PID1心脏组织特异性表达转基因小鼠的构建及表型分析

发布时间：2018-04-20 13:02

本文选题：PID1 + 组织特异性转基因小鼠　；参考：《南京医科大学》2012年硕士论文

【摘要】：PID1 (phosphotyrosine interaction domain containing 1)基因,又称NYGGF4基因,是本研究小组应用抑制性差减杂交(suppression subtractive hybridization, SSH)技术筛选肥胖与正常人网膜脂肪组织中差异表达基因时获得并克隆到的一条人类全长新基因(GenBank登录号：AY317148)。前期研究发现：1)该基因编码的蛋白定位于胞浆,存在多个丝氨酸、苏氨酸的磷酸化位点,另在蛋白的C端存在一个磷酸酪氨酸的作用位点(Phosphotyrosine-binding domain, PTB Domain),提示该蛋白可能参与胞内信号转导；2)PID1基因在心肌、骨骼肌、脂肪等组织中呈现较高表达；3)PID1基因过表达能够抑制信号转导途径PI3K-AKT信号通路的活性。由于基因的表达谱是揭示基因功能的一个重要线索,如果一个基因在某种组织中表达量很高,那么很可能该基因在该组织中行使功能,因此鉴于PID1基因在心脏组织中呈现高表达,同时PI3K-AKT信号通路与心脏发育、心肌肥厚、心脏保护等功能密切相关,提示该基因可能在心脏发育、心肌肥厚、心脏保护中起重要作用,本研究采用转基因技术构建了PIDl心脏组织特异性表达转基因小鼠的模型,以探讨PID1基因在心脏发育、心肌肥厚、心脏保护中的作用。第一部分PID1心脏组织特异性转基因小鼠的构建目的：构建PID1心脏组织特异性表达载体PBSⅡSK-aMHC-PID1,建立PID1心脏组织特异性转基因小鼠。方法：RT-PCR法克隆小鼠PID1基因CDS,将PID1基因插入心脏特异表达基因αMHC启动子的下游,构建心脏组织特异性表达载体。采用Notl将PBSⅡSK-aMHC-PID1线性化,通过显微注射法将线性化的心脏组织特异性表达载体显微注射到小鼠受精卵原核。PCR法检测转基因首建鼠的基因型,Western blot法验证鼠源性PID1基因在转基因小鼠心脏组织中的特异性高表达。结果：1)成功构建了心脏组织特异性表达载体PBSⅡSK-aMHC-PID1; 2)建立了2个株的心脏组织特异性表达PID1转基因小鼠。结论：PID1心脏组织特异性转基因小鼠构建成功。第二部分对PID1心脏组织特异性转基因小鼠的初步观察目的：初步观察PID1心脏组织特异性转基因小鼠的一般状况,评价小鼠在心脏重量指数、整体外观、功能等方面的变化。方法：以PID1心脏组织特异性转基因(transgenic mice, TG)小鼠和同年龄、同性别野生型(wild type, WT)小鼠为研究对象,从出生后开始观察小鼠的生长发育等一般情况；2个月时(性成熟)称量心脏重量和体重,计算心脏重量指数(HW/BW)；光镜下观察心脏的整体外观变化；超声心动图检测评估小鼠心脏功能变化。结果：1)小鼠一般状况：观察期间TG小鼠生长良好、活动自如、饮食正常,与WT小鼠相比无明显异常,可存活一年半以上甚至更长时间；2)TG小鼠与WT小鼠的心脏重量指数无显著性差异；3)TG小鼠和WT小鼠的心脏外观无明显差异；4)超声心动图检测发现TG小鼠的心脏功能也无明显变化。结论：PID1心脏组织特异转基因小鼠与野生型小鼠相比,在生长发育、寿命等方面均无明显变化,心脏重量指数、整体外观、心脏功能与WT小鼠比较也无明显差异。第三部分 PIDl心脏组织特异性转基因小鼠异丙肾上腺素诱导实验后的表型分析目的:比较PID1心脏组织特异性转基因小鼠异丙肾上腺素诱导实验下的心脏重量指数变化。方法：以2月龄、雌性PID1心脏组织特异性转基因(TG)小鼠和野生型(WT)小鼠为研究对象,TG小鼠和WT小鼠均随机分为实验组和对照组。实验组TG小鼠和WT小鼠均腹腔注射异丙肾上腺素(isoproterenol, ISO) 0.06mg/g体重/d,对照组TG小鼠和WT小鼠腹腔注射等体积生理盐水,持续6天,第7天称量小鼠的心脏重量和体重,计算心脏重量指数。结果：1)ISO处理后的WT小鼠和TG小鼠的心脏重量指数分别为6.14±0.39和6.96±1.13,两组间无显著差异(P0.05)；2)生理盐水注射的WT鼠和TG鼠的心脏重量指数分别为4.60±0.46和4.83±0.29,两组间也无显著差异(P0.05); 3) ISO处理后的WT鼠和TG鼠分别与WT鼠和TG鼠对照组(生理盐水组)比较,结果显示心脏重量指数分别增加了33.5%和44.1%,有统计学差异(P0.01)。结论：异丙肾上腺素诱导实验下,PID1心脏组织特异转基因小鼠的心脏重量指数与WT小鼠相比无显著差异。第四部分 PID1心脏组织特异性转基因小鼠主动脉弓缩窄实验后的表型分析目的：观察PID1心脏组织特异性转基因小鼠主动脉弓缩窄实验后心脏结构及功能变化,并初步探讨其可能机制。方法：以2月龄、雄性PID1心脏组织特异性转基因(TG)小鼠和野生型(WT)小鼠为研究对象,TG小鼠和WT小鼠均随机分为主动脉弓缩窄手术(transverse aortic constriction, TAC)组和假手术(sham)组。术前、术后1周、术后4周采用超声心动图检测小鼠心脏功能；术后1周、术后4周分别称量心脏重量和体重,计算心脏重量指数；HE染色观察术后小鼠心脏组织形态学改变；Masson染色法观察心肌纤维化及心脏重构。结果：1)TAC术后1周,TG小鼠的心脏体积明显增大；HE染色显示其左心室明显增大,但室壁厚度尚未呈现显著改变；心脏重量指数(HW/BW)显著高于WT小鼠；Masson染色显示尚未发生心肌纤维化改变；超声心动图未见明显异常,超声心动图中反应心脏功能的指标尚无显著性变化；2)TAC术后4周,TG小鼠的心脏体积出现显著增大；HE染色结果显示左心室明显增大、室壁变薄；Masson染色结果显示心脏组织严重纤维化,纤维化弥漫分布在心肌组织周围,包绕心肌细胞,正常的心肌细胞被大片蓝色的纤维组织代替,心肌细胞有代偿性肥大;心脏重量指数显著增加；超声心动图呈现明显异常,数据分析显示,心脏收缩末期室间隔厚度(IVS;s)、左室收缩末期内径(LVID;s)、收缩末期左心室体积(LV vol;s)、射血分数(%EF)、缩短分数(%FS)等指标均已发生显著改变。结论：1)主动脉缩窄(TAC)术后1周,TG小鼠的心脏结构发生了变化,但心脏功能尚无改变；2)术后4周,TG小鼠的心脏结构发生了显著改变,功能受损。
[Abstract]:The PID1 (phosphotyrosine interaction domain containing 1) gene, also known as the NYGGF4 gene, is a new human full-length gene (GenBank) obtained and cloned when the differential subtractive hybridization (suppression subtractive hybridization, SSH) technique is used to screen the differentially expressed genes in fat and normal human omentum adipose tissue by the suppression subtractive hybridization (SSH) technique. Logon number: AY317148). Previous studies found that: 1) the protein encoded by the gene is located in the cytoplasm, there are several serine, threonine phosphorylation sites, and there is a phosphoric acid tyrosine site (Phosphotyrosine-binding domain, PTB Domain) at the C end of the protein, suggesting that the protein may be involved in intracellular signal transduction; 2) PID1 base High expression in tissues such as myocardium, skeletal muscle and fat; 3) overexpression of PID1 gene can inhibit the activity of PI3K-AKT signaling pathway in