CNN1和Meox1基因对心脏发育及心肌病的调节机理的初步研究
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摘要
背景心血管病已经成为人类健康的头号杀手。利用基因芯片技术,比较肥厚型心肌病和扩张型心肌病转基因小鼠心肌细胞基因表达的变化,研究从基因突变到病理发生过程中参与调控的重要修饰基因,对了解心肌病发生机制、治疗和新药创制具有重要意义。经过基因芯片分析发现,cTnTR92Q和cTnTR141W转基因小鼠心脏中Meox1基因与野生型小鼠相比表达升高,推测编码大量的非簇生的,分离的,控制触角的同源盒基因的Meox1基因有可能参与心肌病的调节。因此本文特建立心脏特异表达Meox1的转基因小鼠,以研究Meox1对心脏发育及心肌病的调节作用。
方法cTnTR92Q及cTnTR141W转基因小鼠基因表达芯片及逆转录PCR和Western blot检测Meox1于肥厚型心肌病及扩张型心肌病模型小鼠心脏中的表达变化。Western blot检测Meox1于野生型C57BL/6J小鼠心脏中的时程表达。将Meox1基因插入心脏特异启动子α-MHC下游,构建转基因表达载体,通过显微注射法建立Meox1转基因C57BL／6J小鼠,PCR鉴定转基因小鼠的基因型,Western blot检测Meox1在心脏组织中的表达,心脏超声检测转基因小鼠及野生型小鼠的心脏结构和功能,病理组织学染色观察转基因小鼠心脏组织学及显微结构改变。逆转录PCR检测Meox1转基因小鼠的心脏肥大分子标志物及心脏转录因子,并记录转基因小鼠的生存状况。
结果cTnTR92Q和cTnTR141W转基因小鼠基因表达芯片及逆转录PCR结果显示Meox1基因在肥厚型心肌病及扩张型心肌病小鼠心脏中的表达升高。Western blot检测结果显示,在生理状态下,Meox1基因只在幼鼠心脏中表达,在病理状态下,Meox1基因在成年肥厚型和扩张型心肌病小鼠的心脏组织中表达均升高。通过显微注射,建立了两个Meox1基因在心脏组织的表达水平明显高于同龄对照小鼠的转基因小鼠品系。M型超声心动图显示,与野生型小鼠相比,两个Meox1转基因小鼠品系收缩期左室内径分别增加15.6%、24.2%(P＜0.01,n=16),舒张期左室内径分别增加7.2%、12.8%(P＜0.01,n=16),收缩期容积分别增加36.8%、65.7%(P＜0.01,n=16),舒张期容积分别增加18.2%、33.8%(P＜0.01,n=16)。射血分数分别减小6.6%、9.3%(P＜0.05,n=16),短轴内径缩短率分别减小9.4%、12.3%(P＜0.05,n=16)。病理分析显示Meox1转基因小鼠心房心室明显大于野生型,心肌细胞不均匀肥大,间隙变大,心肌间质纤维增多。心脏肥大的分子标志物检测显示Nppa表达无明显变化,Nppb, Co11a1, Co13a1和Acta1表达增加,而Atp2a2降低。心脏转录因子GATA-4, NKX2-5, MEF2C表达升高。观察至9月龄,转基因小鼠和同窝阴性对照小鼠均未出现死亡。
结论Meox1在肥厚型和扩张型心肌病心脏中表达升高,Meox1转基因小鼠呈现心脏左室内径增加,收缩期容积和舒张期容积显著增大,射血分数及短轴缩短率减少等扩张性心肌病表型。说明成功建立了心脏特异表达的Meox1转基因小鼠,Meox1是参与心肌病病理发生的基因之一,为研究扩张型心肌病发病机制和药物研发提供了有价值的动物模型。
目的建立心脏特异表达CNN1转基因小鼠,研究CNN1对心脏发育及心肌病的调节作用。
方法Western blot检测碱性调宁蛋白于肥厚型心肌病cTnTR92Q及扩张型心肌病cTnTR141W模型小鼠心脏中的表达变化。Western blot检测碱性调宁蛋白于野生型C57BL／6J小鼠心脏中的时程表达。利用心脏特异启动子α-MHC构建转基因表达载体,显微注射法建立CNN1转基因小鼠,PCR鉴定转基因小鼠的基因型,Western blot检测碱性调宁蛋白在心脏组织中的表达,心脏超声检测转基因小鼠及野生小鼠的心脏结构和功能,H&E染色检测转基因小鼠心脏的病理改变。
结果在生理状态下,碱性调宁蛋白在小鼠心脏中均有较高的表达,且表达随年龄的增长没有明显的变化规律,在病理状态下,碱性调宁蛋白在扩张型心肌病小鼠的心脏组织表达降低。通过显微注射,建立了2个CNN1基因在心脏组织的表达水平明显高于同龄对照小鼠的转基因小鼠品系。与野生型小鼠相比,CNN1转基因小鼠品系收缩期左室内径(LVID, systolic)增加28%(P＜0.01,n=12),舒张期左室内径(LVID, diastolic)增加16.2%(P＜0.01,n=12),收缩期左室后壁厚度(LVPW,systolic)减小15.7%(P＜0.01,n=12),舒张期左室后壁厚度(LVPW, diastolic)减小21%(P＜0.01,n=12),射血分数EF (Ejection fraction)减小11.5%(P＜0.01,n=12),短轴内径缩短率FS (fraction shortening)减小14.6%(P＜0.05,n=12)。转基因小鼠心脏组织学检测显示,心肌细胞不均匀肥大,间隙变大,心室扩张。观察至9月龄转基因小鼠和同窝阴性对照小鼠均未出现死亡。
