本文選題：心肌肥厚 + 賴氨酸特異性組蛋白去甲基化酶1��； 參考：《天津醫(yī)科大學(xué)》2017年碩士論文

【摘要】：背景心血管疾病是醫(yī)學(xué)界始終難以攻克的一大難關(guān)。心肌肥厚是多種心血管疾病發(fā)生發(fā)展的必然結(jié)果,也是心血管疾病中最危險(xiǎn)的因素之一。初期的心肌肥厚是一種適應(yīng)性的反應(yīng),但是長期的心肌肥厚將最終造成心臟功能衰竭,終末期死亡率極高。由于各種信號通路相互交織,匯聚,造成心臟的肥大性生長,引起轉(zhuǎn)錄系統(tǒng)的紊亂。伴隨基因組學(xué)的發(fā)展,表觀遺傳修飾在疾病機(jī)制中的作用日益受到重視,尤其是組蛋白甲基化修飾調(diào)控基因的表達(dá),被認(rèn)為與多種病理性信號通路相關(guān),參與多種疾病的發(fā)生發(fā)展,已經(jīng)成為近些年研究的熱點(diǎn)。組蛋白甲基化修飾研究涉及較多的是組蛋白去甲基化酶LSD1以及組蛋白甲基化酶EZH2。LSD1可以對組蛋白H3第4位賴氨酸(H3K4)以及組蛋白H3第4位賴氨酸(H3K9)的甲基化狀態(tài)進(jìn)行去甲基化,發(fā)揮生物學(xué)功能,在許多疾病類型中,LSD1表達(dá)的增加與其功能的獲得呈正相關(guān),由于LSD1對于H3K4,H3K9的調(diào)節(jié)在正常細(xì)胞以及癌細(xì)胞中都是起關(guān)鍵性作用的基因,故通過改變LSD1表達(dá)引起組蛋白甲基化狀態(tài)的改變,可能在某些疾病的發(fā)生和發(fā)展過程中起重要作用。EZH2通過對組蛋白H3第27位賴氨酸(H3K27)的三甲基化發(fā)揮其功能,研究表明心肌祖細(xì)胞分化為心肌細(xì)胞過程中EZH2及H3K27三甲基化水平增加,EZH2的穩(wěn)定性及功能增強(qiáng)可抑制心肌肥厚,有報(bào)道稱Micro RNA-214通過抑制EZH2引起心肌肥厚,但是關(guān)于EZH2與心肌肥厚的關(guān)系仍不明確。因此,深入探討組蛋白甲基化修飾的狀態(tài)變化與心肌肥厚信號通路之間的關(guān)系十分必要。目的1.探討賴氨酸特異性組蛋白去甲基化酶1(Lysine specific demethylase 1,LSD1)在異丙基腎上腺素(Isoproterenol,ISO)誘導(dǎo)的病理性心肌肥厚發(fā)病機(jī)制中的作用,研究組蛋白甲基化修飾與心肌肥厚的相關(guān)性,為開發(fā)治療和預(yù)防病理性心肌肥厚的有效臨床藥物尋找新靶點(diǎn)。2.構(gòu)建特異性的大鼠EZH2(Enhancer of zeste homolog 2,組蛋白甲基轉(zhuǎn)移酶的一種)過表達(dá)質(zhì)粒以及EZH2催化亞基set domain缺失質(zhì)粒,并在293T細(xì)胞中評估其表達(dá)水平,為本項(xiàng)目后期深入研究組蛋白甲基化修飾對心肌肥厚發(fā)生發(fā)展的潛在作用提供新工具。方法一、LSD1抑制實(shí)驗(yàn):選取健康雄性的Wistar大鼠,體重為140~180 g,SPF級飼養(yǎng)方式飼養(yǎng)48 h。首先,將實(shí)驗(yàn)大鼠24只,隨機(jī)分成4組:正常對照組(CTRL),ISO處理組(ISO),LSD1抑制劑OGL002組(OGL002)和ISO處理+LSD1抑制劑OGL002組(ISO+OGL002),每組6只。ISO處理組及ISO+抑制劑OGL002組動物每天腹腔注射2 mg/kg ISO,CTRL及OGL002組腹腔注射相同體積的生理鹽水,連續(xù)一周。一周后OGL002組及ISO+OGL002組動物每日腹腔注射LSD1抑制劑OGL002溶液,濃度為50μg/kg,溶解方法由公司提供,其余組注射等體積藥品稀釋液,處理連續(xù)一周。