signal transduction pathway. Because gene expression profiles are an important clue to reveal gene function, if a gene is expressed in a certain tissue, it is likely that the gene is in the tissue. In view of the high expression of the PID1 gene in the heart, the PI3K-AKT signaling pathway is closely related to cardiac development, cardiac hypertrophy, and cardiac protection, suggesting that the gene may play an important role in heart development, myocardial hypertrophy, and heart protection. This study used transgenic technology to construct PIDl cardiac tissue. Specific expression of transgenic mice model to explore the role of PID1 gene in heart development, myocardial hypertrophy and heart protection. The first part of the construction of PID1 cardiac specific transgenic mice: constructing a specific expression vector of PID1 cardiac tissue, PBS II SK-aMHC-PID1, and establishing a PID1 cardiac specific transgenic mouse. The mouse PID1 gene CDS was cloned by RT-PCR method, and the PID1 gene was inserted into the downstream of the cardiac specific expression gene alpha MHC promoter to construct the specific expression vector of the cardiac tissue. The PBS II SK-aMHC-PID1 was linearized by Notl, and the linearized cardiac tissue specific expression vector was microinjected into the mouse zygote.PCR method by microinjection. The specific expression of mouse derived PID1 gene in the cardiac tissue of transgenic mice was detected by Western blot assay. Results: 1) the specific expression vector of cardiac tissue, PBS II SK-aMHC-PID1, was successfully constructed, and 2) the specific expression of PID1 transgenic mice in the heart tissue of 2 strains was established. Conclusion: PID1 heart. Tissue specific transgenic mice were constructed successfully. Second preliminary observation on the specific transgenic mice of PID1 heart tissue: preliminary observation of the general condition of PID1 cardiac specific transgenic mice and the evaluation of the changes in the cardiac weight index, overall appearance, function and other aspects of the mice. Methods: the specificity of PID1 heart tissue Transgenic mice (TG) mice and mice of the same age, wild type (wild type, WT) were used to observe the general condition of the growth and development of the mice from birth. At 2 months (sexual maturity), the weight and weight of the heart were weighed and the cardiac weight index (HW/BW) was calculated; the overall appearance of the heart was observed under the light microscope. Echocardiographic assessment of the changes in cardiac function of mice. Results: 1) the general condition of mice: during the observation period, the TG mice grew well, the activity was good, the diet was normal, there was no obvious abnormality compared with the WT mice, and could survive for more than one and a half years or even longer; 2) the heart weight index of the TG mice and the WT mice was not significantly different; 3) the TG mice and the WT were small. There was no significant difference in heart appearance between rats; 4) the cardiac function of TG mice was not significantly changed by echocardiography. Conclusion: compared with wild type, PID1 heart tissue specific transgenic mice have no obvious changes in growth and life, heart weight index, overall appearance, and heart function compared with WT mice. Third part of the phenotypic analysis of PIDl specific transgenic mice induced by isoproterenol: comparison of cardiac weight index changes in PID1 cardiac specific transgenic mice induced by isoproterenol. Methods: 2 month old, female PID1 heart tissue specific transgenic (TG) mice and mice TG mice and WT mice were randomly divided into experimental and control groups. Both TG mice and WT mice in the experimental group were