结论CNN1在心脏高表达引起转基因小鼠心脏左室内径增加,心室壁变薄,收缩期容积和舒张期容积显著增大,射血分数及短轴缩短率减少等扩张性心肌病表型,是参与心肌病病理发生的基因之一。
目的设计并构建靶向小鼠Meox1基因的miRNA干扰质粒,将干扰质粒表达载体显微注射法建立转基因小鼠,即Meox1基因沉默小鼠,为探索Meox1基因功能奠定基础。
方法摇菌扩增pLKO.1-shRNA-Meox1质粒(Open Biosystems,美国TRCN0000070603-TRCN0000070607),大量提取质粒。质粒转染293T细胞,用RT-PCR法验证五个质粒的沉默效果,选择两个沉默效果较好的质粒。按照Invitrogen公司的BLOCK-iTTM PolⅡmiR RNAi Expression Vector Kits说明书和沉默质粒的信息合成含有粘性末端的回文DNA序列,退火复性成双链,将双链片段插入pcDNATM 6.2-GW/EmGFP-miR表达载体,构建两套miRNA真核表达载体pcDNATM 6.2-GW/EmGFP-Meox1-miR1和pcDNATM6.2-GW/EmGFP-Meox1-miR2,并使用该载体带有的串联方法将两载体上的miRNA前体寡核苷酸序列串联成为pcDNATM6.2-GW/EmGFP-Meox1-miR1-miR2。将上述重组载体与pcDNA3.1(+)-Meox1表达质粒共转染293T细胞,RT-PCR鉴定其沉默效果。PCR法扩增EmGFP-Meox1-miR1+2,插入α-MHC启动子的下游,经测序并比对正确无突变碱基后,用NotⅠ将其线性化,SephedexG50柱纯化DNA片段,获得α-MHC启动的EmGFP-Meox1-miR1+2的转基因片段,注射前将转基因片段的浓度调整至5ng／μL,用显微注射法将线性化的转基因表达载体注射到C57BL／6J小鼠的受精卵中,用ICR小鼠作假孕受体,制备基因沉默小鼠。PCR鉴定基因沉默小鼠的基因型,Western blot检测GFP在心脏组织中的表达,RT-PCR检测Meox1在心脏组织中的表达。
结果经酶切及测序鉴定,针对小鼠Meox1基因的miRNA干扰质粒构建成功,并能够有效抑制共转染293T细胞的pcDNA3.1(+)-Meox1质粒的表达。通过转基因小鼠的筛选,得到1个基因沉默小鼠品系心脏GFP蛋白表达量与野生型鼠相比明显增加,Meox1表达量降低。
结论小鼠Meox1基因的靶向miRNA表达载体构建成功,成功建立了心脏特异的Meox1基因沉默小鼠,为进一步和心肌病小鼠模型杂交,研究该基因在心肌病发病中的作用提供了工具。
Background Cardiovascular disease has become the top killer of human health. Compare expression changes of cardiac gene of hypertrophic cardiomyopathy and dilated cardiomyopathy transgenic mice using gene expression microarray analysis, to research important modifier genes involved in regulation from gene mutation to the pathogenesis, which is important to understand the mechanism, treatment and drug research of cardiomyopathy. Gene expression microarray analysis showed that the expression levels of Meoxl gene in the heart tissues of cTnTR92Q and cTnTR141W transgenic mice were improved compared with that of wild-type mice, which suggests that Meox1 that encodes a member of a subfamily of non-clustered, diverged, antennapedia-like homeobox-containing genes may be involved in regulation of cardiomyopathy. This article is to establish the heart-specific expression Meox1 transgenic mice and to investigate its effect on the development of heart and cardiomyopathy.
