E1A激活基因阻遏子在小鼠胚胎发育中的表达

发布时间：2018-06-23 04:59

本文选题：阻遏蛋白 + 腺病毒E1A蛋白　；参考：《第四军医大学》2007年硕士论文

【摘要】： 目的E1A激活基因阻遏子(CREG)是从HeLa细胞cDNA文库中克隆的一种新的转录调控相关因子。它可直接与腺病毒E1A蛋白和转录因子E2F竞争结合靶基因的启动子区,阻遏它们对靶基因的转录调控,从而抑制细胞的增殖,促进细胞分化。CREG蛋白与6-磷酸甘露醇胰岛素样生长因子II受体相关,在多种肿瘤细胞中发挥抑制增殖和促进分化的作用。先前本研究室应用差异显示PCR技术在体外培养的分化表型人VSMCs克隆株HITASY细胞中筛选到高表达差异性CREG基因,进一步研究发现CREG基因表达与HITASY细胞的分化状态密切相关,即去血清培养诱导HITASY细胞由合成表型逆转为分化表型的过程中伴有CREG mRNA和蛋白表达上调。CREG可以与血清反应因子协同作用于平滑肌α-肌动蛋白(SMα-actin)启动子区的CArG元件从而启动SMα-actin的表达,参与体外培养的大鼠原代VSMCs表型转化的调控。大鼠颈动脉拉伤实验证实,CREG蛋白表达与血管损伤后RS过程中平滑肌细胞增殖能力呈负相关。提示CREG基因具有促进分化、抑制增殖等使VSMCs向分化表型转化的功能。为进一步明确CREG表达与组织器官发育的关系,本研究采用免疫组织化学、蛋白印迹(Western blot)及逆转录-聚合酶链反应(RT-PCR)等方法观察了不同小鼠胚胎发育阶段和胚胎E18.5 d时各组织中CREG的表达,以及小鼠血管发育过程中CREG的表达变化,为后续CREG的生物学功能研究提供实验依据。方法①建立小鼠胚胎模型，取不同胎龄的胎鼠制备标本、HE染色、形态学观察。②以免疫组织化学染色方法，检测CREG蛋白在小鼠胚胎的表达时相及在不同脏器的表达定位。③Western blot方法检测不同发育时期小鼠胚胎CREG表达变化及E18.5 d胎鼠各脏器CREG蛋白表达含量。④采用TRIZOL法提取不同胎龄胎鼠总RNA，以RT-PCR法，检测不同胎龄胎鼠CREG mRNA的动态表达。⑤HE染色观察胚胎及新生、成年小鼠血管发育的形态学变化。⑥研究发育不同时期的小鼠胚胎及新生、成年小鼠血管平滑肌分化标志蛋白SMα-actin与CREG蛋白表达的相互关系。⑦观察CREG蛋白在成鼠不同脏器血管的表达变化。结果(1) CREG蛋白在小鼠不同胚胎发育阶段的表达：CREGE5.5 d开始表达，CREG阳性细胞主要分布于原始外胚层。原肠期E6.5 d后三胚层均有CREG表达。以后随着各组织器官的逐渐形成，CREG分布于不同组织器官，但在不同器官的表达时相并不同步。E13.5 d、E15.5 d和E18.5 d胎鼠心脏、脑、肝脏中CREG表达均为阳性，而肺、肾和小肠在E13.5 d和E15.5 d无表达，，E18.5 d时各脏器表达阳性。Western blot结果显示从E9.5 d开始，CREG表达呈明显上升趋势，至出生前即E18.5 d表达最高。对不同发育阶段的小鼠胚胎实施RT-PCR分析显示，E9.5 d~E18.5 dCREG mRNA表达均为阳性，且表达量逐渐增高，到E18.5 d CREG mRNA表达最高，同Western blot结果相似。 (2) CREG在E18.5 d小鼠体内的表达分布：提取E18.5 d小鼠各脏器的蛋白行Western blot分析，结果显示, CREG在脑、心脏、肺、肝、小肠和肾等多个脏器中均有较高表达，且肺和肝脏中的表达弱于其他脏器。免疫组化观察CREG在各脏器细胞中表达分别为脑胶质细胞、心肌细胞、肺泡上皮细胞、肝细胞、小肠粘膜细胞和肾小管细胞，呈均一的深棕色颗粒物质分布于细胞浆内,胞核呈阴性。 (3)小鼠胚胎血管的形态学特征及CREG表达定位:E9.5 d血管的管壁由单层内皮细胞构成,细胞间隙较大,腔内可见血细胞。免疫组化结果显示CREG表达阳性(++),主要定位于血管壁单层内皮细胞中;此时VSMCs分化标志蛋白SMα-actin表达为阴性。胚胎发育至E10.5 d,胚胎血管壁周围开始出现少量双层或多层环绕排列的管壁细胞,细胞间排列松散。免疫组化染色观察到SMα-actin蛋白阳性表达(+),主要定位于内皮细胞外侧的管壁细胞中。同时,CREG蛋白表达阳性(+),定位与SMα-actin蛋白一致。E12.5 d的胚胎动脉壁内VSMCs环绕管腔呈不规则多层排列,细胞为多角形,与周围间充质细胞分界不明显。免疫组化分析显示VSMCs中SMα-actin蛋白表达(++),较E10.5 d明显增强,同时VSMCs中CREG蛋白表达(++)也明显增强。胚胎E15.5 d至E18.5 d,随着血管进一步发育,血管壁内、中、外三层膜结构逐渐清晰,管腔增大,与周围组织分界清楚。管壁各层细胞间排列致密,中膜VSMCs由多角形转化为长梭形,并呈层状排列,细胞核浆比例大。在E15.5 d胚胎血管中SMα-actin蛋白(+++)和CREG蛋白的表达(+++)均较前述各时间点明显增强,并且CREG蛋白在血管壁内、中和外膜三层结构细胞中均明显表达。但胚胎发育至E18.5 d时,SMα-actin蛋白(+++)仍在VSMCs中高表达,而CREG蛋白表达则下调(++)。 (4)新生及成年小鼠主动脉的形态学变化及CREG的表达:新生1 d小鼠主动脉血管壁内、中和外膜结构清晰,管腔大且与周围组织分界清楚。管壁各层细胞间排列紧密,VSMCs在动脉中膜层状排列,细胞细长呈长梭形,胞核明显,核浆比例及平均细胞直径较大。出生28 d后,小鼠主动脉逐渐发育成熟与2 m龄成鼠主动脉的形态相似,细胞外基质增加,VSMCs被增宽的弹力膜和胶原纤维分隔形成成熟的中膜层。VSMCs核浆比例进一步缩小,细胞核近似长杆状。出生后1 d、28 d和2 m小鼠主动脉血管壁中SMα-actin蛋白和CREG表达均为阳性(++)。 (5)不同脏器功能血管中CREG的表达:成年小鼠心、肺、脾和肾脏标本石蜡切片的CREG免疫组化分析显示,上述脏器功能血管中CREG蛋白表达均为阳性,但表达强度不同。其中心脏冠状动脉的CREG蛋白表达呈强阳性(+++)。而在肺小动脉、脾动脉和肾脏小动脉中,CREG蛋白的表达则为弱阳性(+),较冠状动脉明显降低。结论①CREG在小鼠胚胎早期E5.5 d开始表达,持续表达至出生前,表达含量逐渐增多。CREG在胚胎各脏器发生中的表达时相不同,但E18.5 d时胚胎各脏器中均可见CREG蛋白表达,其表达分布与成年小鼠各脏器的表达一致。上述结果提示,CREG可能作为胚胎发育的调控因子,在胚胎发育、分化和成熟过程中起重要作用。②CREG蛋白在小鼠胚胎血管发育极早期即开始表达、持续表达的特点及其在不同脏器功能血管中表达的差异,提示CREG蛋白可能通过调控并维持管壁细胞,特别是VSMCs的分化,参与了胚胎血管发生的调控。
[Abstract]:Objective E1A activated gene repressor (CREG) is a new transcriptional regulation related factor cloned from the cDNA Library of HeLa cells. It can directly compete with the adenovirus E1A protein and transcription factor E2F to combine the promoter region of the target gene to repression of their transcriptional regulation of the target gene, thus inhibiting the proliferation of the cells and promoting the cell differentiation of the.CREG protein. 6- phosphate mannitol insulin-like growth factor II receptor is associated with the inhibition of proliferation and differentiation in a variety of tumor cells. Previously, this laboratory application showed that PCR technology was used to screen the highly expressed CREG gene in human VSMCs cloned HITASY cells cultured in vitro, and to further study the discovery of the CREG gene. The expression is closely related to the differentiation state of HITASY cells, that is, serum-free culture induces the reversal of HITASY cells from the synthetic phenotype to the differentiation phenotype, with CREG mRNA and protein expression up regulation.CREG, which can cooperate with the serum reaction factor in the promoter region of the smooth muscle alpha actin (SM alpha -actin) to start the SM alpha -actin The expression of CREG was involved in the regulation of primary VSMCs phenotypic transformation in rats in vitro. The rat carotid artery strain test confirmed that the expression of CREG protein was negatively correlated with the proliferation ability of smooth muscle cells in the RS process after vascular injury. It suggests that the gene has the function of promoting differentiation, inhibiting proliferation and so on to the differentiation of VSMCs to the differentiated phenotype. The relationship between the expression of G and the development of tissues and organs was observed by immunohistochemistry, Western blot and reverse transcription polymerase chain reaction (RT-PCR). The expression of CREG in various embryonic development stages and E18.5 D in different mice, as well as the changes of CREG expression during the development of rat blood vessels, were observed for the subsequent CREG. The study of biological function provides experimental basis.
