本文關鍵詞：RBBP6敲除小鼠胚胎致死原因的探討　出處：《安徽醫(yī)科大學》2012年碩士論文　論文類型：學位論文

  更多相關文章： RBBP6 DNA損傷 基因敲除 γH2AX 胚胎致死

【摘要】：背景成視網(wǎng)膜瘤結合蛋白6(retinoblastom binding protein6,RBBP6),是可以同時與體內(nèi)兩種重要抑癌基因腫瘤抑制基因p53(tumor protein p53, p53)和視網(wǎng)膜母細胞瘤基因(retinoblastoma gene,Rb)結合的蛋白之一。RBBP6由多個重要的結構域組成，如鋅指環(huán)區(qū)，，絲氨酸/精氨酸富含區(qū)(serine/arginine-rich domain，SR)區(qū)，p53結合區(qū)，Rb結合區(qū)等，提示RBBP6的功能復雜多樣。至從1995年YoshihisaSakai以Rb為探針篩選相互作用蛋白得到此基因后，不同形式的同源體依次被發(fā)現(xiàn)，不同實驗室通過不同角度對其功能進行研究。RBBP6在終末分化及增殖能力喪失的細胞系中，G1期的表達比在G2/M期的表達下降了70%-80%。另外發(fā)現(xiàn)RBBP6的表達量與細胞內(nèi)定位受到細胞周期和細胞分化的調(diào)控，在G0期幾乎檢測不到其表達，而在M期其表達水平可上調(diào)10倍；分化后的RBBP6細胞內(nèi)水平可降至分化前的30%，終末分化時，則降至5%。在分裂間期，RBBP6定位于核仁和核內(nèi)斑點(nuclear speckle)，分裂期則定位于染色體外周。在細胞凋亡方面，RBBP6在MCF-7細胞中參與喜樹堿誘導的死亡途徑，其中發(fā)揮作用的氨基酸區(qū)域也是與p53結合的區(qū)域。由于其具有絲氨酸/精氨酸富含區(qū)(serine/arginine-richdomain，SR)結構域，RBBP6可以調(diào)節(jié)前體mRNA剪接，非有絲分裂細胞中其參與RNA代謝，有絲分裂細胞中為RNA剪接提供空間。在食管癌與肺癌患者的組織中，RBBP6在癌組織的表達高于癌旁組織，其在腫瘤發(fā)生發(fā)展中起到作用。本實驗室建立RBBP6-/-小鼠模型，該小鼠在胚胎期6.5-7.5d死亡，針對RBBP6-/-小鼠胚胎致死的原因進行了研究與探討。 第一部分RBBP6敲低后，分析細胞中DNA損傷標志因子γH2AX的表達情況 目的驗證本實驗室的HeLa細胞在化學藥物刺激下發(fā)生DNA損傷反應，在該細胞系中研究敲低RBBP6后檢測DNA損傷標志因子γH2AX的表達情況。 方法利用誘發(fā)DNA損傷的化學藥物順鉑刺激細胞，通過Western Blot方法檢測DNA損傷標志物γH2AX的表達，驗證該細胞在DNA損傷后發(fā)生雙鏈斷裂反應。合成RBBP6的siRNA，轉染該細胞系，再分別通過實時定量PCR與Western Blot方法檢測DNA損傷標志物γH2AX的表達，驗證RBBP6與γH2AX的相關性。 結果本室的HeLa細胞被誘發(fā)DNA損傷的化學藥物順鉑刺激后，DNA損傷的標志因子γH2AX蛋白表達水平上調(diào)。在該細胞系中利用RBBP6的siRNA敲低RBBP6后，DNA損傷的標志因子γH2AX伴隨著RBBP6蛋白表達水平的下降而上調(diào)。 結論本室的HeLa細胞在DNA損傷后發(fā)生雙鏈斷裂，從細胞水平上證明RBBP6與γH2AX具有相關性，RBBP6可能通過調(diào)控γH2AX參與DNA損傷應答過程。 第二部分分析RBBP6基因敲除小鼠中DNA損傷標志因子γH2AX的表達情況 目的分離本室的7日齡RBBP6-/-小鼠胚胎組織，在不同的解剖層面利用免疫組化分析DNA損傷標志因子γH2AX的表達情況。 方法在顯微鏡下分離7日齡小鼠胚胎組織，通過免疫組化區(qū)分RBBP6敲除型與野生型小鼠胚胎組織，檢測兩者γH2AX的表達情況并進行比較，驗證RBBP6-/-小鼠胚胎組織中γH2AX表達上調(diào)。 結果免疫組化分析RBBP6敲除型小鼠的胚胎組織中DNA損傷標志因子γH2AX的表達水平較RBBP6野生型小鼠的胚胎組織增高。 結論在動物水平解釋了RBBP6-/-小鼠胚胎致死的可能原因，說明了RBBP6基因敲除后γH2AX的上升導致基因組不穩(wěn)定性可能是RBBP6-/-小鼠胚胎致死的原因之一，為RBBP6基因可能通過γH2AX參與DNA損傷提供了線索。
[Abstract]:Background retinal tumorconjugated protein 6 (retinoblastom binding, protein6, RBBP6), and in two at the same time can be an important tumor suppressor gene of tumor suppressor gene p53 (tumor protein p53, p53) and retinoblastoma gene (retinoblastoma gene Rb) binding protein of.RBBP6 by a number of important domains, such as the zinc ring region, serine / arginine rich region (serine/arginine-rich domain, SR), p53 binding domain, Rb binding domain, suggesting that the function of RBBP6 is complicated. From 1995 to YoshihisaSakai to Rb as a probe for screening interaction protein of this gene, a homolog of different forms were found in different laboratories the research of.RBBP6 in terminal differentiation and proliferation of cells in the loss of its function through different angles, the expression of G1 than in the expression of G2/M decreased by 70%-80%. also found that the expression of RBBP6 and fine Intracellular localization is regulated by cell cycle and cell differentiation, almost detection in G0 period is less than its expression, and in the M phase and its expression level can increase 10 times; the level of differentiated RBBP6 cells can be reduced to 30% before differentiation, terminal differentiation, dropped to 5%. in the interphase, RBBP6 localized in the nucleolus and nuclear foci (nuclear speckle), is