Methods The expression levels of Meox1 in the heart tissues of both hypertrophic cardiomyopathy (HCM, cTnTR92Q) and dilated cardiomyopathy (DCM, cTnTR141W) mice were analyzed using gene expression microarray analysis and confirmed by reverse transcriptional polymerase chain reaction (RT-PCR) and western blot. The temporal expression patterns of Meox1 in the heart of WT mice were analyzed by western blot. The transgenic plasmid was constructed by inserting the murine Meox1 gene into the down-stream ofα-MHC promoter. The transgenic mice were generated by the method of microinjection. The genotype of transgenic lines was identified by PCR, and the expression levels of the Meoxl gene in the transgenic mice were detected by western blot. The pathologic and functional changes of heart were analyzed with echocardiography and light microscopy. The hypertrophy markers, Nppa, Nppb, Co11a1, Co13a1, Acta1, and Atp2a2, and the transcription factors, GATA-4, NKX2-5, MEF2C, were analyzed by RT-PCR. Survival datas of the experimental mice were recorded.
Results Gene expression microarray analysis and RT-PCR revealed that a higher expression level of Meox1 in HCM mice and DCM mice. The results of western blot showed that the expression of Meox1 was only detected in the heart of one-week wide-type mice, but its expression was up-regulated in the adult mice with HCM mice and DCM mice. The heart-specific transgenic C57BL/6J mice were established and used to analyze its effect on the heart with echocardiography. Compared with the wild type mice, both of the two lines of Meox1 transgenic mice showed significantly heart remodeling with increased left ventricular systolic diameter (15.6%or 24.2%, P<0.01, n = 16),increased left ventricular diastolic diameter (7.2% or 12.8%, P<0.01, n=16), increased systolic volume(36.8% or 65.7%, P<0.01, n=16), and increased diastolic volume(18.2% or 33.8%, P<0.01, n=16). Compared with the wild type mice, both of the two lines of Meoxl transgenic mice showed weaker heart function with decreased ejection fraction (6.6% or 9.3%, P<0.05, n= 16), and decreased fraction shortening (9.4% or 12.3%, P<0.05, n= 16). The heart of the Meoxl transgenic mice exhibited an enlarged ventricular chamber compared with that of the wild type mice under the light microscope. The expressions of hypertrophy markers, Nppb, Co11a1, Co13a1 and Acta1 were increased, however Atp2a2 was decreased in the heart of transgenic mice, there was no change with Nppa. The expressions of transcription factor GATA-4, NKX2-5, MEF2C were increased in the heart of transgenic mice. There was no death until 9 months of age both in the Meoxl transgenic mice and widetype mice.
Conclusions The expression level of Meoxl was up-regulated during cardiomyopathy. The Meoxl transgenic mice display a similar pathologic phenotype with the light dilated cardiomyopathy. It suggests that we established the heart-specific expression Meox1 transgenic mice successfully, Meoxl may be one of modifier genes which enhance pathogenesis of cardiomyopathy and the heart-specific expression Meox1 transgenic mice maybe a useful animal model for developmet of DCM and drug research.
