本文選題：PID1 + 組織特異性轉(zhuǎn)基因小鼠�。� 參考：《南京醫(yī)科大學(xué)》2012年碩士論文

【摘要】：PID1 (phosphotyrosine interaction domain containing 1)基因,又稱NYGGF4基因,是本研究小組應(yīng)用抑制性差減雜交(suppression subtractive hybridization, SSH)技術(shù)篩選肥胖與正常人網(wǎng)膜脂肪組織中差異表達(dá)基因時(shí)獲得并克隆到的一條人類全長新基因(GenBank登錄號：AY317148)。前期研究發(fā)現(xiàn)：1)該基因編碼的蛋白定位于胞漿,存在多個絲氨酸、蘇氨酸的磷酸化位點(diǎn),另在蛋白的C端存在一個磷酸酪氨酸的作用位點(diǎn)(Phosphotyrosine-binding domain, PTB Domain),提示該蛋白可能參與胞內(nèi)信號轉(zhuǎn)導(dǎo)；2)PID1基因在心肌、骨骼肌、脂肪等組織中呈現(xiàn)較高表達(dá)；3)PID1基因過表達(dá)能夠抑制信號轉(zhuǎn)導(dǎo)途徑PI3K-AKT信號通路的活性。由于基因的表達(dá)譜是揭示基因功能的一個重要線索,如果一個基因在某種組織中表達(dá)量很高,那么很可能該基因在該組織中行使功能,因此鑒于PID1基因在心臟組織中呈現(xiàn)高表達(dá),同時(shí)PI3K-AKT信號通路與心臟發(fā)育、心肌肥厚、心臟保護(hù)等功能密切相關(guān),提示該基因可能在心臟發(fā)育、心肌肥厚、心臟保護(hù)中起重要作用,本研究采用轉(zhuǎn)基因技術(shù)構(gòu)建了PIDl心臟組織特異性表達(dá)轉(zhuǎn)基因小鼠的模型,以探討PID1基因在心臟發(fā)育、心肌肥厚、心臟保護(hù)中的作用。第一部分PID1心臟組織特異性轉(zhuǎn)基因小鼠的構(gòu)建目的：構(gòu)建PID1心臟組織特異性表達(dá)載體PBSⅡSK-aMHC-PID1,建立PID1心臟組織特異性轉(zhuǎn)基因小鼠。方法：RT-PCR法克隆小鼠PID1基因CDS,將PID1基因插入心臟特異表達(dá)基因αMHC啟動子的下游,構(gòu)建心臟組織特異性表達(dá)載體。采用Notl將PBSⅡSK-aMHC-PID1線性化,通過顯微注射法將線性化的心臟組織特異性表達(dá)載體顯微注射到小鼠受精卵原核。PCR法檢測轉(zhuǎn)基因首建鼠的基因型,Western blot法驗(yàn)證鼠源性PID1基因在轉(zhuǎn)基因小鼠心臟組織中的特異性高表達(dá)。結(jié)果：1)成功構(gòu)建了心臟組織特異性表達(dá)載體PBSⅡSK-aMHC-PID1; 2)建立了2個株的心臟組織特異性表達(dá)PID1轉(zhuǎn)基因小鼠。結(jié)論：PID1心臟組織特異性轉(zhuǎn)基因小鼠構(gòu)建成功。第二部分對PID1心臟組織特異性轉(zhuǎn)基因小鼠的初步觀察目的：初步觀察PID1心臟組織特異性轉(zhuǎn)基因小鼠的一般狀況,評價(jià)小鼠在心臟重量指數(shù)、整體外觀、功能等方面的變化。方法：以PID1心臟組織特異性轉(zhuǎn)基因(transgenic mice, TG)小鼠和同年齡、同性別野生型(wild type, WT)小鼠為研究對象,從出生后開始觀察小鼠的生長發(fā)育等一般情況；2個月時(shí)(性成熟)稱量心臟重量和體重,計(jì)算心臟重量指數(shù)(HW/BW)；光鏡下觀察心臟的整體外觀變化；超聲心動圖檢測評估小鼠心臟功能變化。結(jié)果：1)小鼠一般狀況：觀察期間TG小鼠生長良好、活動自如、飲食正常,與WT小鼠相比無明顯異常,可存活一年半以上甚至更長時(shí)間；2)TG小鼠與WT小鼠的心臟重量指數(shù)無顯著性差異；3)TG小鼠和WT小鼠的心臟外觀無明顯差異；4)超聲心動圖檢測發(fā)現(xiàn)TG小鼠的心臟功能也無明顯變化。結(jié)論：PID1心臟組織特異轉(zhuǎn)基因小鼠與野生型小鼠相比,在生長發(fā)育、壽命等方面均無明顯變化,心臟重量指數(shù)、整體外觀、心臟功能與WT小鼠比較也無明顯差異。