本文選題：CUL4B + 細(xì)胞周期調(diào)控��； 參考：《山東大學(xué)》2010年碩士論文

【摘要】： 細(xì)胞周期是細(xì)胞生命活動(dòng)的基本過(guò)程。在進(jìn)化過(guò)程中,細(xì)胞建立并發(fā)展了一系列的調(diào)控機(jī)制,以確保細(xì)胞周期嚴(yán)格有序地交替和各個(gè)時(shí)相依次有序變更。多種細(xì)胞周期蛋白在調(diào)控細(xì)胞周期各個(gè)時(shí)相轉(zhuǎn)換中發(fā)揮作用,其主要包括：細(xì)胞周期蛋白依賴激酶(cyclin-dependent kinases, CDKs)家族蛋白、細(xì)胞周期蛋白(cyclins)和細(xì)胞周期蛋白依賴性激酶抑制劑(CKIs)。細(xì)胞通過(guò)對(duì)三類細(xì)胞周期蛋白的合成、降解以及翻譯后修飾等環(huán)節(jié)進(jìn)行調(diào)控,從而保證細(xì)胞周期正常的進(jìn)行。 泛素依賴的蛋白質(zhì)降解是細(xì)胞調(diào)控蛋白質(zhì)活性的一個(gè)基本機(jī)制,在細(xì)胞周期調(diào)控中起重要的作用,幾乎所有細(xì)胞周期蛋白的降解都依賴于該過(guò)程。CUL4B屬于cullin蛋白家族,該家族成員是泛素依賴降解系統(tǒng)中E3連接酶的成員,在本實(shí)驗(yàn)室發(fā)現(xiàn)的CUL4B突變導(dǎo)致精神發(fā)育遲滯的家系中,女性攜帶者出現(xiàn)X染色體失活偏倚,患者骨骼發(fā)育異常以及CUL4B低表達(dá)的細(xì)胞出現(xiàn)S期停滯提示CUL4B可能在細(xì)胞周期調(diào)控中發(fā)揮重要的作用。然而,目前對(duì)于CUL4B在細(xì)胞周期調(diào)控方面的研究還比較少,僅發(fā)現(xiàn)少數(shù)幾種與細(xì)胞周期調(diào)控相關(guān)的靶蛋白(如Cyclin E、CDT1和p21),因此尋找CUL4B在細(xì)胞周期調(diào)控過(guò)程中的蛋白對(duì)研究CUL4B在細(xì)胞周期調(diào)控中的生物學(xué)功能至關(guān)重要。 首先,我們提取穩(wěn)篩的CUL4B低表達(dá)非同步化的HeLa和HEK293細(xì)胞及其對(duì)照細(xì)胞的總蛋白,利用western blotting分析方法對(duì)p21、p27、p53、CDT1、CDC6、cyclin D1、CHK1、E2F1、CDC25A、CDC25C和CDK2等細(xì)胞周期調(diào)控相關(guān)蛋白的表達(dá)進(jìn)行檢測(cè),結(jié)果發(fā)現(xiàn)這些蛋白的表達(dá)在實(shí)驗(yàn)組和對(duì)照組沒(méi)有顯著的差異。 隨后我們通過(guò)向培養(yǎng)基中加入細(xì)胞周期阻斷藥物的方法將CUL4B低表達(dá)的HeLa和HEK293細(xì)胞及其對(duì)照細(xì)胞分別同步化至G1期、S期、G2/M期,并使用流式細(xì)胞術(shù)對(duì)同步化效果進(jìn)行監(jiān)控。確定合適的處理方法后,我們提取同步化至不同時(shí)期的CUL4B低表達(dá)HeLa和HEK293細(xì)胞及其對(duì)照細(xì)胞的總蛋白,同樣使用western blotting分析檢測(cè)上述蛋白的表達(dá)情況,結(jié)果發(fā)現(xiàn)與對(duì)照細(xì)胞相比,CUL4B低表達(dá)的HeLa細(xì)胞內(nèi)p53、p27和CHK1蛋白在S期有明顯的積累。然而在CUL4B低表達(dá)的HEK293細(xì)胞中p53在G1/S期積累,CHK1在S期積累,p27沒(méi)有明顯的變化。為了明確導(dǎo)致CUL4B低表達(dá)HeLa細(xì)胞內(nèi)p27、p53和CHK1蛋白積累的分子機(jī)制,我們又檢測(cè)了上述基因在S期時(shí)的mRNA的表達(dá)水平,結(jié)果發(fā)現(xiàn)CUL4B低表達(dá)的細(xì)胞中上述基因的mRNA水平與對(duì)照細(xì)胞相比并沒(méi)有明顯的差異,提示這些蛋白在S期內(nèi)CUL4B低表達(dá)HeLa細(xì)胞中的表達(dá)上調(diào)是在轉(zhuǎn)錄后水平而非轉(zhuǎn)錄水平。對(duì)這幾種蛋白半衰期的測(cè)定發(fā)現(xiàn)在CUL4B低表達(dá)的HeLa細(xì)胞中p27和CHK1的半衰期明顯延長(zhǎng),表明CUL4B的表達(dá)下調(diào)導(dǎo)致這兩種蛋白的降解發(fā)生障礙。隨后我們發(fā)現(xiàn)外源性CUL4B的表達(dá)能夠減輕因內(nèi)源性CUL4B表達(dá)下調(diào)而造成的p27、p53和CHK1的積累。以上實(shí)驗(yàn)結(jié)果提示,CUL4B在p27、p53和CHK1的降解中發(fā)揮重要的調(diào)控作用。 綜上所述,我們建立了針對(duì)本實(shí)驗(yàn)室條件下所用細(xì)胞(HeLa和HEK293)的細(xì)胞周期同步化方法,并在此基礎(chǔ)上尋找到CUL4B在細(xì)胞周期調(diào)控中的候選靶蛋白。這些實(shí)驗(yàn)結(jié)果為進(jìn)一步研究CUL4B在細(xì)胞周期調(diào)控中的功能奠定了基礎(chǔ)。
[Abstract]:Cell cycle is the basic process of cell life activity. In the process of evolution, cells have established and developed a series of regulatory mechanisms to ensure that the cell cycle is alternately alternately and orderly in each phase. A variety of cyclin plays a role in the regulation of cell cycle phase transformation, mainly including cell cycle. Phase protein dependent kinase (cyclin-dependent kinases, CDKs) family protein, cyclin protein (cyclins) and cyclin dependent kinase inhibitor (CKIs). Cells are regulated by the synthesis, degradation and post-translational modification of three kinds of cyclin proteins to ensure the normal cell cycle.