Objective To establish the heart-specific CNN1 expression transgenic mice and to investigate its effect on the development of heart and cardiomyopathy.
Methods The expression of h1 calponin in the heart tissues of both hypertrophic cardiomyopathy (cTnTR92Q) and dilated cardiomyopathy (cTnTR141W) mice were analyzed by western blot. The temporal expression patterns of h1 calponin in the heart of WT mice were analyzed by western blot. The transgenic plasmid was constructed by inserting the human CNN1 gene into the down-stream ofα-MHC promoter. The transgenic mice were generated by the method of microinjection. The genotype of transgenic lines was identified by PCR, and the expression levels of the h1 calponin.were detected by western blot. The pathologic and functional changes of heart were analyzed with echocardiography. The pathologic changes were observed by microscopy.
Results The results of western blot showed that the expression of h1 calponin was detected in the heart of different-age-wide-type mice, but its expression was down-regulated in the mice with DCM. The heart-specific transgenic C57BL/6J mice were established and used to analyze its effect on heart with echocardiography. Compared with the wild type mice, CNN1 transgenic mice showed significantly heart remodeling with increased left ventricular systolic diameter (28%, P<0.01, n= 12), increased left ventricular diastolic diameter (16.2%, P<0.01, n= 12), decreased left ventricular posterior wall during systolic (15.7%, P<0.01, n= 12), and decreased left ventricular posterior wall during diastole (21%, P<0.01, n= 12). Compared with the wild type mice, CNN1 transgenic mice showed weaker heart function with decreased Ejection fraction (11.5%, P＜0.01, n= 12), and decreased fraction shortening (14.6%, P <0.05, n= 12). The heart of the CNN1 transgenic mouse exhibited an enlarged ventricular chamber when compared with that of the wild type mouse under the light microscope. There are no death until 9 months of age both of CNN1 transgenic mice and widetype mice.
Conclusions Over-expression of CNN1 in the heart of the transgenic mice caused dilated cardiomyopathy. It suggested that CNN1 may be one of modifier genes which enhance pathogenesis of cardiomyopathy.
Objective To design and construct miRNA expression vector targeting mouse Meoxl gene, and establish the heart specific Meox1 gene silence mice by the method of microinjection for function study of Meoxl gene with RNA interference technology.
Methods Amplify pLKO.1-shRNA-Meoxl plasmid (Open Biosystems, United States, TRCN0000070603-TRCN0000070607). Transfect 293T cell, compare the silence effect of the five plasmids by the RT-PCR to choose two better silence plasmids. According to Invitrogen's BLOCK-iT TM PolⅡmiR RNAi Expression Vector Kits Manual and informations of silence plasmids, synthesize the specific pre-miRNA single strand DNA oligos for mouse Meox1 gene containing palindromic cohesive end, then via annealing and ligating with pcDNATM 6.2-GW/EmGFP-miR in order, the two kinds of miRNA expression vectors are constructed which included pre-miRNA-1 and pre-miRNA-2 separately. Then the pre-miRNA-1 and pre-miRNA-2 are combined using the Chaining technology of the-vector. After the recombined plasmids were cotransfected into 293T cells with the constructed recombined plasmid of pcDNA3.1(+)-Meox1, the suppression effect targeting Meox1 is identified by RT-PCR. Amplify EmGFP-Meox1-miR1+2 by PCR, then inserte it into down-stream of a-MHC promoter, sequence and correct, then lineearized with NotⅠand purified with SephedexG50. Inject the DNA fragmentα-MHC-EmGFP-Meox1-miR1+2 which was adjusted to 5 ng/μL into C57BL/6J mice with microinjection, to prepare Meoxl gene silence mice. The genotype of transgenic lines was identified by PCR, and the expression levels of the GFP protein were detected by western blot, the expression levels of the Meox1 gene were detected by RT-PCR.
Results The miRNA expression plasmids were successfully constructed by the treatment of specific restriction enzymes and identification of sequencing analysis, and they could effectively inhibit the mRNA expressing of Meoxl from the recombined plasmid of pcDNA3.1(+)-Meox1 in vitro. The heart-specific Meox1 gene silence mice were established, and the GFP protein of one line of Meox1 gene silence mice is increased and Meox1 gene was decreaed compaired with the wide-type mice.
