扩张型心肌病转基因小鼠模型的建立和心肌病修饰基因的筛选

【作者】 冯娟；

【导师】 秦川； 张连峰；

【作者基本信息】 中国协和医科大学 ， 病理学与病理生理学， 2008， 博士

【摘要】 目的建立cTnT^R141W扩张型心肌病的转基因小鼠模型。方法把cTnT^R141W基因插入α-MHC启动子下游,构建转基因表达载体,通过显微注射法建立cTnT^R141W转基因C57BL/6J小鼠。PCR鉴定cTnT^R141W转基因小鼠的基因表型,Realtime PCR检测基因的拷贝数,Northern blotting检测基因表达,组织学和电镜检测cTnT^R141W转基因小鼠心脏的病理改变。免疫组织化学检测突变的cTnT蛋白的定位。超声心动图检测cTnT^R141W转基因小鼠心脏的结构和功能。记录cTnT^R141W转基因小鼠的生存状况。逆转录PCR检测cTnT^R141W转基因小鼠的心脏肥大分子标志物。结果建立了3个系的cTnT^R141W转基因小鼠。3个系的基因拷贝数分别是15、20和59拷贝。cTnT^R141W的表达水平是内源性cTnT的1.5倍～2.0倍。cTnT^R141W转基因小鼠心脏组织学检测显示,室壁变薄,心室扩张,间质纤维化。电镜检测显示肌纤维变细,部分溶解。免疫组织化学检测显示,突变的cTnT蛋白聚集在cTnT^R141W转基因小鼠心脏的肌小节中。M型超声心动图检测显示,左室收缩末期内径和左室舒张末期内径明显增加58.3%（p＜0.01）和27.8%（p＜0.01）,左室收缩期容积和左室舒张期容积显著增加191.2%（p＜0.01）和75.9%（p＜0.01）,射血分数和短轴缩短率分别降低39.9%（p＜0.01）和45.4%（p＜0.01）。从2月龄到8月龄,转基因小鼠左室收缩末期容积和左室舒张末期容积增加2倍左右,EF%降低36.7%,FS%降低39.7%。4月龄后转基因小鼠出现早期死亡,至8月龄早期死亡率达11.1%。心脏肥大的分子标记物检测显示Nppb表达增加29%,Actal增加4倍,而Atp2a2降低58%。结论cTnT^R141W转基因小鼠的全心扩大,室壁变薄,心肌细胞肥大,间质纤维化以及心肌收缩力下降,和人类扩张型心肌病具有类似的病理表型。成功建立了cTnT^R141W扩张型心肌病转基因小鼠模型,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。目的比较cTnT^R92Q和cTnT^R141W转基因小鼠,筛选肥厚型心肌病和扩张型心肌病的重要修饰基因。方法M型超声心动图检测cTnT^R92Q和cTnT^R141W转基因小鼠心脏的结构和功能。病理检测心脏组织学。透射电镜检测心脏的超微结构。免疫组织化学检测突变的cTnT蛋白的定位。基因表达芯片分析cTnT^R92Q和cTnT^R141W转基因小鼠心脏的基因表达差异,RT-PCR验证基因芯片分析结果。结果cTnT^R92Q转基因小鼠显示室壁增厚,心腔缩小,心肌细胞排列紊乱,间质纤维化,心肌纤维变粗;射血分数和短轴缩短率增加,左室容积显著下降。cTnT^R141W转基因小鼠显示室壁变薄,心室扩张,间质纤维化,心肌纤维变细,部分溶解;射血分数和短轴缩短率下降,左室容积显著增加。突变的cTnT蛋白在cTnT^R92Q和cTnT^R141W转基因小鼠心肌细胞的肌小节中分布不同。基因表达芯片分析显示1200个基因表现出不同的调控。RT-PCR验证30个表达改变的基因,包括肌小节蛋白基因,细胞骨架蛋白基因,钙调节相关蛋白基因,离子通道蛋白基因,细胞外基质基因,信号传导相关基因和未知基因等。结论cTnT^R92Q和cTnT^R141W突变蛋白的表达导致小鼠HCM和DCM两种相反的病理表型。在相同的遗传背景下,通过全基因表达芯片发现多种基因在HCM和DCM心脏中有相反的表达调控。其中Gas5、Riok1、Ask1、Meox1、Ryk、Dkk3和Wif-1等细胞外因子或表面受体有可能是控制心肌细胞向相反方向分化的重要修饰基因,为进一步认识心脏生长发育和心肌病的发病机制提供线索。目的探讨肝素结合类表皮生长因子对心脏的作用。方法Western blotting检测野生型和DCM模型小鼠心脏中HB-EGF的表达,HE和Masson染色检测DCM模型小鼠心脏组织学,Realtime PCR检测DCM模型小鼠心脏Ⅰ型胶原和Ⅲ型胶原的表达。显微注射法建立HB-EGF转基因C57BL/6J小鼠,PCR鉴定基因表型,Western blotting检测表达水平。病理检测HB-EGF转基因小鼠心脏的组织学,Realtime PCR检测HB-EGF转基因小鼠心脏Ⅰ型胶原和Ⅲ型胶原的表达。原代培养C57BL/6J小鼠心肌成纤维细胞,重组人HB-EGF（10ng/mL）刺激心肌成纤维细胞后,BrdU免疫组织化学法检测心肌成纤维细胞的增殖,Realtime PCR检测Ⅰ型胶原和Ⅲ型胶原的表达,Westernblot检测丝裂原活化蛋白激酶（MAPKs）,ERK1/2和JNK信号通路的变化。M型超声心动图检测HB-EGF转基因小鼠心脏的结构和功能。BrdU免疫组织化学法检测HB-EGF转基因小鼠心肌细胞的增殖。心肌细胞计数检测HB-EGF转基因小鼠心脏细胞总数。双相电泳检测HB-EGF转基因小鼠心脏中ERK1/2和JNK的磷酸化位点。重组人HB-EGF刺激H9c2（2-1）细胞,Western blot检测ERK1/2和JNK信号通路的变化。结果野生型小鼠心脏HB-EGF的表达随月龄增加而逐渐降低,而DCM转基因小鼠心脏中HB-EGF随月龄增加病情加重而增加,并伴随有间质纤维化以及Ⅰ型胶原和Ⅲ型胶原的表达增加（1.7倍和1.6倍）。用HB-EGF转基因小鼠研究HB-EGF的功能,发现HB-EGF可增加心脏间质纤维化,Ⅰ型胶原和Ⅲ型胶原的表达（各1.8倍）。用重组人HB-EGF刺激原代培养心肌成纤维细胞,30 min即引起Ⅰ型胶原和Ⅲ型胶原表达显著增加,24h引起心肌成纤维细胞明显增殖,10 min即可引起ERK1/2,JNK磷酸化水平增加。同野生型心脏比较,HB-EGF转基因小鼠左室收缩末期内径比野生型增加11.5%,左室收缩末期容积增加28.4%。HB-EGF转基因小鼠心肌细胞增殖明显,心脏细胞总数增加。HB-EGF转基因小鼠心脏ERK1/2和JNK的磷酸化位点与野生型不同。用重组人HB-EGF刺激H9c2（2-1）细胞10 min即可引起ERK1/2和JNK磷酸化水平增加。结论结果证实HB-EGF参与DCM的发生。HB-EGF促进间质纤维化及Ⅰ型胶原和Ⅲ型胶原的表达,也促进心肌细胞增殖,导致心脏细胞总数增加。研究结果提示HB-EGF可能通过MAPKs信号通路发挥上述作用。目的建立红色荧光转基因小鼠,为活体荧光影像系统建立重要的实验动物模型。方法把DsRed-Express基因插入chickenβ-actin强启动子下游构建转基因载体,建立红色荧光转基因C57BL/6J小鼠。PCR鉴定红色荧光转基因小鼠的基因表型,活体荧光影像系统分析红色荧光转基因小鼠,荧光显微镜检测红色荧光转基因小鼠全身组织器官的病理改变。结果建立了3个系的红色荧光转基因小鼠。活体荧光成像系统分析转基因小鼠呈现红色荧光。经荧光显微镜观察,DsRed-Express转基因小鼠的红色荧光蛋白在多个组织器官中才表达,尤其在胰腺、肝脏、肾脏和脾脏等器官表达量较高。结论DsRed-Express基因在转基因小鼠中系统性高表达,成功建立了红色荧光转基因小鼠。DsRed-Express转基因小鼠将成为活体荧光影像系统的重要实验动物模型。目的建立神经组织特异表达绿色荧光蛋白的转基因小鼠,为研究神经系统提供可以荧光示踪的工具动物。方法把EGFP基因插入PDGF B-链启动子下游构建转基因载体,用显微注射的方法建立转基因C57BL/6J小鼠。PCR鉴定转基因小鼠的基因表型,对阳性转基因小鼠的脑组织进行矢状面冰冻切片,每两张连续切片为一组,共48组,分别进行HE染色,显微镜观察组织结构,荧光体视镜及荧光显微镜观察绿色荧光蛋白在神经组织的表达。结果在8个首建品系中筛选出一个神经组织高表达绿色荧光蛋白的转基因小鼠系。观察到绿色荧光蛋白在大脑皮层、海马、丘脑、小脑及脑干等部位表达。结论建立了稳定遗传的神经组织特异表达绿色荧光蛋白转基因小鼠品系,为研究神经系统生理及病理提供了可以荧光示踪的模型动物。更多还原

