本文選題：核型Vasohibin-2 + 組織芯片�。� 參考：《南京醫(yī)科大學(xué)》2015年碩士論文

【摘要】：背景：Vasohibin (VASH)是近年來發(fā)現(xiàn)的一族新基因,該基因家族目前由VASH1及VASH2共同組成。VASH2具有與VASH1具有截然相反的功能：對(duì)血管生成起促進(jìn)作用。最近,VASH2還被發(fā)現(xiàn)在多種實(shí)體腫瘤中具有促進(jìn)腫瘤血管生成的作用。因此,VASH家族被認(rèn)為在胚胎發(fā)育及多種實(shí)體腫瘤的發(fā)生、發(fā)展過程中通過調(diào)控血管生成起促瘤作用。我們?cè)诜治鯲ASH2蛋白細(xì)胞內(nèi)定位時(shí)發(fā)現(xiàn)：編碼355個(gè)氨基酸的VASH2蛋白定位于胞漿中,而編碼311個(gè)氨基酸的VASH2蛋白定位于胞核中；根據(jù)VASH2蛋白不同變構(gòu)體的細(xì)胞內(nèi)差異定位,我們將VASH2分為胞漿型及核型�，F(xiàn)有研究主要集中于胞漿型VASH2,而核型VASH2蛋白的相關(guān)研究尚無報(bào)道。我們首次分析了胞核型VASH2在不同胎齡人胚胎組織、多器官腫瘤及正常組織中的表達(dá)及相關(guān)功能。目的：1)研究不同胎齡人胚胎組織中胞核VASH2表達(dá)與細(xì)胞增殖作用的相關(guān)性。2)研究人多器官腫瘤及正常組織中胞核VASH2表達(dá)及促細(xì)胞增殖功能。方法：1)購(gòu)買26個(gè)器官/78點(diǎn)的胚胎組織芯片。免疫組化檢測(cè)核型VASH2的表達(dá),并分析核型VASH2表達(dá)與胚胎組織細(xì)胞增殖的相關(guān)性。進(jìn)一步免疫熒光分析VASH2蛋白在人胚腎細(xì)胞293T的表達(dá)水平和細(xì)胞中的表達(dá)部位。MTT法檢測(cè)核型VASH2對(duì)293T細(xì)胞活性的影響,流式細(xì)胞儀檢測(cè)細(xì)胞周期。2)購(gòu)買547點(diǎn)的人多器官腫瘤及正常組織芯片,免疫組化檢測(cè)核型VASH2的表達(dá)并分析核型VASH2表達(dá)與細(xì)胞增殖的相關(guān)性。進(jìn)一步免疫組化檢測(cè)Ki-67與核型VASH2表達(dá)水平差異,驗(yàn)證核型VASH2與細(xì)胞增殖的相關(guān)性。構(gòu)建核型VASH2過表達(dá)/干擾穩(wěn)轉(zhuǎn)肝臟腫瘤及正常細(xì)胞模型。檢測(cè)穩(wěn)轉(zhuǎn)肝臟腫瘤及正常細(xì)胞BrdU光吸收度；流式細(xì)胞儀檢測(cè)細(xì)胞周期并分析過表達(dá)/干擾穩(wěn)轉(zhuǎn)肝臟腫瘤及正常細(xì)胞周期的變化；結(jié)果：1)購(gòu)買了臨床資料完整的胚胎組織芯片。胞核型VASH2除了在已經(jīng)發(fā)育完善的心臟組織中表達(dá)陰性或弱表達(dá),在其他胚胎組織中顯著高表達(dá)。核型VASH2的表達(dá)與胚胎細(xì)胞核分裂指數(shù)明顯正相關(guān)。轉(zhuǎn)染核型VASH2可促進(jìn)293T細(xì)胞的生長(zhǎng)；轉(zhuǎn)染核型VASH2細(xì)胞的G2+S期較空白組及干擾組增加,比較差異有顯著性。2)胞核型VASH2除了在已經(jīng)發(fā)育完善的成熟組織中表達(dá)陰性或弱表達(dá),在其他處于分裂增殖狀態(tài)正常及腫瘤組織中顯著高表達(dá)。Ki-67檢測(cè)證實(shí)核型VASH2的表達(dá)與細(xì)胞增殖存在相關(guān)性。過表達(dá)核型VASH2上調(diào)肝臟腫瘤及正常細(xì)胞中BrdU水平；干擾核型VASH2下調(diào)肝臟腫瘤及正常細(xì)胞中BrdU水平。轉(zhuǎn)染核型VASH2肝臟腫瘤及正常細(xì)胞的G2+S期較空白組及干擾組增加。結(jié)論：1)核型VASH2促進(jìn)人胚胎、正常組織及腫瘤細(xì)胞增殖。2)核型VASH2通過促進(jìn)細(xì)胞周期G0/G1向S轉(zhuǎn)化來促進(jìn)細(xì)胞增殖。
[Abstract]:Background: Vasohibin (VASH) is a novel gene found in recent years. The gene family consists of VASH1 and VASH2. VASH2 has the opposite function to VASH1: it promotes angiogenesis. Recently VASH2 has also been found to promote tumor angiogenesis in a variety of solid tumors. Therefore, the VASH family is thought to play a role in promoting tumor formation by regulating angiogenesis during embryonic development and the development of various solid tumors. When we analyzed the intracellular localization of VASH2 protein, we found that the VASH2 protein encoding 355 amino acids was located in the cytoplasm, while the VASH2 protein encoding 311 amino acids was located in the nucleus. We divided VASH2 into cytoplasm and karyotype. The present studies are mainly focused on cytoplasmic VASH2, but the study of karyotype VASH2 protein has not been reported. We first analyzed the expression