二、LSD1過表達(dá)實(shí)驗(yàn):另外選取實(shí)驗(yàn)大鼠24只,隨機(jī)分為四組:正常對照組(CTRL),ISO處理組(ISO),正常大鼠LSD1過表達(dá)病毒濃縮組(LSD1overexpression virus,LSD1-OV),和ISO處理大鼠+LSD1過表達(dá)病毒濃縮組(ISO+LSD1 overexpression virus,ISO+LSD1-OV),每組6只。ISO及ISO+LSD1過表達(dá)病毒濃縮組大鼠每天腹腔注射2 mg/kg ISO,CTRL及正常大鼠LSD1過表達(dá)病毒濃縮組大鼠腹腔注射相同體積的生理鹽水,連續(xù)一周,一周后正常大鼠LSD1過表達(dá)病毒濃縮組及ISO處理+LSD1過表達(dá)病毒濃縮組動物每日心室注射LSD1過表達(dá)濃縮病毒,CTRL和ISO處理組大鼠每日心室內(nèi)注射等體積培養(yǎng)液,處理連續(xù)一周。上述各組實(shí)驗(yàn)動物取心肌組織,通過心重比結(jié)果以及HE染色方法,檢測各組大鼠心肌肥厚的表型發(fā)生情況;采用實(shí)時(shí)熒光定量PCR檢測上述各組大鼠心肌組織中三種肥厚因子(MLC-2V,MHC,ANP)以及LSD1的m RNA表達(dá)情況;采用Western Blot、免疫組化、免疫熒光法檢測LSD1及其作用下游的四種組蛋白H3K4me1,H3K4me2,H3K9me1,H3K9me2的蛋白表達(dá)水平。三、構(gòu)建EZH2基因過表達(dá)質(zhì)粒:通過普通PCR與重疊PCR法,質(zhì)粒與載體雙酶切以及T載體連接等基因方法構(gòu)建大鼠EZH2過表達(dá)質(zhì)粒以及SET domain缺失質(zhì)粒,并通過脂質(zhì)體轉(zhuǎn)染法轉(zhuǎn)入293T細(xì)胞中實(shí)現(xiàn)其表達(dá),采用實(shí)時(shí)熒光定量PCR以及Western Blot對EZH2以及H3K27me3在RNA水平以及蛋白質(zhì)表達(dá)水平進(jìn)行驗(yàn)證。結(jié)果一、LSD1抑制實(shí)驗(yàn)(1)生理學(xué)指標(biāo)顯示ISO組、LSD1抑制劑(OGL002)組以及ISO+OGL002組與對照組相比,心率和血壓均明顯下調(diào)(n=6,P0.01)。(2)心肌表型及心重比顯示ISO組、OGL002組以及ISO+OGL002組明顯高于對照組,其中ISO+OGL002組最高,約為對照組的2倍(n=6,P0.05)。(3)HE染色結(jié)果顯示結(jié)果表明ISO組、OGL002組及ISO+OGL002組與對照組相比細(xì)胞橫截面積明顯增大。(4)心肌肥厚標(biāo)志物q PCR檢測結(jié)果顯示,ISO組、OGL002組及ISO+OGL002組與對照組相比,三種肥厚因子(MLC-2V,MHC,ANP)表達(dá)水平均明顯上調(diào):其中ISO+OGL002組肥厚因子表達(dá)水平最高,約為對照組的4-5倍。(n=6,P0.05)。(5)LSD1表達(dá)水平q PCR、Western Blot、免疫組化和免疫熒光檢測顯示,ISO組、OGL002組及ISO+OGL002組LSD1的m RNA和蛋白表達(dá)水平均明顯下調(diào);對照組LSD1的m RNA約為ISO組和OGL002組4倍左右,約為ISO+OGL002組高6倍左右。(n=6,P0.01)。(6)LSD1下游四種組蛋白(H3K4me1,H3K4me2,H3K9me1,H3K9me2)Western Blot、免疫組化和免疫熒光檢測顯示,ISO組、OGL002組及ISO+OGL002組蛋白表達(dá)水平均明顯上調(diào),ISO+OGL002組LSD1蛋白表達(dá)水平最高。二、LSD1過表達(dá)實(shí)驗(yàn):(1)生理學(xué)指標(biāo)顯示ISO組與對照組相比HR、Bp均明顯下調(diào),LSD1-OV組及ISO+LSD1-OV組與ISO組相比HR、Bp均明顯上調(diào)(n=6,P0.01)(2)心肌表型及心重比顯示ISO組明顯高于對照組;LSD1-OV組及ISO+LSD1-OV組明顯低于ISO組(n=6,P0.05)(3)HE染色結(jié)果顯示結(jié)果表明ISO組與對照組相比細(xì)胞橫截面積明顯增大;LSD1-OV組及ISO+LSD1-OV組與ISO組相比,細(xì)胞橫截面積顯著減小。