Conclusion The effective miRNA expression vector targeting mouse Meox1 has been successfully constructed, and the heart-specific Meox1 gene silence mice were successfully established. It can be used to cross with the DCM and HCM models to investigate the function of Meox1 gene on the development of cardiomyopathy.
方法cTnTR92Q及cTnTR141W转基因小鼠基因表达芯片及逆转录PCR和Western blot检测Meox1于肥厚型心肌病及扩张型心肌病模型小鼠心脏中的表达变化。Western blot检测Meox1于野生型C57BL/6J小鼠心脏中的时程表达。将Meox1基因插入心脏特异启动子α-MHC下游,构建转基因表达载体,通过显微注射法建立Meox1转基因C57BL／6J小鼠,PCR鉴定转基因小鼠的基因型,Western blot检测Meox1在心脏组织中的表达,心脏超声检测转基因小鼠及野生型小鼠的心脏结构和功能,病理组织学染色观察转基因小鼠心脏组织学及显微结构改变。逆转录PCR检测Meox1转基因小鼠的心脏肥大分子标志物及心脏转录因子,并记录转基因小鼠的生存状况。
结果cTnTR92Q和cTnTR141W转基因小鼠基因表达芯片及逆转录PCR结果显示Meox1基因在肥厚型心肌病及扩张型心肌病小鼠心脏中的表达升高。Western blot检测结果显示,在生理状态下,Meox1基因只在幼鼠心脏中表达,在病理状态下,Meox1基因在成年肥厚型和扩张型心肌病小鼠的心脏组织中表达均升高。通过显微注射,建立了两个Meox1基因在心脏组织的表达水平明显高于同龄对照小鼠的转基因小鼠品系。M型超声心动图显示,与野生型小鼠相比,两个Meox1转基因小鼠品系收缩期左室内径分别增加15.6%、24.2%(P＜0.01,n=16),舒张期左室内径分别增加7.2%、12.8%(P＜0.01,n=16),收缩期容积分别增加36.8%、65.7%(P＜0.01,n=16),舒张期容积分别增加18.2%、33.8%(P＜0.01,n=16)。射血分数分别减小6.6%、9.3%(P＜0.05,n=16),短轴内径缩短率分别减小9.4%、12.3%(P＜0.05,n=16)。病理分析显示Meox1转基因小鼠心房心室明显大于野生型,心肌细胞不均匀肥大,间隙变大,心肌间质纤维增多。心脏肥大的分子标志物检测显示Nppa表达无明显变化,Nppb, Co11a1, Co13a1和Acta1表达增加,而Atp2a2降低。心脏转录因子GATA-4, NKX2-5, MEF2C表达升高。观察至9月龄,转基因小鼠和同窝阴性对照小鼠均未出现死亡。
结论Meox1在肥厚型和扩张型心肌病心脏中表达升高,Meox1转基因小鼠呈现心脏左室内径增加,收缩期容积和舒张期容积显著增大,射血分数及短轴缩短率减少等扩张性心肌病表型。说明成功建立了心脏特异表达的Meox1转基因小鼠,Meox1是参与心肌病病理发生的基因之一,为研究扩张型心肌病发病机制和药物研发提供了有价值的动物模型。
目的建立心脏特异表达CNN1转基因小鼠,研究CNN1对心脏发育及心肌病的调节作用。
方法Western blot检测碱性调宁蛋白于肥厚型心肌病cTnTR92Q及扩张型心肌病cTnTR141W模型小鼠心脏中的表达变化。Western blot检测碱性调宁蛋白于野生型C57BL／6J小鼠心脏中的时程表达。利用心脏特异启动子α-MHC构建转基因表达载体,显微注射法建立CNN1转基因小鼠,PCR鉴定转基因小鼠的基因型,Western blot检测碱性调宁蛋白在心脏组织中的表达,心脏超声检测转基因小鼠及野生小鼠的心脏结构和功能,H&E染色检测转基因小鼠心脏的病理改变。
结果在生理状态下,碱性调宁蛋白在小鼠心脏中均有较高的表达,且表达随年龄的增长没有明显的变化规律,在病理状态下,碱性调宁蛋白在扩张型心肌病小鼠的心脏组织表达降低。通过显微注射,建立了2个CNN1基因在心脏组织的表达水平明显高于同龄对照小鼠的转基因小鼠品系。与野生型小鼠相比,CNN1转基因小鼠品系收缩期左室内径(LVID, systolic)增加28%(P＜0.01,n=12),舒张期左室内径(LVID, diastolic)增加16.2%(P＜0.01,n=12),收缩期左室后壁厚度(LVPW,systolic)减小15.7%(P＜0.01,n=12),舒张期左室后壁厚度(LVPW, diastolic)减小21%(P＜0.01,n=12),射血分数EF (Ejection fraction)减小11.5%(P＜0.01,n=12),短轴内径缩短率FS (fraction shortening)减小14.6%(P＜0.05,n=12)。转基因小鼠心脏组织学检测显示,心肌细胞不均匀肥大,间隙变大,心室扩张。观察至9月龄转基因小鼠和同窝阴性对照小鼠均未出现死亡。