Methods the mouse embryo model was established and the specimens of fetal mice of different gestational age were prepared, HE staining and morphological observation were taken. The expression of CREG protein in mouse embryos and expression in different organs were detected by immunohistochemical staining. (3) Western blot method was used to detect the changes of CREG expression and E18.5 D fetus in different developmental stages of mouse embryos. The expression of CREG protein in every organ of rats. (4) using TRIZOL to extract the total RNA of fetal rats of different gestational age and to detect the dynamic expression of CREG mRNA in fetal rats of different gestational age by RT-PCR method. 5. HE staining was used to observe the morphological changes of the embryo and newborn and the vascular development in adult mice. The relationship between the expression of SM protein -actin and CREG protein expression was observed. The expression of CREG protein in different organs of adult rats was observed.
Results (1) the expression of CREG protein in different embryonic development stages of mice: CREGE5.5 D began to express, CREG positive cells were mainly distributed in the original ectoderm. After E6.5 D in the primary intestinal stage, the three embryo layer had CREG expression. With the gradual formation of various tissues and organs, CREG was distributed in different tissues and organs, but the phase of the expression of different organs was not synchronized. The expressions of CREG in the heart, brain and liver of.E13.5 D, E15.5 D and E18.5 D were all positive, while the lungs, kidneys and small intestine were not expressed in E13.5 D and E15.5 D. RT-PCR analysis showed that the expression of E9.5 d~E18.5 dCREG mRNA was positive, and the expression increased gradually, and the expression of E18.5 D CREG mRNA was the highest, similar to Western blot.
(2) expression distribution of CREG in E18.5 D mice: the protein of each organ of E18.5 D mice was extracted by Western blot analysis. The results showed that CREG was highly expressed in many organs such as brain, heart, lung, liver, small intestine and kidney, and the expression in lung and liver was weaker than that of other organs. For glial cells, cardiac myocytes, alveolar epithelial cells, hepatocytes, small intestinal mucosa cells and renal tubular cells, the homogeneous dark brown particles are distributed in the cytoplasm, and the nuclei are negative.