(4)心肌肥厚標(biāo)志物q PCR檢測結(jié)果顯示,ISO組與對照組相比,三種肥厚因子表達(dá)(MLC-2V、MHC、ANP)均明顯上調(diào);ISO+LSD1-OV組與ISO組相比,三種肥厚因子表達(dá)水平下調(diào);LSD1-OV組與對照組相比,ANP表達(dá)水平下調(diào)(n=6,P0.05)。(5)LSD1表達(dá)水平q PCR、Western Blot、免疫組化和免疫熒光檢測顯示,ISO組的LSD1m RNA和蛋白表達(dá)水平均明顯下調(diào);ISO+LSD1-OV組與ISO組相比,LSD1-OV組與對照組相比,LSD1表達(dá)水平顯著上調(diào)(n=6,P0.01)。(6)LSD1下游組蛋白(H3K4me1,H3K4me2,H3K9me1,H3K9me2)Western Blot、免疫組化和免疫熒光檢測顯示,ISO組四種組蛋白表達(dá)水平均明顯上調(diào),ISO+LSD1-OV組與ISO組相比,LSD1-OV組與對照組相比均下調(diào)(n=6,P0.01)。三、構(gòu)建EZH2基因過表達(dá)質(zhì)粒:EZH2過表達(dá)質(zhì)粒以及SET domain缺失質(zhì)粒Western和實(shí)時(shí)熒光定量PCR檢測結(jié)果顯示在293T細(xì)胞中成功實(shí)現(xiàn)了EZH2兩種質(zhì)粒的過表達(dá)。由于SET domain位置的缺失,在驗(yàn)證構(gòu)建的缺失SET domain區(qū)域的過表達(dá)質(zhì)粒組中H3K27me3蛋白表達(dá)水平無改變,說明SET domain位置對于EZH2實(shí)現(xiàn)甲基化功能的重要性。結(jié)論1.本研究初步證明了組蛋白甲基化修飾水平變化與心肌肥厚的相關(guān)性。ISO可能通過抑制LSD1的表達(dá)增加組蛋白甲基化水平,誘導(dǎo)肥厚因子上調(diào),導(dǎo)致病理性心肌肥厚的發(fā)生。過表達(dá)LSD1可以部分逆轉(zhuǎn)ISO誘導(dǎo)的病理性心肌肥厚,為LSD1作為心肌肥厚臨床治療的新靶點(diǎn)提供理論依據(jù)。2.成功構(gòu)建了特異性大鼠EZH2基因過表達(dá)質(zhì)粒以及SET domain缺失質(zhì)粒,為今后研究組蛋白甲基化修飾以及心肌肥厚信號通路之間的關(guān)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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R54

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 武利軍;趙連友;鄭強(qiáng)蓀;薛玉生;牛曉琳;黃志剛;尚福軍;;心肌營養(yǎng)素-1誘導(dǎo)心肌細(xì)胞肥大及辛伐他汀的干預(yù)作用[J];中華高血壓雜志;2006年09期

2 ;Effects of combination of irbesartan and perindopril on calcineurin expression and sarcoplasmic reticulum Ca~(2+)-ATPase activity in rat cardiac pressure-overload hypertrophy[J];Journal of Zhejiang University Science;2006年03期

3 沈小梅;張巨艷;成蓓;;Construction of Rat Calcineurin A α cDNA Recombinant Adenovirus Vector and Its Identification[J];華中科技大學(xué)學(xué)報(bào)(醫(yī)學(xué)英德文版);2006年01期

，

本文編號：1996236