结论CNN1在心脏高表达引起转基因小鼠心脏左室内径增加,心室壁变薄,收缩期容积和舒张期容积显著增大,射血分数及短轴缩短率减少等扩张性心肌病表型,是参与心肌病病理发生的基因之一。
目的设计并构建靶向小鼠Meox1基因的miRNA干扰质粒,将干扰质粒表达载体显微注射法建立转基因小鼠,即Meox1基因沉默小鼠,为探索Meox1基因功能奠定基础。
方法摇菌扩增pLKO.1-shRNA-Meox1质粒(Open Biosystems,美国TRCN0000070603-TRCN0000070607),大量提取质粒。质粒转染293T细胞,用RT-PCR法验证五个质粒的沉默效果,选择两个沉默效果较好的质粒。按照Invitrogen公司的BLOCK-iTTM PolⅡmiR RNAi Expression Vector Kits说明书和沉默质粒的信息合成含有粘性末端的回文DNA序列,退火复性成双链,将双链片段插入pcDNATM 6.2-GW/EmGFP-miR表达载体,构建两套miRNA真核表达载体pcDNATM 6.2-GW/EmGFP-Meox1-miR1和pcDNATM6.2-GW/EmGFP-Meox1-miR2,并使用该载体带有的串联方法将两载体上的miRNA前体寡核苷酸序列串联成为pcDNATM6.2-GW/EmGFP-Meox1-miR1-miR2。将上述重组载体与pcDNA3.1(+)-Meox1表达质粒共转染293T细胞,RT-PCR鉴定其沉默效果。PCR法扩增EmGFP-Meox1-miR1+2,插入α-MHC启动子的下游,经测序并比对正确无突变碱基后,用NotⅠ将其线性化,SephedexG50柱纯化DNA片段,获得α-MHC启动的EmGFP-Meox1-miR1+2的转基因片段,注射前将转基因片段的浓度调整至5ng／μL,用显微注射法将线性化的转基因表达载体注射到C57BL／6J小鼠的受精卵中,用ICR小鼠作假孕受体,制备基因沉默小鼠。PCR鉴定基因沉默小鼠的基因型,Western blot检测GFP在心脏组织中的表达,RT-PCR检测Meox1在心脏组织中的表达。
结果经酶切及测序鉴定,针对小鼠Meox1基因的miRNA干扰质粒构建成功,并能够有效抑制共转染293T细胞的pcDNA3.1(+)-Meox1质粒的表达。通过转基因小鼠的筛选,得到1个基因沉默小鼠品系心脏GFP蛋白表达量与野生型鼠相比明显增加,Meox1表达量降低。
结论小鼠Meox1基因的靶向miRNA表达载体构建成功,成功建立了心脏特异的Meox1基因沉默小鼠,为进一步和心肌病小鼠模型杂交,研究该基因在心肌病发病中的作用提供了工具。
Background Cardiovascular disease has become the top killer of human health. Compare expression changes of cardiac gene of hypertrophic cardiomyopathy and dilated cardiomyopathy transgenic mice using gene expression microarray analysis, to research important modifier genes involved in regulation from gene mutation to the pathogenesis, which is important to understand the mechanism, treatment and drug research of cardiomyopathy. Gene expression microarray analysis showed that the expression levels of Meoxl gene in the heart tissues of cTnTR92Q and cTnTR141W transgenic mice were improved compared with that of wild-type mice, which suggests that Meox1 that encodes a member of a subfamily of non-clustered, diverged, antennapedia-like homeobox-containing genes may be involved in regulation of cardiomyopathy. This article is to establish the heart-specific expression Meox1 transgenic mice and to investigate its effect on the development of heart and cardiomyopathy.