【Abstract】 Objective To establish the transgenic mouse of cTnT^R141W gene to make an animal model of dilated cardiomyopathy.Methods A transgenic plasmid was constructed by inserting the cTnT^R141W gene driven by theα-MHC promoter and the genotyping of the transgenic mice was detected by the polymerase chain reaction（PCR）.The copy number of the transgenic gene was determined by Realtime PCR and the expression level of the gene was determined with Northern blotting and confirmed by HpaⅡrestriction endonuclease digestion Of reverse transcriptional PCR product made with a pair of primers amplifying both the transgenic and endogenous gene.Pathologic changes were observed by light microscopy and transmission electronic microscopy and analyzed with echocardiography.The localization of the mutant human cTnT protein was detected by Immunohistochemistry.The hypertrophy markers,Nppa,Nppb,Actal, and Atp2a2 were analyzed by reverse transcriptional PCR.Survival data of the experimental mice were recorded.Results Transgenic mice carrying the cTnT^R141W mutation were established and the copy number of the transgenic gene was found to be 15,20 and 59 in the three respective lines identified.The expression level of the cTnT^R141W was 1.5-to 2.0-fold that of the endogenous cTnT gene.The heart of the cTnT^R141W transgenic mouse exhibited a thinner ventricular wall and an enlarged ventricular chamber when compared with these aspects of the wild type mouse under the light microscope.Interstitial fibrosis was also observed in the 4 months old cTnT^R141W heart tissue.In the transgenic heart tissue,the elongated and lysed myofrils were observed under the transmission electronic microscope.The mutant human cTnT protein was’assembled in the sarcomere of the transgenic heart.The ejection fraction （EF%）,fractional shortening（FS%） and movement of the ventricular wall were significantly decreased in the cTnT^R141W mice.Furthermore,the LV volume was increased,and EF%and FS%were decreased continuously in the age range of 2 to 8 months.Immature death occurred after 4 months of age and the immature death rate was 11.1%before 8 months of age in the cTnT^R141W mice.Nppb was shown to be increased 29%,and Actal increased 4 fold,however Atp2a2 was decreased 58% in transgenic heart.Conclusions The expression of mutant cTnT^R141W in the mouse heart caused ventricular chamber enlargement,systolic dysfunction,myocardial hypertrophy,and interstitial fibrosis.This mouse model can display a similar pathologic phenotype with the human DCM.Our results suggest that the cTnT^R141W gene is a causal factor for DCM and that the cTnT^R141W transgenic mouse is a useful animal model for the study of human DCM. Objective To compare the transgenic mice of cTnT^R92Q gene and cTnT^R141W gene and screen the modifier genes in the pathogenesis of hypertrophic cardiomyopathy and dilated cardiomyopathy.Methods The cardiac structure and function of the transgenic mice of cTnT^R92Q and cTnT^R141W were compared with M-mode echocardiography and by light microscopy and transmission electronic microscopy oberservation.The localization of the mutant human cTnT protein was detected by Immunohistochemistry.The gene expression patterns of cTnT^R92Q and cTnT^R141W hearts were analyzed using Gene expression microarray analysis and confirmed by reverse transcriptional