核型Vasohibin-2在人胚胎组织、多器官肿瘤及正常组织中的表达及促增殖功能的初步研究

发布时间：2018-06-27 18:46

本文选题：核型Vasohibin-2 + 组织芯片　；参考：《南京医科大学》2015年硕士论文

【摘要】：背景：Vasohibin (VASH)是近年来发现的一族新基因,该基因家族目前由VASH1及VASH2共同组成。VASH2具有与VASH1具有截然相反的功能：对血管生成起促进作用。最近,VASH2还被发现在多种实体肿瘤中具有促进肿瘤血管生成的作用。因此,VASH家族被认为在胚胎发育及多种实体肿瘤的发生、发展过程中通过调控血管生成起促瘤作用。我们在分析VASH2蛋白细胞内定位时发现：编码355个氨基酸的VASH2蛋白定位于胞浆中,而编码311个氨基酸的VASH2蛋白定位于胞核中；根据VASH2蛋白不同变构体的细胞内差异定位,我们将VASH2分为胞浆型及核型。现有研究主要集中于胞浆型VASH2,而核型VASH2蛋白的相关研究尚无报道。我们首次分析了胞核型VASH2在不同胎龄人胚胎组织、多器官肿瘤及正常组织中的表达及相关功能。目的：1)研究不同胎龄人胚胎组织中胞核VASH2表达与细胞增殖作用的相关性。2)研究人多器官肿瘤及正常组织中胞核VASH2表达及促细胞增殖功能。方法：1)购买26个器官/78点的胚胎组织芯片。免疫组化检测核型VASH2的表达,并分析核型VASH2表达与胚胎组织细胞增殖的相关性。进一步免疫荧光分析VASH2蛋白在人胚肾细胞293T的表达水平和细胞中的表达部位。MTT法检测核型VASH2对293T细胞活性的影响,流式细胞仪检测细胞周期。2)购买547点的人多器官肿瘤及正常组织芯片,免疫组化检测核型VASH2的表达并分析核型VASH2表达与细胞增殖的相关性。进一步免疫组化检测Ki-67与核型VASH2表达水平差异,验证核型VASH2与细胞增殖的相关性。构建核型VASH2过表达/干扰稳转肝脏肿瘤及正常细胞模型。检测稳转肝脏肿瘤及正常细胞BrdU光吸收度；流式细胞仪检测细胞周期并分析过表达/干扰稳转肝脏肿瘤及正常细胞周期的变化；结果：1)购买了临床资料完整的胚胎组织芯片。胞核型VASH2除了在已经发育完善的心脏组织中表达阴性或弱表达,在其他胚胎组织中显著高表达。核型VASH2的表达与胚胎细胞核分裂指数明显正相关。转染核型VASH2可促进293T细胞的生长；转染核型VASH2细胞的G2+S期较空白组及干扰组增加,比较差异有显著性。2)胞核型VASH2除了在已经发育完善的成熟组织中表达阴性或弱表达,在其他处于分裂增殖状态正常及肿瘤组织中显著高表达。Ki-67检测证实核型VASH2的表达与细胞增殖存在相关性。过表达核型VASH2上调肝脏肿瘤及正常细胞中BrdU水平；干扰核型VASH2下调肝脏肿瘤及正常细胞中BrdU水平。转染核型VASH2肝脏肿瘤及正常细胞的G2+S期较空白组及干扰组增加。结论：1)核型VASH2促进人胚胎、正常组织及肿瘤细胞增殖。2)核型VASH2通过促进细胞周期G0/G1向S转化来促进细胞增殖。
[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.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.7

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