组蛋白甲基化修饰相关蛋白在ISO诱导的心肌肥厚中的作用

发布时间：2018-06-08 15:15

本文选题：心肌肥厚 + 赖氨酸特异性组蛋白去甲基化酶1　；参考：《天津医科大学》2017年硕士论文

【摘要】：背景心血管疾病是医学界始终难以攻克的一大难关。心肌肥厚是多种心血管疾病发生发展的必然结果,也是心血管疾病中最危险的因素之一。初期的心肌肥厚是一种适应性的反应,但是长期的心肌肥厚将最终造成心脏功能衰竭,终末期死亡率极高。由于各种信号通路相互交织,汇聚,造成心脏的肥大性生长,引起转录系统的紊乱。伴随基因组学的发展,表观遗传修饰在疾病机制中的作用日益受到重视,尤其是组蛋白甲基化修饰调控基因的表达,被认为与多种病理性信号通路相关,参与多种疾病的发生发展,已经成为近些年研究的热点。组蛋白甲基化修饰研究涉及较多的是组蛋白去甲基化酶LSD1以及组蛋白甲基化酶EZH2。LSD1可以对组蛋白H3第4位赖氨酸(H3K4)以及组蛋白H3第4位赖氨酸(H3K9)的甲基化状态进行去甲基化,发挥生物学功能,在许多疾病类型中,LSD1表达的增加与其功能的获得呈正相关,由于LSD1对于H3K4,H3K9的调节在正常细胞以及癌细胞中都是起关键性作用的基因,故通过改变LSD1表达引起组蛋白甲基化状态的改变,可能在某些疾病的发生和发展过程中起重要作用。EZH2通过对组蛋白H3第27位赖氨酸(H3K27)的三甲基化发挥其功能,研究表明心肌祖细胞分化为心肌细胞过程中EZH2及H3K27三甲基化水平增加,EZH2的稳定性及功能增强可抑制心肌肥厚,有报道称Micro RNA-214通过抑制EZH2引起心肌肥厚,但是关于EZH2与心肌肥厚的关系仍不明确。因此,深入探讨组蛋白甲基化修饰的状态变化与心肌肥厚信号通路之间的关系十分必要。目的1.探讨赖氨酸特异性组蛋白去甲基化酶1(Lysine specific demethylase 1,LSD1)在异丙基肾上腺素(Isoproterenol,ISO)诱导的病理性心肌肥厚发病机制中的作用,研究组蛋白甲基化修饰与心肌肥厚的相关性,为开发治疗和预防病理性心肌肥厚的有效临床药物寻找新靶点。2.构建特异性的大鼠EZH2(Enhancer of zeste homolog 2,组蛋白甲基转移酶的一种)过表达质粒以及EZH2催化亚基set domain缺失质粒,并在293T细胞中评估其表达水平,为本项目后期深入研究组蛋白甲基化修饰对心肌肥厚发生发展的潜在作用提供新工具。方法一、LSD1抑制实验:选取健康雄性的Wistar大鼠,体重为140~180 g,SPF级饲养方式饲养48 h。首先,将实验大鼠24只,随机分成4组:正常对照组(CTRL),ISO处理组(ISO),LSD1抑制剂OGL002组(OGL002)和ISO处理+LSD1抑制剂OGL002组(ISO+OGL002),每组6只。ISO处理组及ISO+抑制剂OGL002组动物每天腹腔注射2 mg/kg ISO,CTRL及OGL002组腹腔注射相同体积的生理盐水,连续一周。一周后OGL002组及ISO+OGL002组动物每日腹腔注射LSD1抑制剂OGL002溶液,浓度为50μg/kg,溶解方法由公司提供,其余组注射等体积药品稀释液,处理连续一周。二、LSD1过表达实验:另外选取实验大鼠24只,随机分为四组:正常对照组(CTRL),ISO处理组(ISO),正常大鼠LSD1过表达病毒浓缩组(LSD1overexpression virus,LSD1-OV),和ISO处理大鼠+LSD1过表达病毒浓缩组(ISO+LSD1 overexpression virus,ISO+LSD1-OV),每组6只。ISO及ISO+LSD1过表达病毒浓缩组大鼠每天腹腔注射2 mg/kg ISO,CTRL及正常大鼠LSD1过表达病毒浓缩组大鼠腹腔注射相同体积的生理盐水,连续一周,一周后正常大鼠LSD1过表达病毒浓缩组及ISO处理+LSD1过表达病毒浓缩组动物每日心室注射LSD1过表达浓缩病毒,CTRL和ISO处理组大鼠每日心室内注射等体积培养液,处理连续一周。上述各组实验动物取心肌组织,通过心重比结果以及HE染色方法,检测各组大鼠心肌肥厚的表型发生情况;采用实时荧光定量PCR检测上述各组大鼠心肌组织中三种肥厚因子(MLC-2V,MHC,ANP)以及LSD1的m RNA表达情况;采用Western Blot、免疫组化、免疫荧光法检测LSD1及其作用下游的四种组蛋白H3K4me1,H3K4me2,H3K9me1,H3K9me2的蛋白表达水平。三、构建EZH2基因过表达质粒:通过普通PCR与重叠PCR法,质粒与载体双酶切以及T载体连接等基因方法构建大鼠EZH2过表达质粒以及SET domain缺失质粒,并通过脂质体转染法转入293T细胞中实现其表达,采用实时荧光定量PCR以及Western Blot对EZH2以及H3K27me3在RNA水平以及蛋白质表达水平进行验证。结果一、LSD1抑制实验(1)生理学指标显示ISO组、LSD1抑制剂(OGL002)组以及ISO+OGL002组与对照组相比,心率和血压均明显下调(n=6,P0.01)。(2)心肌表型及心重比显示ISO组、OGL002组以及ISO+OGL002组明显高于对照组,其中ISO+OGL002组最高,约为对照组的2倍(n=6,P0.05)。(3)HE染色结果显示结果表明ISO组、OGL002组及ISO+OGL002组与对照组相比细胞横截面积明显增大。(4)心肌肥厚标志物q PCR检测结果显示,ISO组、OGL002组及ISO+OGL002组与对照组相比,三种肥厚因子(MLC-2V,MHC,ANP)表达水平均明显上调:其中ISO+OGL002组肥厚因子表达水平最高,约为对照组的4-5倍。(n=6,P0.05)。(5)LSD1表达水平q PCR、Western Blot、免疫组化和免疫荧光检测显示,ISO组、OGL002组及ISO+OGL002组LSD1的m RNA和蛋白表达水平均明显下调;对照组LSD1的m RNA约为ISO组和OGL002组4倍左右,约为ISO+OGL002组高6倍左右。(n=6,P0.01)。(6)LSD1下游四种组蛋白(H3K4me1,H3K4me2,H3K9me1,H3K9me2)Western Blot、免疫组化和免疫荧光检测显示,ISO组、OGL002组及ISO+OGL002组蛋白表达水平均明显上调,ISO+OGL002组LSD1蛋白表达水平最高。二、LSD1过表达实验:(1)生理学指标显示ISO组与对照组相比HR、Bp均明显下调,LSD1-OV组及ISO+LSD1-OV组与ISO组相比HR、Bp均明显上调(n=6,P0.01)(2)心肌表型及心重比显示ISO组明显高于对照组;LSD1-OV组及ISO+LSD1-OV组明显低于ISO组(n=6,P0.05)(3)HE染色结果显示结果表明ISO组与对照组相比细胞横截面积明显增大;LSD1-OV组及ISO+LSD1-OV组与ISO组相比,细胞横截面积显著减小。(4)心肌肥厚标志物q PCR检测结果显示,ISO组与对照组相比,三种肥厚因子表达(MLC-2V、MHC、ANP)均明显上调;ISO+LSD1-OV组与ISO组相比,三种肥厚因子表达水平下调;LSD1-OV组与对照组相比,ANP表达水平下调(n=6,P0.05)。(5)LSD1表达水平q PCR、Western Blot、免疫组化和免疫荧光检测显示,ISO组的LSD1m RNA和蛋白表达水平均明显下调;ISO+LSD1-OV组与ISO组相比,LSD1-OV组与对照组相比,LSD1表达水平显著上调(n=6,P0.01)。(6)LSD1下游组蛋白(H3K4me1,H3K4me2,H3K9me1,H3K9me2)Western Blot、免疫组化和免疫荧光检测显示,ISO组四种组蛋白表达水平均明显上调,ISO+LSD1-OV组与ISO组相比,LSD1-OV组与对照组相比均下调(n=6,P0.01)。三、构建EZH2基因过表达质粒:EZH2过表达质粒以及SET domain缺失质粒Western和实时荧光定量PCR检测结果显示在293T细胞中成功实现了EZH2两种质粒的过表达。由于SET domain位置的缺失,在验证构建的缺失SET domain区域的过表达质粒组中H3K27me3蛋白表达水平无改变,说明SET domain位置对于EZH2实现甲基化功能的重要性。结论1.本研究初步证明了组蛋白甲基化修饰水平变化与心肌肥厚的相关性。ISO可能通过抑制LSD1的表达增加组蛋白甲基化水平,诱导肥厚因子上调,导致病理性心肌肥厚的发生。过表达LSD1可以部分逆转ISO诱导的病理性心肌肥厚,为LSD1作为心肌肥厚临床治疗的新靶点提供理论依据。2.成功构建了特异性大鼠EZH2基因过表达质粒以及SET domain缺失质粒,为今后研究组蛋白甲基化修饰以及心肌肥厚信号通路之间的关系提供了工具。