PCR.Results The cTnT^R920 transgenic mice showed hypertrophic ventricular wall, reduced ventricular chamber,myocyte disarray,interstitial fibrosis,hypertrophic cardiomyocytes,increased Ejection fraction（EF%） and increased Fractional shortening（FS%） and decreased LV volume.However the cTnT^R141W transgenic heart exhibited a thinner ventricular wall,an enlarged ventricular chamber, interstitial fibrosis,elongated and lysed myofrils,decreased EF%,decreased FS% and increased LV volume.The two different mutant human cTnT proteins were also showed different localization in the sarcomere.Gene expression microarray analysis revealed that more than 1200 genes were showed opposite regulation compared the two transgenic hearts.The opposite regulated genes included the genes of sarcomeric proteins,cytoskeletal proteins,calcium-regulation proteins,ion channel proteins and extracellular matrix proteins,the genes associated with signal transduction and unknown genes.Thirty of them were verified by RT-PCR.Conclusions The expression of mutant cTnT^R92Q and cTnT^R141W iri the mouse heart caused opposite pathologic phenotypes.The gene expression microarry analysis using the same genetic background mice models indicated that a certain number of genes were regulated in an opposite way.The extracellular factor genes and receptor genes,as Gas5,Riokl,Askl,Meoxl,Ryk,Dkk3,and Wif-1 were strongly showed different expression in the opposite model hearts.These results suggest that those genes could be the important modifer genes,which caused the myocyte differentiation in a different way and play role in the HCM and DCM. Objective To investigate the function of the heparin-binding EGF-like growth factor （HB-EGF） on heart.Methods The expression level of HB-EGF was detected by Western blotting between the wild type and DCM transgenic hearts.The pathologic changes of DCM transgenic heart were observed under the microscope,and the expression of collagenⅠand collagenⅢwas detected by Realtime PCR.The HB-EGF transgenic mice were produced by microinjection method,and the expression of HB-EGF was detected with PCR and Western blotting.The histology of the HB-EGF transgenic heart was observed,and the expression of collagenⅠand collagenⅢwas also detected.The primary mouse cardiac fibroblasts were cultured,then stimulated with the recombinant human HB-EGF.The proliferation of primary mouse cardiac fibroblasts was examined by BrdU staining.The expression of collagenⅠand collagenⅢwas detected by Realtime PCR,and the phorsphoralation of ERK1/2 and JNK were detected by western blotting.The structure and function of the HB-EGF transgenic heart were analyzed with M-mode echocardiography.The proliferation of cardiomyocytes of HB-EGF transgenic mice was examined by BrdU staining.The total cell number of heart was counted according to the genomic DNA amount from the heart tissue.The phosphoraltion sites of ERK1/2 and JNK in wild type and transgenic hearts were detected by two-dimensional gel electrophoresis.The recombinant human HB-EGF stimulated the H9c2（2-1） cells,and then the phosphoraltion of ERK 1/2 and JNK were detected by westem blotting.Results The expression level of HB-EGF was decreased in the wild type heart with aging while the expression level of HB-EGF was increased in the heart with development of the DCM following increased interstitial content and increased collagenⅠand collagenⅢby 1.7-fold and 1.6-fold.The