Ubiquitin dependent protein degradation is a basic mechanism for cell regulation of protein activity, which plays an important role in the regulation of cell cycle. The degradation of almost all cell cycle proteins depends on the process.CUL4B belongs to the Cullin protein family. The family members are members of the E3 ligase in the ubiquitin dependent degradation system and are in our laboratory. X chromosome inactivation bias, skeletal dysplasia and S stagnation of CUL4B low expression cells in the patients with mental retardation in the family of mental retardation were found to play an important role in the regulation of cell cycle. However, the study on the regulation of cell cycle in CUL4B is still more than that of CUL4B in the regulation of cell cycle. Few, only a few target proteins related to cell cycle regulation (such as Cyclin E, CDT1 and p21) are found, so it is essential to find the protein in the process of cell cycle regulation of CUL4B to study the biological function of CUL4B in the regulation of cell cycle.
First, we extracted the low expression of CUL4B and the total protein of the non synchronous HeLa and HEK293 cells and their control cells. The expression of the cell cycle regulation related proteins, such as p21, p27, p53, CDT1, CDC6, cyclin D1, p27, CDC6, cyclin D1, etc., was detected by western blotting analysis. There was no significant difference between the experimental group and the control group.
Then we synchronize CUL4B low expression HeLa and HEK293 cells and their control cells to G1, S, G2/M phase, respectively by adding cell cycle blocking drugs into the medium, and monitor the synchronization effect by flow cytometry. After determining the appropriate treatment, we extract the synchronization to a different period of CUL4B low. The expression of the HeLa and HEK293 cells and their control cells was also expressed by Western blotting analysis. The results showed that the p53, p27 and CHK1 protein in the HeLa cells with low CUL4B expression were accumulated in S phase compared with the control cells. In S accumulation, there was no obvious change in p27. In order to clarify the molecular mechanism of the accumulation of p27, p53 and CHK1 protein in CUL4B low expression HeLa cells, we also detected the expression level of mRNA at S phase, and found that the mRNA level of the above genes in the CUL4B low expressed cells was not significantly different from that of the control cells. The up-regulated expression of these proteins in CUL4B low expression HeLa cells during S period was at post transcriptional level but not transcriptional level. The determination of the half-life of these proteins showed that the half-life of p27 and CHK1 in HeLa cells with low expression of CUL4B significantly prolonged, indicating that the downregulation of CUL4B expression led to the degradation of these two proteins. We found that the expression of exogenous CUL4B could reduce the accumulation of p27, p53 and CHK1 resulting from the downregulation of endogenous CUL4B expression. These results suggest that CUL4B plays an important role in the degradation of p27, p53 and CHK1.
In summary, we set up a cell cycle synchronization method for the cells (HeLa and HEK293) used in our laboratory, and on this basis, we find the candidate target proteins in the cell cycle regulation of CUL4B. These results provide a basis for further study of the function of CUL4B in cell cycle control.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

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