intraperitoneally injected with isoproterenol (isoproterenol, ISO) 0.06mg/g weight /d, the control group of TG mice and WT mice were intraperitoneally injected with isovolumetric saline for 6 days, and the heart weight of the mice was weighed on seventh days for 6 days. The cardiac weight index was calculated. Results: 1) the cardiac weight index of WT mice and TG mice after ISO treatment was 6.14 + 0.39 and 6.96 + 1.13 respectively, and there was no significant difference between the two groups (P0.05); 2) the cardiac weight index of WT and TG rats injected by physiological saline was 4.60 + 0.46 and 4.83 +, respectively, and there was no significant difference between the two groups (P0.05); 3 IS O treated WT rats and TG mice were compared with WT rats and TG mice (normal saline group). The results showed that the cardiac weight index increased by 33.5% and 44.1%, respectively, and there was a statistical difference (P0.01). Conclusion: the cardiac weight index of PID1 heart tissue specific transgenic mice was not significantly different from that of WT mice. Fourth part of the phenotypic analysis of the aortic arch constriction in PID1 specific transgenic mice Objective: To observe the changes of cardiac structure and function after aortic arch constriction in PID1 cardiac specific transgenic mice, and to explore its possible mechanism. Methods: 2 month old, male PID1 cardiac tissue specific transgene (T G) mice and wild type (WT) mice were studied. Both TG mice and WT mice were randomly divided into aortic arch constriction operation (transverse aortic constriction, TAC) and sham operation (sham). Preoperative, 1 weeks after operation, 4 weeks after operation, echocardiography was used to detect the cardiac function of mice; 1 weeks after the operation, the weight and weight of the heart were weighed at 4 weeks after the operation, respectively. Cardiac weight index was calculated; HE staining was used to observe the changes of cardiac histomorphology in mice after operation; Masson staining was used to observe myocardial fibrosis and cardiac remodeling. Results: 1) 1 weeks after TAC, the heart volume of TG mice increased obviously; HE staining showed that the left ventricle was obviously enlarged, but the wall thickness had not been significantly changed; cardiac weight index (H) W/BW) was significantly higher than that of WT mice; Masson staining showed no changes in myocardial fibrosis, no obvious abnormalities in echocardiography, no significant changes in cardiac function in echocardiography; 2) the heart volume of TG mice increased significantly at 4 weeks after TAC, and the result of HE staining showed that the left ventricle was obviously enlarged and the ventricular wall became thinner. The results of Masson staining showed severe fibrosis in the heart tissue, fibrosis diffuse around the myocardium, wrapped around cardiac myocytes, normal cardiac myocytes were replaced by large blue fibrous tissue, cardiomyocytes had compensatory hypertrophy, cardiac weight index increased significantly, echocardiography showed obvious abnormalities, data analysis showed, heart End systolic interventricular septum thickness (IVS; s), left ventricular end systolic diameter (LVID; s), end systolic left ventricular volume (LV Vol; s), ejection fraction (%EF), shortened fraction (%FS) and other indexes have been significantly changed. Conclusion: 1) 1 weeks after aortic coarctation (TAC), the cardiac structure of TG mice has changed, but the heart function has not changed; 2) 4 weeks after operation, 2 The heart structure of TG mice has changed significantly, and its function has been impaired.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R3416;Q78

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