本文選題：結(jié)節(jié)性硬化癥 + 結(jié)節(jié)性硬化基因1��； 參考：《北京協(xié)和醫(yī)學院》2013年博士論文

【摘要】：背景：結(jié)節(jié)性硬化癥(Tuberous Sclerosis Complex,TSC)是由于抑癌基因TSC1或TSC2突變引起的常染色體顯性遺傳病。TSC1/TSC2構(gòu)成的異二聚體復合物是雷帕霉素靶蛋白(mTOR)的負性調(diào)節(jié)因子。該復合物的突變可導致下游mTOR信號通路的活化。mTOR的活化是TSC的主要發(fā)病機制,在臨床上常有癲癇、智力減退、面部血管纖維瘤等常見癥狀,此外還可并發(fā)心臟錯構(gòu)瘤,肺血管平滑肌瘤,腎臟血管肌脂瘤和肝血管瘤等血管相關(guān)癥狀。但目前對TSC患者血管受累的研究仍較少,血管受累在TSC病變進展中的作用尚未清楚。 目的：為了探討TSC1/2-mTOR信號通路在TSC相關(guān)血管形成與胚胎發(fā)育的生理作用。運用Cre/LoxP條件性基因敲除技術(shù)使小鼠血管內(nèi)皮細胞Tsc1基因缺失,構(gòu)建血管內(nèi)皮細胞mTOR活化模型,闡明TSC血管受累的病變機理。 方法和結(jié)果：血管內(nèi)皮細胞Tsc1基因敲除后內(nèi)皮細胞mTOR過度活化。突變小鼠可出現(xiàn)嚴重的心血管缺陷并死于胚胎期的第14.5天。這些異常的表型缺陷主要包括皮下水腫、出血和血管形成缺陷。全胚胎免疫組化和病理形態(tài)分析表明與同窩對照相比,突變胚胎血管分級不精細,血管走行紊亂,外周血管數(shù)目嚴重減少,血管成熟不完善。并伴心臟受累,心肌小梁發(fā)育不良、心室壁變薄。另外,Tscl突變的胚胎血管內(nèi)皮細胞增殖與凋亡調(diào)控異常,凋亡細胞數(shù)明顯增多,正常發(fā)育所需的增殖受影響。同時,血管形成相關(guān)的細胞生成因子表達水平與正常對照相比有顯著差異。電鏡分析顯示異常的血管內(nèi)皮細胞還伴有內(nèi)質(zhì)網(wǎng)結(jié)構(gòu)的擴張。為逆轉(zhuǎn)以上血管缺陷,我們在血管發(fā)育E12-13天使用雷帕霉素預治療,結(jié)果發(fā)現(xiàn)通過降低mTOR的活性,挽救了心血管缺陷和胚胎的致死性并能顯著改善突變胚胎生存期,使Tie2-Cre/Tsc1-/-小鼠在出生后存活,最長者可達到出生后22天。雖能存活,但基因突變的小鼠伴有嚴重的發(fā)育不良,部分突變小鼠在剛出生時與同窩對照相比無明顯差別,而隨其正常的生長發(fā)育,Tie2-Cre/Tsc1-/-小鼠發(fā)育緩慢體重較野生型顯著減少,最終全身消瘦而死亡。 結(jié)論：TSC1/2-mTOR信號通路對血管發(fā)育和胚胎形成起至關(guān)重要的作用。通過上調(diào)mTOR信號能夠干擾正常的血管形成過程。對TSC的血管受累的研究有助于深入了解腎臟血管肌脂瘤和肺淋巴管肌瘤病等血管受累的發(fā)病機理。 肺泡Ⅱ型上皮細胞(ABCⅡ s)對于維持肺的生理和病理功能有至關(guān)重要的作用。AECⅡ s被認為是肺泡Ⅰ型上皮細胞(AEC Ⅰ s)的祖細胞并具有調(diào)節(jié)免疫功能的作用。AECⅡ s的功能障礙與多種肺疾病有關(guān),但其具體調(diào)控機制仍未清楚。因此有必要建立肺上皮轉(zhuǎn)基因工具鼠模型。已有多種研究顯示可誘導Cre轉(zhuǎn)基因表達系統(tǒng)被應用于研究靶基因在AECⅡ s功能的模型中,如(SPC-rtTetO-Cre, SPC-Cre-ERT2)。盡管如此,某些局限性的存在限制了目的基因在這些系統(tǒng)中的研究。非誘導條件性Cre基因表達系統(tǒng)是研究正常肺發(fā)育和肺部疾病較好的工具模型。雖然SPC-Cre在先前的研究中己被應用,但是該小鼠的具體制作過程尚未報道過。另外,有研究指出SPC-Cre可并有肺大泡表型缺陷,因此,SPC-Cre未能被廣泛應用于疾病模型。在本研究中,我們通過原核注射制作了C57BL/6J背景的SPC-Cre小鼠。為檢測Cre重組酶在AECⅡ s細胞中的特異性活性,SPC-Cre小鼠分別與ROSA26R報告小鼠和Tsclfx/fx小鼠雜交。X-Ga1染色和PCR分析結(jié)果顯示β-半乳糖苷酶的藍染細胞表達在SPC-Cre/ROSA26R的AECⅡ s細胞中,在SPC-Cre/Tscl-/-小鼠的肺組織中可檢測到Tsc1的突變帶。因此它是研究AECⅡ s細胞在肺發(fā)育和肺相關(guān)疾病中的有用工具。另外對SPC-Cre/Tscl-/-小鼠肺發(fā)育的初步研究發(fā)現(xiàn),SPC-Cre/Tscl-/-小鼠大體形態(tài)表型與正常小鼠相似。但病理學分析顯示突變小鼠肺泡腔擴增,肺泡間隔數(shù)減少。參與肺發(fā)育的轉(zhuǎn)錄因子水平與同窩對照相比表達調(diào)控異常,但具體的調(diào)控機制還需更深入的研究。
[Abstract]:Background: Tuberous Sclerosis Complex (TSC) is an abnormal two polymer complex composed of autosomal dominant hereditary disease.TSC1/TSC2 caused by the TSC1 or TSC2 mutation of the tumor suppressor gene. The negative regulator of the rapamycin target protein (mTOR) is a negative regulator of the target protein of rapamycin (mTOR). The mutagenesis of the complex can lead to the activation of the activation.MTOR of the downstream mTOR signaling pathway. The main pathogenesis of TSC is the common symptoms of epilepsy, hypointelligence, facial angiofibroma and other common symptoms such as cardiac hamartoma, pulmonary vascular leiomyoma, renal angiomyolipoma and hepatic hemangioma, but the study of vascular involvement in TSC patients is still less, and vascular involvement is involved in TSC lesions. The role of the exhibition is not clear.