[Abstract]:Background cardiovascular disease is a difficult problem in the medical community. Myocardial hypertrophy is an inevitable result of the development of various cardiovascular diseases. It is also one of the most dangerous factors in cardiovascular disease. Early cardiac hypertrophy is an adaptive response, but long-term cardiac hypertrophy will eventually cause heart failure and end stage. The death rate is very high. Due to the interweaving and aggregation of various signal pathways, the hypertrophic growth of the heart and the disorder of the transcriptional system are caused. With the development of genomics, epigenetic modification has been paid more and more attention in the mechanism of disease, especially the expression of the histone methylation modifier gene, which is considered to be with a variety of pathological signals. The pathway related and participation in the development of a variety of diseases has become a hot spot in recent years. Histone methylation modification is involved in the methylation of histone H3 fourth - bit lysine (H3K4) and histone H3 fourth - lysine (H3K9), which is involved in the methylation of histone methylation enzyme LSD1 and histone methylation enzyme. In many types of disease, the increase of LSD1 expression is positively related to the acquisition of its function in many types of diseases. Due to the regulation of LSD1 to H3K4, H3K9 is a key gene in normal cells and cancer cells, so by changing the expression of LSD1 expression, the change of the methylation status of histone may be in some cases. .EZH2 plays an important role in the occurrence and development of the disease..EZH2 plays its function through the trimylation of the histone H3 twenty-seventh lysine (H3K27). The study shows that the level of EZH2 and H3K27 trimethylation of cardiac progenitor cells is increased during the differentiation of myocardial cells into cardiomyocytes. The stability and function of EZH2 can inhibit the hypertrophy of the myocardium. It is reported that Micro RNA-21 is known as Micro RNA-21. 4 myocardial hypertrophy is caused by inhibiting EZH2, but the relationship between EZH2 and cardiac hypertrophy is still not clear. Therefore, it is necessary to explore the relationship between the state changes of histone methylation modification and the signal pathway of myocardial hypertrophy. Objective 1. to investigate the lysine specific histone normethylation enzyme 1 (Lysine specific demethylase 1, LSD1) The role of isoproterenol (Isoproterenol, ISO) induced pathological myocardial hypertrophy in the pathogenesis of myocardial hypertrophy, study the correlation between methylation of protein methylation and myocardial hypertrophy, and find a new target for the development of effective clinical drugs for the development of therapeutic and preventive myocardial hypertrophy by finding a specific EZH2 (Enhancer of zeste homolog 2, group Enhancer, group 2, group). A protein methyltransferase, an overexpressed plasmid and a EZH2 catalyzed subunit SET domain deletion plasmid, and evaluate its expression in 293T cells, provide a new tool for the further study of the potential role of histone methylation modification for the development of myocardial hypertrophy in the later stage of the project. A formula 1, LSD1 inhibition experiment: a healthy male Wistar Rats, body weight of 140~180 g, and SPF class feeding 48 h. first, 24 rats were randomly divided into 4 groups: normal control group (CTRL), ISO treatment group (ISO), LSD1 inhibitor OGL002 group (OGL002) and