Tsc1基因在小鼠血管发育和肺发育中的调控机制研究

发布时间：2018-07-05 16:13

本文选题：结节性硬化症 + 结节性硬化基因1　；参考：《北京协和医学院》2013年博士论文

【摘要】：背景：结节性硬化症(Tuberous Sclerosis Complex,TSC)是由于抑癌基因TSC1或TSC2突变引起的常染色体显性遗传病。TSC1/TSC2构成的异二聚体复合物是雷帕霉素靶蛋白(mTOR)的负性调节因子。该复合物的突变可导致下游mTOR信号通路的活化。mTOR的活化是TSC的主要发病机制,在临床上常有癫痫、智力减退、面部血管纤维瘤等常见症状,此外还可并发心脏错构瘤,肺血管平滑肌瘤,肾脏血管肌脂瘤和肝血管瘤等血管相关症状。但目前对TSC患者血管受累的研究仍较少,血管受累在TSC病变进展中的作用尚未清楚。目的：为了探讨TSC1/2-mTOR信号通路在TSC相关血管形成与胚胎发育的生理作用。运用Cre/LoxP条件性基因敲除技术使小鼠血管内皮细胞Tsc1基因缺失,构建血管内皮细胞mTOR活化模型,阐明TSC血管受累的病变机理。方法和结果：血管内皮细胞Tsc1基因敲除后内皮细胞mTOR过度活化。突变小鼠可出现严重的心血管缺陷并死于胚胎期的第14.5天。这些异常的表型缺陷主要包括皮下水肿、出血和血管形成缺陷。全胚胎免疫组化和病理形态分析表明与同窝对照相比,突变胚胎血管分级不精细,血管走行紊乱,外周血管数目严重减少,血管成熟不完善。并伴心脏受累,心肌小梁发育不良、心室壁变薄。另外,Tscl突变的胚胎血管内皮细胞增殖与凋亡调控异常,凋亡细胞数明显增多,正常发育所需的增殖受影响。同时,血管形成相关的细胞生成因子表达水平与正常对照相比有显著差异。电镜分析显示异常的血管内皮细胞还伴有内质网结构的扩张。为逆转以上血管缺陷,我们在血管发育E12-13天使用雷帕霉素预治疗,结果发现通过降低mTOR的活性,挽救了心血管缺陷和胚胎的致死性并能显著改善突变胚胎生存期,使Tie2-Cre/Tsc1-/-小鼠在出生后存活,最长者可达到出生后22天。虽能存活,但基因突变的小鼠伴有严重的发育不良,部分突变小鼠在刚出生时与同窝对照相比无明显差别,而随其正常的生长发育,Tie2-Cre/Tsc1-/-小鼠发育缓慢体重较野生型显著减少,最终全身消瘦而死亡。结论：TSC1/2-mTOR信号通路对血管发育和胚胎形成起至关重要的作用。通过上调mTOR信号能够干扰正常的血管形成过程。对TSC的血管受累的研究有助于深入了解肾脏血管肌脂瘤和肺淋巴管肌瘤病等血管受累的发病机理。肺泡Ⅱ型上皮细胞(ABCⅡ s)对于维持肺的生理和病理功能有至关重要的作用。AECⅡ s被认为是肺泡Ⅰ型上皮细胞(AEC Ⅰ s)的祖细胞并具有调节免疫功能的作用。AECⅡ s的功能障碍与多种肺疾病有关,但其具体调控机制仍未清楚。因此有必要建立肺上皮转基因工具鼠模型。已有多种研究显示可诱导Cre转基因表达系统被应用于研究靶基因在AECⅡ s功能的模型中,如(SPC-rtTetO-Cre, SPC-Cre-ERT2)。尽管如此,某些局限性的存在限制了目的基因在这些系统中的研究。非诱导条件性Cre基因表达系统是研究正常肺发育和肺部疾病较好的工具模型。虽然SPC-Cre在先前的研究中己被应用,但是该小鼠的具体制作过程尚未报道过。另外,有研究指出SPC-Cre可并有肺大泡表型缺陷,因此,SPC-Cre未能被广泛应用于疾病模型。在本研究中,我们通过原核注射制作了C57BL/6J背景的SPC-Cre小鼠。为检测Cre重组酶在AECⅡ s细胞中的特异性活性,SPC-Cre小鼠分别与ROSA26R报告小鼠和Tsclfx/fx小鼠杂交。X-Ga1染色和PCR分析结果显示β-半乳糖苷酶的蓝染细胞表达在SPC-Cre/ROSA26R的AECⅡ s细胞中,在SPC-Cre/Tscl-/-小鼠的肺组织中可检测到Tsc1的突变带。因此它是研究AECⅡ s细胞在肺发育和肺相关疾病中的有用工具。另外对SPC-Cre/Tscl-/-小鼠肺发育的初步研究发现,SPC-Cre/Tscl-/-小鼠大体形态表型与正常小鼠相似。但病理学分析显示突变小鼠肺泡腔扩增,肺泡间隔数减少。参与肺发育的转录因子水平与同窝对照相比表达调控异常,但具体的调控机制还需更深入的研究。
[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.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R563

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