PTEN在胚胎早期血管发生中的作用研究

发布时间：2018-04-18 12:46

本文选题：血管发生 + 胚胎发育　；参考：《暨南大学》2011年硕士论文

【摘要】：目的胚胎早期血管发生(vasculogenesis)和肿瘤血管新生(angiogenesis)具有相似相关性,通过研究抑癌基因PTEN在早期胚胎血管发生中对细胞迁移、分化的影响,为进一步对肿瘤的治疗提供基础理论支持。方法胚胎原位杂交：利用分子生物学手段合成chick PTEN和VE-Cadherin的RNA探针,并且检测其在早期鸡胚胎中原位表达情况。利用这一技术我们过表达PTEN后检测胚胎基因的表达。标记造血前体细胞：利用早期鸡胚胎体外培养的方法(EC Culture),将早期胚胎在体外培养至HH3期左右,利用DiI和GFP标记示踪原条后部造血前体细胞的迁移模式。示踪造血前体细胞迁移：为了研究PTEN基因是否参与调节血管发生,采用显微注射和电穿孔导入基因法将pEGFP-N1和Wt PTEN-GFP质粒导入早期胚胎中,构建过表达PTEN基因的胚胎模型。通过荧光表达情况,观察GFP阳性细胞迁移情况。另外,采用异体移植后部原条的方法,提供进一步的实验证明。将转染有pEGFP-N1和Wt PTEN-GFP的供体胚胎的原条后部切下,分别移植到HH3期左右的原条后部,观察过表达PTEN基因的细胞在后部原条迁移EMT过程中的作用。该方法排除后部原条周围GFP的干扰,更直观的观察细胞迁移轨迹。 VE-Cadherin表达标记血岛：过表达PTEN基因的胚胎通过VE-Cadherin的原位杂交显示血岛形成的形态变化,分析过表达PTEN基因的细胞在血岛发生中的作用,并对转染pEGFP-N1和Wt PTEN-GFP后的胚胎血岛密度及基因参与血岛形成情况进行IPP软件计算和SPSS统计软件分析。抑制PI3K信号：采用半侧LY294002处理胚胎的方法,半侧阻断PI3K信号通路,另半侧作为正常对照,观察血岛形态及密度的变化情况,并做统计分析。结果 chick PTEN在早期鸡胚胎的上胚层和中胚层开始表达,之后在胚体及暗区(胚外血管发生部位)血岛上均有表达。VE-Cadherin在早期鸡胚胎的血岛结构中表达明显,于血管内皮前体细胞和造血干细胞中均有表达。DiI标记后部原条细胞迁移至暗区并参与血管发生过程。转染和异体移植pEGFP-N1和Wt PTEN-GFP的原条后发现,过表达PTEN基因的细胞在后部原条迁移的EMT过程受到抑制,转染Wt PTEN-GFP的细胞大都停留在胚胎的上胚层而没有进入中胚层。过表达PTEN基因的胚胎对VE-Cadherin的表达基本没有影响,对血岛的密度的影响也不大(p0.05),但是过表达PTEN基因的细胞很多都没有参与到血岛的形成中,与转染pEGFP-N1组的胚胎相比有显著差异(p0.01)。阻断PI3K信号通路后,血岛密度影响不大(p0.05),但是对血岛的形态有明显的影响,血岛大都聚集成簇,没有明显的岛状及正常的血岛之间明显的连接缝隙。结论过表达PTEN基因后对后部原条向后迁移的EMT过程有抑制作用,同时在血管发生过程中对血岛的形成有抑制作用。阻断PI3K信号通路影响了血岛的形态,但对血岛密度影响不大。
[Abstract]:PurposeEarly embryonic angiogenesis (Vasculogenesis) and tumor angiogenesis (Angiogenesis) have similar correlation. By studying the effect of tumor suppressor gene PTEN on cell migration and differentiation in early embryonic angiogenesis, this study provides basic theoretical support for the further treatment of tumor.MethodIn situ hybridization (ish): the RNA probes of chick PTEN and VE-Cadherin were synthesized by molecular biology and their expression was detected in the early chicken embryos.We use this technique to detect the expression of embryonic genes after overexpression of PTEN.Labelled hematopoietic progenitor cells: using the method of early chicken embryo culture in vitro, the early embryo was cultured to HH3 stage in vitro, and DiI and GFP were used to trace the migration pattern of hematopoietic progenitor cells in the back of the original strip.Tracing hematopoietic progenitor cell migration: in order to study whether PTEN gene is involved in angiogenesis, pEGFP-N1 and Wt PTEN-GFP plasmids were introduced into early embryos by microinjection and electroporation to construct an embryo model of overexpression of PTEN gene.The migration of GFP positive cells was observed by fluorescence expression.In addition, the method of allograft posterior original was used to provide further experimental proof.The donor embryos transfected with pEGFP-N1 and Wt PTEN-GFP were cut off and transplanted to the posterior of the HH3 phase, respectively. The role of the cells expressing PTEN gene in the process of EMT migration was observed.This method eliminates the interference of GFP around the back strip, and more intuitively observe the migration path of cells.VE-Cadherin expression marker blood island: embryos with overexpression of PTEN gene show the morphological changes of blood island formation by in situ hybridization of VE-Cadherin, and analyze the role of cells expressing PTEN gene in the development of blood island.After transfection of pEGFP-N1 and Wt PTEN-GFP, the density of embryonic blood island and the involvement of gene in the formation of blood island were calculated by IPP software and analyzed by SPSS software.Inhibition of PI3K signal: half side LY294002 was used to treat embryo, half side blocked PI3K signal pathway, the other half was used as normal control to observe the changes of blood island morphology and density, and to do statistical analysis.ResultChick PTEN was expressed in the epiderm and mesoderm of the early chicken embryo, and then in both the embryonic body and the dark area (the location of extraembryonic angiogenesis). VE-Cadherin was expressed significantly in the blood island structure of the early chicken embryo.Both endothelial precursor cells and hematopoietic stem cells expressed. Dii labeled the posterior original cells migrated to the dark area and participated in the angiogenesis process.After transfection and allotransplantation of pEGFP-N1 and Wt PTEN-GFP, it was found that the cells overexpression of PTEN gene were inhibited in the EMT migration process of the posterior strand, and most of the cells transfected with Wt PTEN-GFP remained in the embryonic ectoderm but did not enter the mesoderm.The overexpression of PTEN gene had no effect on the expression of VE-Cadherin, nor did it affect the density of the blood island. However, many of the cells overexpression of PTEN gene did not participate in the formation of the blood island, and there was a significant difference between the embryos transfected with pEGFP-N1 and the embryos transfected with pEGFP-N1.After blocking the PI3K signaling pathway, the density of the blood island had little effect on the morphology of the blood island, but it had a significant effect on the morphology of the blood island. Most of the blood islands were clustered into clusters, and there was no obvious connection gap between the blood island and the normal blood island.ConclusionOverexpression of PTEN gene inhibited the process of backward migration of EMT and the formation of blood island during angiogenesis.Blocking the PI3K signaling pathway affected the morphology of the blood island, but had little effect on the density of the blood island.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R321

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