Objective: To explore the physiological role of TSC1/2-mTOR signaling pathway in TSC related angiogenesis and embryonic development, the Tsc1 gene deletion of vascular endothelial cells in mice was deleted by Cre/LoxP conditional knockout technique, and the mTOR activation model of vascular endothelial cells was constructed to elucidate the pathological mechanism of vascular involvement of TSC.
Methods and results: endothelial cell mTOR was overactivated after Tsc1 gene knockout in vascular endothelial cells. The mutant mice could have serious cardiovascular defects and died of the embryonic day 14.5 days. These abnormal phenotypic defects mainly include hypodermic edema, bleeding and vascular defects. Compared with the comparison, the vascular classification of the mutant embryos was not fine, the blood vessels were disorganized, the number of peripheral blood vessels was greatly reduced, the vascular maturity was not perfect. With the heart involvement, the cardiac trabecula was dysplasia and the ventricular wall thinned. In addition, the proliferation and apoptosis control of the Tscl mutant fetal vascular endothelial cells were abnormal, the number of apoptotic cells increased obviously, and the normal development needed to be found. The proliferation is affected. Meanwhile, the expression level of angiogenesis related factor of angiogenesis is significantly different from that of normal. The electron microscopic analysis shows that abnormal vascular endothelial cells are accompanied by endoplasmic reticulum expansion. In order to reverse the above vascular defects, we use rapamycin pretreatment at E12-13 days for vascular development, and the results have been found to be passed. Reducing the activity of mTOR, saving the mortality of cardiovascular defects and embryos and significantly improving the survival period of the mutant embryo, the Tie2-Cre/Tsc1-/- mice survived after birth, the longest could reach 22 days after birth. Although it could survive, the mutant mice were accompanied by severe dysplasia, and some mutant mice were photographed with the same nest at birth. Compared with the normal growth and development, Tie2-Cre/Tsc1-/- mice developed slower weight than wild type, and eventually became thinner and died.
Conclusion: TSC1/2-mTOR signaling pathway plays a vital role in vascular development and embryogenesis. The regulation of mTOR signals can interfere with the normal angiogenesis process. The study of vascular involvement of TSC helps to understand the pathogenesis of vascular involvement in renal angiomyoma and pulmonary lymphangiomyomatosis.
The pulmonary alveolar type II epithelial cells (ABC II s) play an important role in maintaining the physiological and pathological functions of the lung..AEC II s is considered to be the progenitor of the alveolar type I epithelial cell (AEC i s) and has the function of regulating the immune function. The dysfunction of.AEC II s is related to a variety of lung diseases, but its specific regulatory mechanism is still not clear. Therefore, it is necessary. A variety of studies have shown that the inducible Cre gene expression system has been used to study the target gene in the AEC II s function model, such as (SPC-rtTetO-Cre, SPC-Cre-ERT2). However, some limitations restrict the study of the target based on these systems. Non inducible conditioned Cre Gene expression system is a good tool model for the study of normal lung development and lung disease. Although SPC-Cre has been used in previous studies, the specific process of the mouse has not been reported. In addition, there have been studies that SPC-Cre can have phenotypic defects of the pulmonary bullae. Therefore, SPC-Cre has not been widely used in the disease model. In the study, we made the C57BL/6J background SPC-Cre mice by the prokaryotic injection. In order to detect the specific activity of the Cre recombinant enzyme in AEC II s cells, SPC-Cre mice were hybridized with ROSA26R reported mice and Tsclfx/fx mice by.X-Ga1 staining and PCR analysis. The expression of beta galactosidase in the SPC-Cre/ROSA26R AEC II cells was shown in SPC-Cre/ROSA26R AEC II. In s cells, the mutant band of Tsc1 can be detected in the lung tissue of SPC-Cre/Tscl-/- mice. Therefore, it is a useful tool to study the lung development and lung related diseases of AEC II s cells. In addition to the preliminary study of lung development in SPC-Cre/Tscl-/- mice, the large body shape phenotype of SPC-Cre/Tscl-/- mice is similar to that of normal mice. But the pathological scores are similar to those of the normal mice. The analysis showed that the alveolar cavity amplification and the number of alveolar septum in the mutant mice were reduced. The level of transcription factors involved in lung development and the ratio of the same fossa to photographic expression were abnormal, but the specific regulatory mechanism needed to be further studied.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R563

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