發(fā)布時間：2018-06-23 04:59

  本文選題：阻遏蛋白 + 腺病毒E1A蛋白　； 參考：《第四軍醫(yī)大學》2007年碩士論文

【摘要】： 目的E1A激活基因阻遏子(CREG)是從HeLa細胞cDNA文庫中克隆的一種新的轉錄調控相關因子。它可直接與腺病毒E1A蛋白和轉錄因子E2F競爭結合靶基因的啟動子區(qū),阻遏它們對靶基因的轉錄調控,從而抑制細胞的增殖,促進細胞分化。CREG蛋白與6-磷酸甘露醇胰島素樣生長因子II受體相關,在多種腫瘤細胞中發(fā)揮抑制增殖和促進分化的作用。先前本研究室應用差異顯示PCR技術在體外培養(yǎng)的分化表型人VSMCs克隆株HITASY細胞中篩選到高表達差異性CREG基因,進一步研究發(fā)現CREG基因表達與HITASY細胞的分化狀態(tài)密切相關,即去血清培養(yǎng)誘導HITASY細胞由合成表型逆轉為分化表型的過程中伴有CREG mRNA和蛋白表達上調。CREG可以與血清反應因子協同作用于平滑肌α-肌動蛋白(SMα-actin)啟動子區(qū)的CArG元件從而啟動SMα-actin的表達,參與體外培養(yǎng)的大鼠原代VSMCs表型轉化的調控。大鼠頸動脈拉傷實驗證實,CREG蛋白表達與血管損傷后RS過程中平滑肌細胞增殖能力呈負相關。提示CREG基因具有促進分化、抑制增殖等使VSMCs向分化表型轉化的功能。為進一步明確CREG表達與組織器官發(fā)育的關系,本研究采用免疫組織化學、蛋白印跡(Western blot)及逆轉錄-聚合酶鏈反應(RT-PCR)等方法觀察了不同小鼠胚胎發(fā)育階段和胚胎E18.5 d時各組織中CREG的表達,以及小鼠血管發(fā)育過程中CREG的表達變化,為后續(xù)CREG的生物學功能研究提供實驗依據。 方法①建立小鼠胚胎模型，取不同胎齡的胎鼠制備標本、HE染色、形態(tài)學觀察。②以免疫組織化學染色方法，檢測CREG蛋白在小鼠胚胎的表達時相及在不同臟器的表達定位。③Western blot方法檢測不同發(fā)育時期小鼠胚胎CREG表達變化及E18.5 d胎鼠各臟器CREG蛋白表達含量。④采用TRIZOL法提取不同胎齡胎鼠總RNA，以RT-PCR法，檢測不同胎齡胎鼠CREG mRNA的動態(tài)表達。⑤HE染色觀察胚胎及新生、成年小鼠血管發(fā)育的形態(tài)學變化。⑥研究發(fā)育不同時期的小鼠胚胎及新生、成年小鼠血管平滑肌分化標志蛋白SMα-actin與CREG蛋白表達的相互關系。⑦觀察CREG蛋白在成鼠不同臟器血管的表達變化。 結果(1) CREG蛋白在小鼠不同胚胎發(fā)育階段的表達：CREGE5.5 d開始表達，CREG陽性細胞主要分布于原始外胚層。原腸期E6.5 d后三胚層均有CREG表達。以后隨著各組織器官的逐漸形成，CREG分布于不同組織器官，但在不同器官的表達時相并不同步。E13.5 d、E15.5 d和E18.5 d胎鼠心臟、腦、肝臟中CREG表達均為陽性，而肺、腎和小腸在E13.5 d和E15.5 d無表達，，E18.5 d時各臟器表達陽性。Western blot結果顯示從E9.5 d開始，CREG表達呈明顯上升趨勢，至出生前即E18.5 d表達最高。對不同發(fā)育階段的小鼠胚胎實施RT-PCR分析顯示，E9.5 d~E18.5 dCREG mRNA表達均為陽性，且表達量逐漸增高，到E18.5 d CREG mRNA表達最高，同Western blot結果相似。 (2) CREG在E18.5 d小鼠體內的表達分布：提取E18.5 d小鼠各臟器的蛋白行Western blot分析，結果顯示, CREG在腦、心臟、肺、肝、小腸和腎等多個臟器中均有較高表達，且肺和肝臟中的表達弱于其他臟器。