第三部分 PIDl心臟組織特異性轉(zhuǎn)基因小鼠異丙腎上腺素誘導(dǎo)實(shí)驗(yàn)后的表型分析目的:比較PID1心臟組織特異性轉(zhuǎn)基因小鼠異丙腎上腺素誘導(dǎo)實(shí)驗(yàn)下的心臟重量指數(shù)變化。方法：以2月齡、雌性PID1心臟組織特異性轉(zhuǎn)基因(TG)小鼠和野生型(WT)小鼠為研究對象,TG小鼠和WT小鼠均隨機(jī)分為實(shí)驗(yàn)組和對照組。實(shí)驗(yàn)組TG小鼠和WT小鼠均腹腔注射異丙腎上腺素(isoproterenol, ISO) 0.06mg/g體重/d,對照組TG小鼠和WT小鼠腹腔注射等體積生理鹽水,持續(xù)6天,第7天稱量小鼠的心臟重量和體重,計(jì)算心臟重量指數(shù)。結(jié)果：1)ISO處理后的WT小鼠和TG小鼠的心臟重量指數(shù)分別為6.14±0.39和6.96±1.13,兩組間無顯著差異(P0.05)；2)生理鹽水注射的WT鼠和TG鼠的心臟重量指數(shù)分別為4.60±0.46和4.83±0.29,兩組間也無顯著差異(P0.05); 3) ISO處理后的WT鼠和TG鼠分別與WT鼠和TG鼠對照組(生理鹽水組)比較,結(jié)果顯示心臟重量指數(shù)分別增加了33.5%和44.1%,有統(tǒng)計(jì)學(xué)差異(P0.01)。結(jié)論：異丙腎上腺素誘導(dǎo)實(shí)驗(yàn)下,PID1心臟組織特異轉(zhuǎn)基因小鼠的心臟重量指數(shù)與WT小鼠相比無顯著差異。第四部分 PID1心臟組織特異性轉(zhuǎn)基因小鼠主動脈弓縮窄實(shí)驗(yàn)后的表型分析目的：觀察PID1心臟組織特異性轉(zhuǎn)基因小鼠主動脈弓縮窄實(shí)驗(yàn)后心臟結(jié)構(gòu)及功能變化,并初步探討其可能機(jī)制。方法：以2月齡、雄性PID1心臟組織特異性轉(zhuǎn)基因(TG)小鼠和野生型(WT)小鼠為研究對象,TG小鼠和WT小鼠均隨機(jī)分為主動脈弓縮窄手術(shù)(transverse aortic constriction, TAC)組和假手術(shù)(sham)組。術(shù)前、術(shù)后1周、術(shù)后4周采用超聲心動圖檢測小鼠心臟功能；術(shù)后1周、術(shù)后4周分別稱量心臟重量和體重,計(jì)算心臟重量指數(shù)；HE染色觀察術(shù)后小鼠心臟組織形態(tài)學(xué)改變；Masson染色法觀察心肌纖維化及心臟重構(gòu)。結(jié)果：1)TAC術(shù)后1周,TG小鼠的心臟體積明顯增大；HE染色顯示其左心室明顯增大,但室壁厚度尚未呈現(xiàn)顯著改變；心臟重量指數(shù)(HW/BW)顯著高于WT小鼠；Masson染色顯示尚未發(fā)生心肌纖維化改變；超聲心動圖未見明顯異常,超聲心動圖中反應(yīng)心臟功能的指標(biāo)尚無顯著性變化；2)TAC術(shù)后4周,TG小鼠的心臟體積出現(xiàn)顯著增大；HE染色結(jié)果顯示左心室明顯增大、室壁變薄；Masson染色結(jié)果顯示心臟組織嚴(yán)重纖維化,纖維化彌漫分布在心肌組織周圍,包繞心肌細(xì)胞,正常的心肌細(xì)胞被大片藍(lán)色的纖維組織代替,心肌細(xì)胞有代償性肥大;心臟重量指數(shù)顯著增加；超聲心動圖呈現(xiàn)明顯異常,數(shù)據(jù)分析顯示,心臟收縮末期室間隔厚度(IVS;s)、左室收縮末期內(nèi)徑(LVID;s)、收縮末期左心室體積(LV vol;s)、射血分?jǐn)?shù)(%EF)、縮短分?jǐn)?shù)(%FS)等指標(biāo)均已發(fā)生顯著改變。結(jié)論：1)主動脈縮窄(TAC)術(shù)后1周,TG小鼠的心臟結(jié)構(gòu)發(fā)生了變化,但心臟功能尚無改變；2)術(shù)后4周,TG小鼠的心臟結(jié)構(gòu)發(fā)生了顯著改變,功能受損。
[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.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R3416;Q78