ISO processing +LSD1 inhibitor group. Every group of 6 treated groups and inhibitors were injected into the abdomen of 2 each day. SO, CTRL and group OGL002 were intraperitoneally injected with the same volume of normal saline for one week. One week later, the OGL002 and ISO+OGL002 groups were intraperitoneally injected with LSD1 inhibitor OGL002 solution, the concentration was 50 mu g/kg, the dissolution method was provided by the company, the other groups were injected with equal volume drug diluents, and the treatment for one week. Two, LSD1 overexpression experiment: and the other selection. 24 rats were randomly divided into four groups: normal control group (CTRL), ISO treatment group (ISO), normal rat LSD1 overexpression virus concentration group (LSD1overexpression virus, LSD1-OV), and ISO treated rat +LSD1 overexpression virus concentration group (ISO+LSD1 overexpression virus,), each group was 6 and expressed virus concentrated rats. Daily intraperitoneal injection of 2 mg/kg ISO, CTRL and normal rat LSD1 overexpression virus concentrated rats intraperitoneal injection of the same volume of normal saline, one week, one week later, normal rat LSD1 overexpressed virus concentration group and ISO treatment +LSD1 overexpressed virus concentration group animals daily ventricular injection of LSD1 overexpression concentrated virus, CTRL and ISO treatment group large Rats were injected intraventricular volume culture medium daily for one week. The experimental animals were treated with myocardial tissue. The phenotypic occurrence of myocardial hypertrophy was detected by cardiac weight ratio and HE staining. Real-time fluorescence quantitative PCR was used to detect three kinds of hypertrophic factors (MLC-2V, MHC, ANP) in the myocardium of the rats. And the expression of M RNA of LSD1; use Western Blot, immunohistochemistry and immunofluorescence to detect the protein expression level of four histones, LSD1, H3K4me2, H3K9me1, H3K9me2. Three, construct EZH2 gene overexpressed plasmids: through common PCR and overlapping method, plasmid and carrier double enzyme cut and connection vector connection The EZH2 overexpression plasmid and SET domain deletion plasmid were constructed and transferred into 293T cells by liposome transfection. Real-time fluorescent quantitative PCR and Western Blot were used to verify EZH2 and H3K27me3 at RNA level and protein expression level. Results 1, LSD1 inhibition experiment (1) physiological indexes showed ISO group. The heart rate and blood pressure of the D1 inhibitor (OGL002) group and the ISO+OGL002 group were significantly down (n=6, P0.01). (2) the myocardial phenotype and cardiac weight ratio showed ISO group, OGL002 group and ISO+OGL002 group were significantly higher than the control group, among which the ISO+OGL002 group was the highest, about 2 times of the control group (n=6, P0.05). (3) HE staining results showed the results showed groups The cross section area of the 2 groups and ISO+OGL002 groups was significantly increased compared with the control group. (4) the results of Q PCR detection of myocardial hypertrophy markers showed that the expression level of three hypertrophy factors (MLC-2V, MHC, ANP) in the ISO group, OGL002 and ISO+OGL002 groups were obviously up: the highest expression level of the hypertrophy factor in the middle ISO+OGL002 group was about 4 of the control group. -5 times. (n=6, P0.05). (5) LSD1 expression level Q PCR, Western Blot, immunohistochemistry and immunofluorescence detection showed that the ISO group, OGL002 group and ISO+OGL002 group were obviously down regulated and the protein expression levels were