HB-EGF transg’enic C57BL/6J mouse was established for invetagation of the effect of HB-EGF on heart, which showed increased interstitial fibrosis,increased expression of collagenⅠand collagenⅢby 1.8-fold.Isolated the mouse cardiac fibroblasts and stimulated with the recombinant human HB-EGF,the primary cardiac fibroblasts increased the expression of collagenⅠand collagenⅢfor 30 min and proliferated for 24h after stimulation.A strong phosphorylation both of ERK1/2 and JNK was also induced by the stimulati6n at 10 min.Compared with the wild type heart,the HB-EGF transgenic heart showed enlarged ventricular chamber with increased proliferation of cardiomyocytes and resulted in the increasing of total cell number of heart.The phosphoraltion sites of ERK1/2 and JNK in wild type and transgenic hearts were different.While the rat cardiac myocytes cell line,H9c2（2-1） cell,was also showed a strong phosphorylation of ERK1/2 and JNK following the HB-EGF sitimaulation for 10 min.Conclusions The results indicated that HB-EGF was involed in the development of the DCM.The HB-EGF increased the interstitial fibrosis and the expression of collagenⅠand collagenⅢ.The HB-EGF was also stimulated the proliferation of the myocytes and resulted in the increasing of total cell number of heart.The results suggest that the HB-EGF play roles in heart through the MAPKs signaling pathway. Objective The transgenic mice of DsRed-Express gene was constructed for establishment of the mouse model,which will be used for in vivo fluorescence imaging analysis.Methods The transgenic plasmid was constructed by inserting the DsRed-Express gene under the control of chickenβ-actin powerful promoter.The transgenic mice were genotyped by PCR.The expression of the DsRed-Express gene was observed under in vivo fluorescence imaging system and fluorescent microscope.Results Three lines of DsRed-Express transgenic C57BL/6J mice were established. The red fluorescent protein was expressed in a certain number of tissues,such as heart,liver,spleen,-pancreas,kidney,lung and uterus in the DsRed-Express transgenic mouse.Conclusions The red fluorescent expression analysis indicated that the DsRed-Express transgenic mouse was established and it will be an important mouse model for in vivo fluorescence imaging analysis. Objective The nervous tissues specific expressed green fluorescent protein transgenic mouse was to establish and to make an animal model for the research of nervous system by fluorescent imaging.Methods The transgenic plasmid was constructed by inserting the EGFP gene under the control of PDGF B-chain promoter and the transgenic C57BL/6J mice was established by microinjection method.The transgenic mice were genotyped by PCR. The total of 48 pairs of freezing sections of transgenic mice were prepared in the sagittal plane,and the every two adjacent sections were observed under microscopes and compared with H&E staining and fluorescent imaging.Results One transgenic line with high levels of green fluorescent protein expression was detected from eight transgenic lines.Green fluorescent protein expression was observed in the tissues of cerebrum,hippocampus,thalamencephalon,cerebellum, and brain stem.Conclusions The results indicated that a brain tissue specific EGFP transgenic mouse line was established.The results suggested that it will be an available animal model for the research of nervous tissues by fluorescent imaging.更多还原