(3) the morphological characteristics and CREG expression of the mouse embryonic blood vessels: the wall of the E9.5 D vessel was composed of the monolayer endothelial cells, the cell space was large and the blood cells were seen in the cavity. The immunohistochemical results showed that the expression of CREG was positive (+ +), mainly located in the monolayer endothelial cells of the vascular wall; at this time, the expression of VSMCs differentiation marker protein SM alpha -actin was negative. The fetus developed to E10.5 D, and a small number of bilayer or multilayer surrounding tube wall cells began to appear around the vascular wall of the embryo, and the cells were arranged loosely. Immunohistochemical staining observed that the positive expression of SM alpha -actin protein (+) was located mainly in the tube wall cells outside the endothelial cells. At the same time, the expression of CREG protein was positive (+), and the location was consistent with the SM alpha -actin protein. In the fetal artery wall of.E12.5 D, the VSMCs encircling canal was arranged in irregular multilayer, and the cells were polygonal, and the boundary of the peripheral mesenchyme cells was not obvious. Immunohistochemical analysis showed that the expression of SM alpha -actin protein (+ +) in VSMCs was obviously enhanced, and the expression of CREG protein (+ +) in VSMCs was also obviously enhanced. In the further development, the three layers of the wall, middle and outer layer of the vascular wall are clear, the cavity is enlarged and the boundary of the surrounding tissue is clear. The cells in each layer of the tube wall are arranged dense, the middle membrane VSMCs is transformed from polygonal to long spindle shape, and the proportion of the cell nuclear plasma is large. The expression of SM alpha -actin protein (+ + +) and the expression of CREG protein (+ + +) in the E15.5 D embryo blood vessels are both Compared with the previous time points, the CREG protein was clearly expressed in the vascular wall and in the three layers of the outer membrane, but when the embryo developed to E18.5 D, the SM alpha -actin protein (+ + +) was still highly expressed in VSMCs, while the expression of CREG protein was down (+ +).
(4) the morphological changes of the aorta and the expression of CREG in the newborn and adult mice: in the aortic vessel wall of the newborn 1 D mice, the structure of the aorta was clear, the cavity was large and the boundary of the surrounding tissue was clear. The cells in each layer of the wall of the tube were arranged closely, the VSMCs was arranged in the middle layer of the artery, the thin cell was long spindle shaped, the nucleus was obvious, the ratio of nuclear plasma and average finer The diameter of the cell was larger. After 28 d, the mouse aorta was gradually developed and mature, and the morphology of the aorta was similar to that of the 2 m old rat aorta. The extracellular matrix was increased. The VSMCs was separated by the widened elastic membrane and collagen fiber to form the mature middle layer of the middle layer, and the proportion of the.VSMCs nucleolus was further narrowed, and the nucleus was similar to the long rod. 1 D, 28 d and 2 m mouse aortic vessels were born after birth. The expression of SM alpha -actin protein and CREG was positive (+ +) in the wall.
(5) expression of CREG in the functional vessels of different organs: the CREG immunohistochemical analysis of the paraffin sections of the heart, lung, spleen and kidney of adult mice showed that the expression of CREG protein in the functional vessels of the above viscera were all positive, but the expression intensity was different. The expression of CREG protein in the coronary artery was strongly positive (+ + +). In the small pulmonary artery, spleen artery and kidney In the arterioles, the expression of CREG protein was weakly positive (+), which was significantly lower than that of the coronary artery.
Conclusion (1) CREG was expressed in the early E5.5 D of mouse embryo, and continued to be expressed before birth, and the expression level was gradually increasing in the expression of.CREG in the embryonal organs of the embryo, but the expression of CREG protein was found in all the organs of the embryo at E18.5 D, and the expression distribution was consistent with the expression of the organs of adult mice. The results suggested that CREG might be possible. As a regulator of embryonic development, it plays an important role in the process of embryonic development, differentiation and maturation. (2) the expression of CREG protein in the early development of the mouse embryonic blood vessels, the characteristics of the continuous expression and the difference in the expression of the functional vessels in different organs, suggest that the CREG protein can regulate and maintain the tube wall cells, especially the VSMCs. The differentiation is involved in the regulation of embryonic angiogenesis.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R321

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