Methods The expression levels of Meox1 in the heart tissues of both hypertrophic cardiomyopathy (HCM, cTnTR92Q) and dilated cardiomyopathy (DCM, cTnTR141W) mice were analyzed using gene expression microarray analysis and confirmed by reverse transcriptional polymerase chain reaction (RT-PCR) and western blot. The temporal expression patterns of Meox1 in the heart of WT mice were analyzed by western blot. The transgenic plasmid was constructed by inserting the murine Meox1 gene into the down-stream ofα-MHC promoter. The transgenic mice were generated by the method of microinjection. The genotype of transgenic lines was identified by PCR, and the expression levels of the Meoxl gene in the transgenic mice were detected by western blot. The pathologic and functional changes of heart were analyzed with echocardiography and light microscopy. The hypertrophy markers, Nppa, Nppb, Co11a1, Co13a1, Acta1, and Atp2a2, and the transcription factors, GATA-4, NKX2-5, MEF2C, were analyzed by RT-PCR. Survival datas of the experimental mice were recorded.
Results Gene expression microarray analysis and RT-PCR revealed that a higher expression level of Meox1 in HCM mice and DCM mice. The results of western blot showed that the expression of Meox1 was only detected in the heart of one-week wide-type mice, but its expression was up-regulated in the adult mice with HCM mice and DCM mice. The heart-specific transgenic C57BL/6J mice were established and used to analyze its effect on the heart with echocardiography. Compared with the wild type mice, both of the two lines of Meox1 transgenic mice showed significantly heart remodeling with increased left ventricular systolic diameter (15.6%or 24.2%, P<0.01, n = 16),increased left ventricular diastolic diameter (7.2% or 12.8%, P<0.01, n=16), increased systolic volume(36.8% or 65.7%, P<0.01, n=16), and increased diastolic volume(18.2% or 33.8%, P<0.01, n=16). Compared with the wild type mice, both of the two lines of Meoxl transgenic mice showed weaker heart function with decreased ejection fraction (6.6% or 9.3%, P<0.05, n= 16), and decreased fraction shortening (9.4% or 12.3%, P<0.05, n= 16). The heart of the Meoxl transgenic mice exhibited an enlarged ventricular chamber compared with that of the wild type mice under the light microscope. The expressions of hypertrophy markers, Nppb, Co11a1, Co13a1 and Acta1 were increased, however Atp2a2 was decreased in the heart of transgenic mice, there was no change with Nppa. The expressions of transcription factor GATA-4, NKX2-5, MEF2C were increased in the heart of transgenic mice. There was no death until 9 months of age both in the Meoxl transgenic mice and widetype mice.
Conclusions The expression level of Meoxl was up-regulated during cardiomyopathy. The Meoxl transgenic mice display a similar pathologic phenotype with the light dilated cardiomyopathy. It suggests that we established the heart-specific expression Meox1 transgenic mice successfully, Meoxl may be one of modifier genes which enhance pathogenesis of cardiomyopathy and the heart-specific expression Meox1 transgenic mice maybe a useful animal model for developmet of DCM and drug research.
Objective To establish the heart-specific CNN1 expression transgenic mice and to investigate its effect on the development of heart and cardiomyopathy.