obviously down; the control group was about 6 times higher than that of the group and about 6 times higher. (6) four kinds of downstream. Histone (H3K4me1, H3K4me2, H3K9me1, H3K9me2) Western Blot, immunohistochemistry and immunofluorescence test showed that the expression level of ISO group, OGL002 group and ISO+OGL002 group were obviously up, the LSD1 protein expression level in ISO+OGL002 group was the highest. Two, LSD1 over expression experiment: (1) physiological indexes showed that both group and control group were obviously down regulated. Group 1-OV and group ISO+LSD1-OV were significantly higher than group ISO (n=6, P0.01) (n=6, P0.01) (2) the myocardial phenotype and cardiac weight ratio showed that ISO group was significantly higher than that of the control group, and LSD1-OV group and ISO+LSD1-OV group were significantly lower than that of ISO group (n=6, 3). The results showed that the cell cross section area was obviously increased compared with the control group. The cross section area of the OV group decreased significantly compared with the ISO group. (4) the results of Q PCR detection of myocardial hypertrophy showed that the expression of three hypertrophic factors (MLC-2V, MHC, ANP) in ISO group was up obviously compared with the control group, and the three kinds of hypertrophic factor tables were downregulated in the ISO+LSD1-OV group compared with the ISO group, and the ANP expression level was downregulated in LSD1-OV group compared with the control group. N=6, P0.05). (5) LSD1 expression level Q PCR, Western Blot, immunohistochemistry and immunofluorescence detection showed that the LSD1m RNA and protein expression level of ISO group were obviously down; ISO+LSD1-OV group compared with the ISO group, the level of expression was significantly up. (6) downstream histone Estern Blot, immunohistochemistry and immunofluorescence test showed that the expression level of four groups of histone in group ISO was obviously up-regulated. Compared with the ISO group, the LSD1-OV group decreased (n=6, P0.01) compared with the ISO group. Three, the EZH2 gene overexpressed plasmids were constructed: EZH2 overexpressed plasmids, SET domain deletion plasmids and real-time quantitative fluorescence quantitative detection. The results showed that the overexpression of two plasmids of EZH2 was successfully realized in 293T cells. The expression level of H3K27me3 protein in the overexpressed plasmid group that verified the deletion of the constructed SET domain region was not changed because of the deletion of the SET domain location, indicating the importance of the domain position of SET to the realization of the methylation of EZH2. Conclusion 1. preliminary evidence of the study The correlation between the level of histone methylation modification and myocardial hypertrophy.ISO may increase the level of histone methylation by inhibiting the expression of LSD1, inducing the up-regulation of hypertrophy factors and leading to the occurrence of pathological myocardial hypertrophy. Overexpression of LSD1 can partly reverse the pathological cardiac hypertrophy induced by ISO, which is a clinical treatment for LSD1 as a cardiac hypertrophy. The new target provides a theoretical basis for the successful construction of the EZH2 gene overexpressed plasmid and SET domain deletion plasmid of the specific rat, which provides a tool for the future study of the relationship between the methylation modification of protein and the signal pathway of cardiac hypertrophy in the study group.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54

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