RBBP6敲除小鼠胚胎致死原因的探讨

发布时间：2018-01-17 17:22

本文关键词：RBBP6敲除小鼠胚胎致死原因的探讨　出处：《安徽医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：背景成视网膜瘤结合蛋白6(retinoblastom binding protein6,RBBP6),是可以同时与体内两种重要抑癌基因肿瘤抑制基因p53(tumor protein p53, p53)和视网膜母细胞瘤基因(retinoblastoma gene,Rb)结合的蛋白之一。RBBP6由多个重要的结构域组成，如锌指环区，，丝氨酸/精氨酸富含区(serine/arginine-rich domain，SR)区，p53结合区，Rb结合区等，提示RBBP6的功能复杂多样。至从1995年YoshihisaSakai以Rb为探针筛选相互作用蛋白得到此基因后，不同形式的同源体依次被发现，不同实验室通过不同角度对其功能进行研究。RBBP6在终末分化及增殖能力丧失的细胞系中，G1期的表达比在G2/M期的表达下降了70%-80%。另外发现RBBP6的表达量与细胞内定位受到细胞周期和细胞分化的调控，在G0期几乎检测不到其表达，而在M期其表达水平可上调10倍；分化后的RBBP6细胞内水平可降至分化前的30%，终末分化时，则降至5%。在分裂间期，RBBP6定位于核仁和核内斑点(nuclear speckle)，分裂期则定位于染色体外周。在细胞凋亡方面，RBBP6在MCF-7细胞中参与喜树碱诱导的死亡途径，其中发挥作用的氨基酸区域也是与p53结合的区域。由于其具有丝氨酸/精氨酸富含区(serine/arginine-richdomain，SR)结构域，RBBP6可以调节前体mRNA剪接，非有丝分裂细胞中其参与RNA代谢，有丝分裂细胞中为RNA剪接提供空间。在食管癌与肺癌患者的组织中，RBBP6在癌组织的表达高于癌旁组织，其在肿瘤发生发展中起到作用。本实验室建立RBBP6-/-小鼠模型，该小鼠在胚胎期6.5-7.5d死亡，针对RBBP6-/-小鼠胚胎致死的原因进行了研究与探讨。第一部分RBBP6敲低后，分析细胞中DNA损伤标志因子γH2AX的表达情况目的验证本实验室的HeLa细胞在化学药物刺激下发生DNA损伤反应，在该细胞系中研究敲低RBBP6后检测DNA损伤标志因子γH2AX的表达情况。方法利用诱发DNA损伤的化学药物顺铂刺激细胞，通过Western Blot方法检测DNA损伤标志物γH2AX的表达，验证该细胞在DNA损伤后发生双链断裂反应。合成RBBP6的siRNA，转染该细胞系，再分别通过实时定量PCR与Western Blot方法检测DNA损伤标志物γH2AX的表达，验证RBBP6与γH2AX的相关性。结果本室的HeLa细胞被诱发DNA损伤的化学药物顺铂刺激后，DNA损伤的标志因子γH2AX蛋白表达水平上调。在该细胞系中利用RBBP6的siRNA敲低RBBP6后，DNA损伤的标志因子γH2AX伴随着RBBP6蛋白表达水平的下降而上调。结论本室的HeLa细胞在DNA损伤后发生双链断裂，从细胞水平上证明RBBP6与γH2AX具有相关性，RBBP6可能通过调控γH2AX参与DNA损伤应答过程。第二部分分析RBBP6基因敲除小鼠中DNA损伤标志因子γH2AX的表达情况目的分离本室的7日龄RBBP6-/-小鼠胚胎组织，在不同的解剖层面利用免疫组化分析DNA损伤标志因子γH2AX的表达情况。方法在显微镜下分离7日龄小鼠胚胎组织，通过免疫组化区分RBBP6敲除型与野生型小鼠胚胎组织，检测两者γH2AX的表达情况并进行比较，验证RBBP6-/-小鼠胚胎组织中γH2AX表达上调。结果免疫组化分析RBBP6敲除型小鼠的胚胎组织中DNA损伤标志因子γH2AX的表达水平较RBBP6野生型小鼠的胚胎组织增高。结论在动物水平解释了RBBP6-/-小鼠胚胎致死的可能原因，说明了RBBP6基因敲除后γH2AX的上升导致基因组不稳定性可能是RBBP6-/-小鼠胚胎致死的原因之一，为RBBP6基因可能通过γH2AX参与DNA损伤提供了线索。
[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.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

【参考文献】