免疫組化觀察CREG在各臟器細胞中表達分別為腦膠質細胞、心肌細胞、肺泡上皮細胞、肝細胞、小腸粘膜細胞和腎小管細胞，呈均一的深棕色顆粒物質分布于細胞漿內,胞核呈陰性。 (3)小鼠胚胎血管的形態(tài)學特征及CREG表達定位:E9.5 d血管的管壁由單層內皮細胞構成,細胞間隙較大,腔內可見血細胞。免疫組化結果顯示CREG表達陽性(++),主要定位于血管壁單層內皮細胞中;此時VSMCs分化標志蛋白SMα-actin表達為陰性。胚胎發(fā)育至E10.5 d,胚胎血管壁周圍開始出現少量雙層或多層環(huán)繞排列的管壁細胞,細胞間排列松散。免疫組化染色觀察到SMα-actin蛋白陽性表達(+),主要定位于內皮細胞外側的管壁細胞中。同時,CREG蛋白表達陽性(+),定位與SMα-actin蛋白一致。E12.5 d的胚胎動脈壁內VSMCs環(huán)繞管腔呈不規(guī)則多層排列,細胞為多角形,與周圍間充質細胞分界不明顯。免疫組化分析顯示VSMCs中SMα-actin蛋白表達(++),較E10.5 d明顯增強,同時VSMCs中CREG蛋白表達(++)也明顯增強。胚胎E15.5 d至E18.5 d,隨著血管進一步發(fā)育,血管壁內、中、外三層膜結構逐漸清晰,管腔增大,與周圍組織分界清楚。管壁各層細胞間排列致密,中膜VSMCs由多角形轉化為長梭形,并呈層狀排列,細胞核漿比例大。在E15.5 d胚胎血管中SMα-actin蛋白(+++)和CREG蛋白的表達(+++)均較前述各時間點明顯增強,并且CREG蛋白在血管壁內、中和外膜三層結構細胞中均明顯表達。但胚胎發(fā)育至E18.5 d時,SMα-actin蛋白(+++)仍在VSMCs中高表達,而CREG蛋白表達則下調(++)。 (4)新生及成年小鼠主動脈的形態(tài)學變化及CREG的表達:新生1 d小鼠主動脈血管壁內、中和外膜結構清晰,管腔大且與周圍組織分界清楚。管壁各層細胞間排列緊密,VSMCs在動脈中膜層狀排列,細胞細長呈長梭形,胞核明顯,核漿比例及平均細胞直徑較大。出生28 d后,小鼠主動脈逐漸發(fā)育成熟與2 m齡成鼠主動脈的形態(tài)相似,細胞外基質增加,VSMCs被增寬的彈力膜和膠原纖維分隔形成成熟的中膜層。VSMCs核漿比例進一步縮小,細胞核近似長桿狀。出生后1 d、28 d和2 m小鼠主動脈血管壁中SMα-actin蛋白和CREG表達均為陽性(++)。 (5)不同臟器功能血管中CREG的表達:成年小鼠心、肺、脾和腎臟標本石蠟切片的CREG免疫組化分析顯示,上述臟器功能血管中CREG蛋白表達均為陽性,但表達強度不同。其中心臟冠狀動脈的CREG蛋白表達呈強陽性(+++)。而在肺小動脈、脾動脈和腎臟小動脈中,CREG蛋白的表達則為弱陽性(+),較冠狀動脈明顯降低。 結論①CREG在小鼠胚胎早期E5.5 d開始表達,持續(xù)表達至出生前,表達含量逐漸增多。CREG在胚胎各臟器發(fā)生中的表達時相不同,但E18.5 d時胚胎各臟器中均可見CREG蛋白表達,其表達分布與成年小鼠各臟器的表達一致。上述結果提示,CREG可能作為胚胎發(fā)育的調控因子,在胚胎發(fā)育、分化和成熟過程中起重要作用。②CREG蛋白在小鼠胚胎血管發(fā)育極早期即開始表達、持續(xù)表達的特點及其在不同臟器功能血管中表達的差異,提示CREG蛋白可能通過調控并維持管壁細胞,特別是VSMCs的分化,參與了胚胎血管發(fā)生的調控。
[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.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R321

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