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 吳連連;袁紅花;朱孝榮;;腦組織特異性表達(dá)胰島素樣生長因子-1轉(zhuǎn)基因小鼠制備[J];實(shí)驗(yàn)動物科學(xué);2012年06期

2 汪兆琦,章靜波;轉(zhuǎn)基因小鼠的研究及其遺傳學(xué)意義[J];國外醫(yī)學(xué).遺傳學(xué)分冊;1987年04期

3 王曉東;潘漪清;陳鴻書;;轉(zhuǎn)基因小鼠生化標(biāo)志基因檢測[J];上海畜牧獸醫(yī)通訊;2006年05期

4 杜靜;全雄志;趙海平;董偉;張連峰;秦川;;腦組織特異表達(dá)膽囊收縮素轉(zhuǎn)基因小鼠的建立[J];中國比較醫(yī)學(xué)雜志;2007年11期

5 李秀梅;劉光澤;黎經(jīng)緯;陳媚娟;陳文吟;周軍輝;孔祥平;;慢病毒載體介導(dǎo)的綠色熒光蛋白轉(zhuǎn)基因小鼠的制備[J];中國比較醫(yī)學(xué)雜志;2010年10期

6 全雄志;高翔;張旭;葛文萍;關(guān)菲菲;董偉;張連峰;;人載脂蛋白C3基因轉(zhuǎn)基因小鼠的建立及血脂變化分析[J];中國比較醫(yī)學(xué)雜志;2012年10期

7 席曉霞;白德成;魏虎來;吳潤;;兩種轉(zhuǎn)基因小鼠生長繁殖方法的研究[J];實(shí)驗(yàn)室科學(xué);2013年03期

8 李來記;;轉(zhuǎn)基因小鼠——一種新型實(shí)驗(yàn)動物[J];上海實(shí)驗(yàn)動物科學(xué);1989年04期

9 史瀛仙;季肖東;李光三;舒幼敏;;轉(zhuǎn)基因小鼠的研究[J];生物工程學(xué)報(bào);1990年03期

10 孫國鳳;;轉(zhuǎn)基因小鼠作為牛基因重組系的模型[J];生物技術(shù)通報(bào);1990年11期

相關(guān)會議論文 前10條

1 全雄志;董偉;曹興水;張連峰;;24-脫氫膽固醇還原酶基因轉(zhuǎn)基因小鼠血生化指標(biāo)分析[A];實(shí)驗(yàn)動物與藥理學(xué)、毒理學(xué)研究學(xué)術(shù)交流會論文匯編[C];2009年

2 黃黎珍;劉光澤;李秀梅;胡蓮美;顧為望;;HBV蛋白在乙肝轉(zhuǎn)基因小鼠中的表達(dá)研究[A];中國實(shí)驗(yàn)動物學(xué)會第七屆學(xué)術(shù)年會論文集[C];2006年

3 王冬梅;李萬波;袁樹民;全雄志;張海濤;馬春梅;曹興水;張連峰;;APP695K595N/M596L突變轉(zhuǎn)基因小鼠建立和病理表型動態(tài)分析[A];2011全國老年癡呆與衰老相關(guān)疾病學(xué)術(shù)會議第三屆山東省神經(jīng)內(nèi)科醫(yī)師（學(xué)術(shù)）論壇論文匯編[C];2011年

4 李岳美;劉素芳;段萍;韓雪飛;許燕;鄢文海;邢瑩;;APP轉(zhuǎn)基因小鼠腦組織中4.1蛋白的表達(dá)及意義[A];Proceedings of the 8th Biennial Conference of the Chinese Society for Neuroscience[C];2009年

5 訾曉淵;張南;熊俊;巴月;姚玉成;黃潔;李建秀;朱海英;王新民;馬鳳云;張樹忠;胡以平;;乙型肝炎病毒全基因組轉(zhuǎn)基因小鼠的生物學(xué)特性[A];中國細(xì)胞生物學(xué)學(xué)會2005年學(xué)術(shù)大會、青年學(xué)術(shù)研討會論文摘要集[C];2005年

6 寧云山;周明乾;王小寧;黃淑幀;曾溢滔;;重組人血小板生成素在轉(zhuǎn)基因小鼠乳腺中的表達(dá)研究[A];中國生物工程學(xué)會第三次全國會員代表大會暨學(xué)術(shù)討論會論文摘要集[C];2001年

7 康愛君;田楓;鄭杰;董宇紅;王美華;郭長占;周淑佩;王兆綽;;乙型肝炎病毒轉(zhuǎn)基因小鼠病理學(xué)觀察[A];中國實(shí)驗(yàn)動物學(xué)會第五屆學(xué)術(shù)年會論文匯編[C];2000年

8 張慶玲;楊玉芳;丁彥青;;構(gòu)建熒光轉(zhuǎn)基因小鼠模型整體水平觀察B-LMP1基因功能的相關(guān)研究[A];中華醫(yī)學(xué)會病理學(xué)分會2007年學(xué)術(shù)年會暨第九屆全國病理大會論文匯編[C];2007年

9 賈永林;賈延R，

本文編號：1777858