located in the mitotic chromosome periphery. In cell apoptosis, RBBP6 in MCF-7 cells involved in the death pathway induced by camptothecin, which also play role in combination with p53 amino acid region area. Because of its serine / arginine rich region (serine/arginine-richdomain. SR) domain, RBBP6 can regulate the pre mRNA splicing, non mitotic cells in their participation in the metabolism of RNA, mitotic cells provide space for RNA splicing. In patients with esophageal cancer and lung cancer tissues, higher expression of RBBP6 in cancer tissue Paracancerous tissue plays an important role in the development of tumor. The RBBP6-/- mouse model was established in this laboratory, and the 6.5-7.5d died in the embryo stage, and the cause of embryo death in RBBP6-/- mice was studied.
In part 1, after RBBP6 knockout, the expression of DNA damage marker factor H2AX in cells is analyzed.
Objective to verify the DNA damage response of HeLa cells in the laboratory under the stimulation of chemical drugs, and detect the expression of DNA damage factor DNA H2AX after knocking down the RBBP6 in this cell line.
By using the method of chemical drugs of cisplatin induced DNA damage in cells stimulated by Western and Blot method to detect DNA damage markers expression of gamma H2AX, verify that the cells undergo double strand breaks in DNA reaction after injury. The synthesis of siRNA RBBP6, the transfected cell lines, respectively by real-time quantitative PCR and Western Blot method to detect the expression of DNA damage complex gamma H2AX, correlation verification of RBBP6 and gamma H2AX.
The results of this room HeLa cell DNA damage induced by cisplatin by chemical stimulation, the expression of DNA damage markers gamma H2AX protein levels were up-regulated. In this cell line using RBBP6 siRNA knockdown of RBBP6 after DNA damage markers of gamma H2AX with RBBP6 protein expression level decreased and increased.
Conclusion the double strand breaks of HeLa cells in DNA cells after injury were observed. It is proved that RBBP6 is correlated with H2AX, and RBBP6 may participate in DNA damage response process by regulating gamma H2AX.
The second part analyses the expression of DNA damage marker factor gamma H2AX in RBBP6 gene knockout mice
Objective to isolate the 7 day old RBBP6-/- mouse embryonic tissue in this room, and to analyze the expression of DNA damage marker factor gamma H2AX by immunohistochemistry at different anatomical levels.
Methods the 7 day old mouse embryo tissue was separated under microscope. The expression of H2AX was detected by immunohistochemistry. The expression of H2AX was detected and compared. The expression of H2AX was increased in RBBP6-/- mouse embryo.
Results the expression of DNA damage marker H2AX in the embryonic tissue of RBBP6 knockout mice was higher than that of the RBBP6 wild type mouse.
Conclusion RBBP6-/- may explain why the embryonic lethality in animal level, the deletion of RBBP6 led to a rise in gamma H2AX after possible genomic instability is one of the causes of RBBP6-/- mouse embryonic lethal, RBBP6 gene may be involved in DNA damage by gamma H2AX provides a clue.

【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R363

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本文編號：1437217