列參與幾乎全部蛋白功能的發(fā)揮。參考以上的研究結(jié)果,我們開展豬BCL 10的研究工作。實(shí)驗(yàn)室前期工作:通過電子克隆得到豬BCL 10的全長基因,通過特異性引物在豬脾臟克隆到該基因片段,并得到該基因原核表達(dá)蛋白,掌握了其原核表達(dá)特點(diǎn)。同時,檢測結(jié)果顯示,豬BCL 10基因mRNA在脾臟中表達(dá)最高,胸腺、大腦和下頜淋巴結(jié)表達(dá)次之,肝臟只有微量表達(dá),腎臟沒有表達(dá),初步獲得該基因在豬體內(nèi)的表達(dá)情況。本研究進(jìn)一步探討豬BCL 10的CARD結(jié)構(gòu)域相關(guān)功能,通過該基因序列分析和真核表達(dá)觀測,取得以下結(jié)果:1.根據(jù)豬BCL 10基因的特點(diǎn),通過激酶的作用位點(diǎn)和空間結(jié)構(gòu)的預(yù)測,找到了可能與其

3、核內(nèi)表達(dá)相關(guān)的位點(diǎn),并成功構(gòu)建出完整豬BCL 10基因的真核表達(dá)載體pEGFP-B。同時,通過大引物擴(kuò)增法構(gòu)建出真核表達(dá)載體pEGFP-D(缺失CARD結(jié)構(gòu)域的豬BCL 10基因)和pEGFP-P(缺失Thr-Ser-Ser位點(diǎn)的豬BCL 10基因)。2.轉(zhuǎn)染豬臍靜脈血管內(nèi)皮細(xì)胞系(SUVEC)后,Western Blot檢測表明融合蛋白得到表達(dá)。分析發(fā)現(xiàn),TSS位點(diǎn)(Thr-Ser-Ser)參與豬BCL 10的CARD結(jié)構(gòu)域與其它蛋白的相互作用,是該基因的重要位點(diǎn)。同時,TSS位點(diǎn)的缺失會導(dǎo)致BCL 10出現(xiàn)核內(nèi)表達(dá)。這些結(jié)果證實(shí),TSS在豬BCL 10基因中具有重要作用。3.細(xì)胞周期變化測

4、定表明,豬BCL 10的CARD結(jié)構(gòu)域會引起細(xì)胞的G 1期縮短,S期和G 2/M期延長,表明該結(jié)構(gòu)域在細(xì)胞生長過程中影響DNA的復(fù)制以及細(xì)胞有絲分裂,過度表達(dá)會導(dǎo)致細(xì)胞在有絲分裂前期停滯積累,但不會導(dǎo)致細(xì)胞凋亡。從而為進(jìn)一步研究豬BCL 10蛋白在信號轉(zhuǎn)導(dǎo)中的作用特點(diǎn),以及淋巴瘤等疾病的診斷與治療奠定基礎(chǔ)?！居⑽恼縉F-B pathway can activate or inhibit the expression of nucleus genes to transfer the external stimuli into the cell signal during the proces

5、s of cell signal transduction. In the immune system, NF-B plays a similar role in the controlling the pathway of antigen receptor signaling transduction as well as the regulation and induction of nucleus transcription factors. BCL 10 is an important functional gene involving in NF-B pathway and its

6、normal expression can promote the production of immune factors, enhance innate immunity and defence capacity. Conversely, the abnormal expression of BCL 10, such as over-expression, over-phosphorylation and nucleus expression will lead to abnormal expression of a series of cytokines, thereby affecti

7、ng the physiological function of normal cells and even resulting in various diseases including lymphoma. It is well known that CARD domain, phosphorylation sites, and the C terminal sequence of BCL10 are involved in the fuctions of almost all of the protein. And the research of our group on swine BC

8、L 10 was carried out based on the previous research.In a previous work of our group, full-length gene of swine BCL 10 was obtained by in silico cloning and coding sequence of this gene were cloned from swine spleen using specific primer. And its protein of prokaryotic expression was obtained and cha