Methods The expression of h1 calponin in the heart tissues of both hypertrophic cardiomyopathy (cTnTR92Q) and dilated cardiomyopathy (cTnTR141W) mice were analyzed by western blot. The temporal expression patterns of h1 calponin in the heart of WT mice were analyzed by western blot. The transgenic plasmid was constructed by inserting the human CNN1 gene into the down-stream ofα-MHC promoter. The transgenic mice were generated by the method of microinjection. The genotype of transgenic lines was identified by PCR, and the expression levels of the h1 calponin.were detected by western blot. The pathologic and functional changes of heart were analyzed with echocardiography. The pathologic changes were observed by microscopy.
Results The results of western blot showed that the expression of h1 calponin was detected in the heart of different-age-wide-type mice, but its expression was down-regulated in the mice with DCM. The heart-specific transgenic C57BL/6J mice were established and used to analyze its effect on heart with echocardiography. Compared with the wild type mice, CNN1 transgenic mice showed significantly heart remodeling with increased left ventricular systolic diameter (28%, P<0.01, n= 12), increased left ventricular diastolic diameter (16.2%, P<0.01, n= 12), decreased left ventricular posterior wall during systolic (15.7%, P<0.01, n= 12), and decreased left ventricular posterior wall during diastole (21%, P<0.01, n= 12). Compared with the wild type mice, CNN1 transgenic mice showed weaker heart function with decreased Ejection fraction (11.5%, P＜0.01, n= 12), and decreased fraction shortening (14.6%, P <0.05, n= 12). The heart of the CNN1 transgenic mouse exhibited an enlarged ventricular chamber when compared with that of the wild type mouse under the light microscope. There are no death until 9 months of age both of CNN1 transgenic mice and widetype mice.
Conclusions Over-expression of CNN1 in the heart of the transgenic mice caused dilated cardiomyopathy. It suggested that CNN1 may be one of modifier genes which enhance pathogenesis of cardiomyopathy.
Objective To design and construct miRNA expression vector targeting mouse Meoxl gene, and establish the heart specific Meox1 gene silence mice by the method of microinjection for function study of Meoxl gene with RNA interference technology.
Methods Amplify pLKO.1-shRNA-Meoxl plasmid (Open Biosystems, United States, TRCN0000070603-TRCN0000070607). Transfect 293T cell, compare the silence effect of the five plasmids by the RT-PCR to choose two better silence plasmids. According to Invitrogen's BLOCK-iT TM PolⅡmiR RNAi Expression Vector Kits Manual and informations of silence plasmids, synthesize the specific pre-miRNA single strand DNA oligos for mouse Meox1 gene containing palindromic cohesive end, then via annealing and ligating with pcDNATM 6.2-GW/EmGFP-miR in order, the two kinds of miRNA expression vectors are constructed which included pre-miRNA-1 and pre-miRNA-2 separately. Then the pre-miRNA-1 and pre-miRNA-2 are combined using the Chaining technology of the-vector. After the recombined plasmids were cotransfected into 293T cells with the constructed recombined plasmid of pcDNA3.1(+)-Meox1, the suppression effect targeting Meox1 is identified by RT-PCR. Amplify EmGFP-Meox1-miR1+2 by PCR, then inserte it into down-stream of a-MHC promoter, sequence and correct, then lineearized with NotⅠand purified with SephedexG50. Inject the DNA fragmentα-MHC-EmGFP-Meox1-miR1+2 which was adjusted to 5 ng/μL into C57BL/6J mice with microinjection, to prepare Meoxl gene silence mice. The genotype of transgenic lines was identified by PCR, and the expression levels of the GFP protein were detected by western blot, the expression levels of the Meox1 gene were detected by RT-PCR.
Results The miRNA expression plasmids were successfully constructed by the treatment of specific restriction enzymes and identification of sequencing analysis, and they could effectively inhibit the mRNA expressing of Meoxl from the recombined plasmid of pcDNA3.1(+)-Meox1 in vitro. The heart-specific Meox1 gene silence mice were established, and the GFP protein of one line of Meox1 gene silence mice is increased and Meox1 gene was decreaed compaired with the wide-type mice.
Conclusion The effective miRNA expression vector targeting mouse Meox1 has been successfully constructed, and the heart-specific Meox1 gene silence mice were successfully established. It can be used to cross with the DCM and HCM models to investigate the function of Meox1 gene on the development of cardiomyopathy.
引文
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