and related functions of VASH2 in human embryonic tissues, multiple organ tumors and normal tissues at different gestational ages. Objective: to study the relationship between VASH2 expression and cell proliferation in human embryonic tissues of different gestational ages. Methods: 1) buy 26 organs / 78 point embryonic tissue chip. The expression of VASH2 was detected by immunohistochemistry, and the correlation between the expression of VASH2 and the proliferation of embryonic tissues was analyzed. The expression level of VASH2 protein in human embryonic kidney cell line 293T and the expression site of VASH2 protein in human embryonic kidney cells were analyzed by immunofluorescence. MTT assay was used to detect the effect of VASH2 on the activity of 293T cells. Flow cytometry was used to detect the expression of VASH2 and the correlation between the expression of VASH2 and cell proliferation. The expression levels of Ki-67 and VASH2 were detected by immunohistochemistry to verify the correlation between VASH2 and cell proliferation. To construct a karyotype VASH2 overexpression / interference stable liver tumor and normal cell model. BrdU light absorption of stable liver tumor and normal cells was detected. Flow cytometry was used to detect cell cycle and analyze the changes of overexpression / interference in stable liver tumor and normal cell cycle. Results: 1) embryo tissue microarray with complete clinical data was purchased. In addition to negative or weak expression of VASH2 in developed heart tissues, VASH2 was significantly overexpressed in other embryonic tissues. The expression of VASH2 was positively correlated with the nuclear mitosis index. Transfection of VASH2 could promote the growth of 293T cells, and the G2-S phase of transfected VASH2 cells was significantly higher than that of blank group and interference group, the difference was significant (.2) the expression of VASH2 was negative or weakly expressed in mature tissues. The expression of VASH2 was correlated with cell proliferation in other tissues with normal mitotic proliferation and significantly higher expression of Ki-67. Overexpression of VASH2 upregulated BrdU levels in liver tumors and normal cells, and interfered with VASH2 down-regulation of BrdU levels in liver tumors and normal cells. The G 2 S phase transfected with VASH2 liver tumor and normal cells was significantly higher than that in control group and interference group. ConclusionVASH2 promotes proliferation of human embryos, normal tissues and tumor cells. 2) VASH2 promotes cell proliferation by promoting cell cycle from G _ 0 / G _ 1 to S.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R735.7

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