本文選題：血管發(fā)生 + 胚胎發(fā)育��； 參考：《暨南大學(xué)》2011年碩士論文

【摘要】：目的 胚胎早期血管發(fā)生(vasculogenesis)和腫瘤血管新生(angiogenesis)具有相似相關(guān)性,通過(guò)研究抑癌基因PTEN在早期胚胎血管發(fā)生中對(duì)細(xì)胞遷移、分化的影響,為進(jìn)一步對(duì)腫瘤的治療提供基礎(chǔ)理論支持。 方法 胚胎原位雜交：利用分子生物學(xué)手段合成chick PTEN和VE-Cadherin的RNA探針,并且檢測(cè)其在早期雞胚胎中原位表達(dá)情況。利用這一技術(shù)我們過(guò)表達(dá)PTEN后檢測(cè)胚胎基因的表達(dá)。 標(biāo)記造血前體細(xì)胞：利用早期雞胚胎體外培養(yǎng)的方法(EC Culture),將早期胚胎在體外培養(yǎng)至HH3期左右,利用DiI和GFP標(biāo)記示蹤原條后部造血前體細(xì)胞的遷移模式。 示蹤造血前體細(xì)胞遷移：為了研究PTEN基因是否參與調(diào)節(jié)血管發(fā)生,采用顯微注射和電穿孔導(dǎo)入基因法將pEGFP-N1和Wt PTEN-GFP質(zhì)粒導(dǎo)入早期胚胎中,構(gòu)建過(guò)表達(dá)PTEN基因的胚胎模型。通過(guò)熒光表達(dá)情況,觀察GFP陽(yáng)性細(xì)胞遷移情況。另外,采用異體移植后部原條的方法,提供進(jìn)一步的實(shí)驗(yàn)證明。將轉(zhuǎn)染有pEGFP-N1和Wt PTEN-GFP的供體胚胎的原條后部切下,分別移植到HH3期左右的原條后部,觀察過(guò)表達(dá)PTEN基因的細(xì)胞在后部原條遷移EMT過(guò)程中的作用。該方法排除后部原條周?chē)鶪FP的干擾,更直觀的觀察細(xì)胞遷移軌跡。 VE-Cadherin表達(dá)標(biāo)記血島：過(guò)表達(dá)PTEN基因的胚胎通過(guò)VE-Cadherin的原位雜交顯示血島形成的形態(tài)變化,分析過(guò)表達(dá)PTEN基因的細(xì)胞在血島發(fā)生中的作用,并對(duì)轉(zhuǎn)染pEGFP-N1和Wt PTEN-GFP后的胚胎血島密度及基因參與血島形成情況進(jìn)行IPP軟件計(jì)算和SPSS統(tǒng)計(jì)軟件分析。 抑制PI3K信號(hào)：采用半側(cè)LY294002處理胚胎的方法,半側(cè)阻斷PI3K信號(hào)通路,另半側(cè)作為正常對(duì)照,觀察血島形態(tài)及密度的變化情況,并做統(tǒng)計(jì)分析。 結(jié)果 chick PTEN在早期雞胚胎的上胚層和中胚層開(kāi)始表達(dá),之后在胚體及暗區(qū)(胚外血管發(fā)生部位)血島上均有表達(dá)。VE-Cadherin在早期雞胚胎的血島結(jié)構(gòu)中表達(dá)明顯,于血管內(nèi)皮前體細(xì)胞和造血干細(xì)胞中均有表達(dá)。DiI標(biāo)記后部原條細(xì)胞遷移至暗區(qū)并參與血管發(fā)生過(guò)程。轉(zhuǎn)染和異體移植pEGFP-N1和Wt PTEN-GFP的原條后發(fā)現(xiàn),過(guò)表達(dá)PTEN基因的細(xì)胞在后部原條遷移的EMT過(guò)程受到抑制,轉(zhuǎn)染W(wǎng)t PTEN-GFP的細(xì)胞大都停留在胚胎的上胚層而沒(méi)有進(jìn)入中胚層。過(guò)表達(dá)PTEN基因的胚胎對(duì)VE-Cadherin的表達(dá)基本沒(méi)有影響,對(duì)血島的密度的影響也不大(p0.05),但是過(guò)表達(dá)PTEN基因的細(xì)胞很多都沒(méi)有參與到血島的形成中,與轉(zhuǎn)染pEGFP-N1組的胚胎相比有顯著差異(p0.01)。阻斷PI3K信號(hào)通路后,血島密度影響不大(p0.05),但是對(duì)血島的形態(tài)有明顯的影響,血島大都聚集成簇,沒(méi)有明顯的島狀及正常的血島之間明顯的連接縫隙。 結(jié)論 過(guò)表達(dá)PTEN基因后對(duì)后部原條向后遷移的EMT過(guò)程有抑制作用,同時(shí)在血管發(fā)生過(guò)程中對(duì)血島的形成有抑制作用。阻斷PI3K信號(hào)通路影響了血島的形態(tài),但對(duì)血島密度影響不大。
[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.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R321

【共引文獻(xiàn)】

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