9、racterized. Scanning the expression profile of BCL 10 in vivo in swine indicated that the highest mRNA expression of swine BCL 10 gene was observed in spleen, followed by thymus, brain, and mandibular lymph node. Comparatively, there was only a trace of mRNA expression and no expression in kidneys.O

10、ur present study explored CARD domain-related functions of swine BCL 10 gene through the analysis of gene sequence and eukaryotic expression. And our results obtained in the present work are as follows:1. According to the characteristics of swine BCL10 gene, the possible sites related to nuclear exp

11、ression was found through the prediction of the kinase action sites and the spatial structure prediction. And the vector pEGFP-B was successfully constructed for complete swine BCL10 gene, meanwhile the vector pEGFP-D(deleting swine BCL 10 gene in CARD structure domain) and pEGFP-P(deleting swine BC

12、L 10 gene at Thr- Ser-Ser site) were constructed through site-mutant PCR method (large primers amplification method). 2. After the above three vectors were transfected into swine umbilical vein endothelial cells (SUVEC), the analysis of Western Blot showed that the fusion protein was expressed. Our

13、analysis showed that TSS sites (Thr-Ser-Ser) involved in CARD domain of swine BCL 10 and interacted with other proteins, which are important sites of the gene. Meanwhile, deleting TSS sites will lead to nucleus expression of BCL 10, thus proving the function of TSS in swine BCL 10 gene.3. The analys

14、is of cell cycle showed that CARD domain of swine BCL 10 would cause shortening of G 1 phase but prolonging of S phase and G 2/M phase, which indicates that domain structure affects DNA replication and cell mitosis, and excessive expression will lead to cell arrest in early stage of cell division, b

15、ut will not lead to cell apoptosis. This lays a good foundation for swine BCL 10 function in cell signal transduction as well as diagnosis and treatment of disease such as lymphoma, etc.【關(guān)鍵詞】NF-B通路 BCL 10基因 核內(nèi)表達(dá) TSS位點(diǎn) CARD結(jié)構(gòu)域【英文關(guān)鍵詞】NF-B pathway BCL 10 gene nuclear expression TSS sites CARD domain【目錄

16、】CARD結(jié)構(gòu)域?qū)ωiBCL 10表達(dá)及細(xì)胞周期的影響 摘要 6-7 ABSTRACT 7-8 文獻(xiàn)綜述 11-20 第一章 BCL 10 的研究進(jìn)展 11-20 1.1 BCL 10 蛋白結(jié)構(gòu)與功能特點(diǎn) 11-12 1.2 BCL 10 與 NF-B 信號通路 12-16 1.2.1 NF-B 相關(guān)蛋白分子 12-13 1.2.2 NF-B 信號通路 13-15 1.2.3 BCL10 參與NF-B 通路 15-16 1.3 BCL10 與細(xì)胞凋亡 16-17 1.3.1 細(xì)胞凋亡 16 1.3.2 蛋白的磷酸化 16-17 1.3.3 BCL 10 的磷酸化參與細(xì)胞凋亡 17 1.4 BCL

17、10 與粘膜相關(guān)組織淋巴瘤 17-20 試驗(yàn)研究 20-38 第二章 CARD 結(jié)構(gòu)域?qū)ωiBCL 10 表達(dá)及細(xì)胞周期的影響 20-38 2.1 材料 20 2.1.1 菌株、細(xì)胞和質(zhì)粒 20 2.1.2 主要試劑 20 2.2 方法 20-29 2.2.1 豬BCL 10 CARD 結(jié)構(gòu)域相關(guān)三個真核表達(dá)載體的構(gòu)建 21-26 2.2.2 豬BCL 10 CARD 結(jié)構(gòu)域相關(guān)三個真核表達(dá)載體在豬血管內(nèi)皮細(xì)胞中表達(dá) 26-29 2.3 結(jié)果與分析 29-36 2.3.1 PCR 結(jié)果 29-31 2.3.2 豬BCL 10 CARD 結(jié)構(gòu)域相關(guān)三個克隆載體構(gòu)建結(jié)果 31 2.3.3 豬BCL 10 CARD 結(jié)構(gòu)域相關(guān)三個真核表達(dá)載體構(gòu)建結(jié)果 31 2.3.4 豬BCL 10 CARD 結(jié)構(gòu)域相關(guān)三