【摘要】：豬既是重要經(jīng)濟(jì)動(dòng)物,又是一種理想的醫(yī)學(xué)模型動(dòng)物,獲得豬多能干細(xì)胞對(duì)動(dòng)物育種和再生醫(yī)學(xué)研究有重要意義。但是,目前沒(méi)有豬ESCs建系的報(bào)道,已報(bào)道建系的(包括本實(shí)驗(yàn)室之前報(bào)道的)豬iPS細(xì)胞依然存在諸多問(wèn)題。深入研究豬多能性相關(guān)基因,發(fā)掘豬與小鼠和人的信號(hào)通路差異,能夠?yàn)槲覀兝斫庳i多能干細(xì)胞的分子調(diào)控機(jī)制提供幫助。國(guó)內(nèi)外已經(jīng)建系的豬iPSCs細(xì)胞中,EpCAM(epithelial cell adhesion molecule)呈現(xiàn)高表達(dá)。在人和小鼠中,EpCAM也可作為指示多能性的標(biāo)記物而用于iPS和其他多能細(xì)胞的鑒定和篩選。除了作為多能性標(biāo)記物外,EpCAM的胞內(nèi)水解產(chǎn)物EpICD可進(jìn)入細(xì)胞核,調(diào)節(jié)某些基因的表達(dá)。本研究以豬EpCAM基因和蛋白作為研究對(duì)象,通過(guò)檢測(cè)EpCAM在豬組織和已經(jīng)建系的豬iPS細(xì)胞中的表達(dá)情況,明確了EpCAM和豬多能性之間存在的聯(lián)系;通過(guò)在iPS細(xì)胞中敲低和超表達(dá)EpCAM,建立了EpCAM與豬多能基因的調(diào)控關(guān)系;通過(guò)在豬體細(xì)胞向iPS誘導(dǎo)重編程過(guò)程中敲低或超表達(dá)EpCAM,發(fā)現(xiàn)了EpCAM及其裂解產(chǎn)物EpICD對(duì)細(xì)胞重編程過(guò)程起重要調(diào)控作用;最后,通過(guò)進(jìn)一步對(duì)EpCAM蛋白調(diào)控和信號(hào)通路的研究,明確了EpCAM在豬多能細(xì)胞中的作用方式和途徑。1.EpCAM基因的轉(zhuǎn)錄譜及其表達(dá)調(diào)控本研究通過(guò)檢測(cè)豬組織、iPS細(xì)胞、早期胚胎發(fā)育過(guò)程中和細(xì)胞重編程過(guò)程中EpCAM的表達(dá)變化,試圖建立EpCAM與豬多能性之間的聯(lián)系;通過(guò)構(gòu)建豬EpCAM啟動(dòng)子報(bào)告載體,嘗試了解豬EpCAM基因在iPS細(xì)胞中的表達(dá)調(diào)控情況。結(jié)果表明,豬各組織中均能檢測(cè)到EpCAM的表達(dá),而在上皮細(xì)胞豐富的器官組織中,EpCAM表達(dá)量更高。在對(duì)建系的豬iPS細(xì)胞和豬成纖維細(xì)胞的比較中,EpCAM在各株iPS細(xì)胞中的表達(dá)水平均高于其前體細(xì)胞PEF,說(shuō)明在重編程過(guò)程中EpCAM被激活;EpCAM的表達(dá)模式與關(guān)鍵的多能基因呈正相關(guān),而與胚層分化基因呈現(xiàn)負(fù)相關(guān);EpCAM的表達(dá)還與MET的相關(guān)基因呈現(xiàn)一定的相關(guān)性。對(duì)早期胚胎中的多能基因表達(dá)分析結(jié)果則說(shuō)明,EpCAM在豬早期胚胎中的表達(dá)模式與關(guān)鍵多能基因的表達(dá)模式類似。OCT4和SOX2蛋白對(duì)EpCAM啟動(dòng)子具有下調(diào)作用,而KLF4、c-MYC、NANOG、LIN28、SALL4和ESRRB等多能轉(zhuǎn)錄因子對(duì)EpCAM啟動(dòng)子具有上調(diào)作用。2.EpCAM在細(xì)胞重編程過(guò)程中和iPS細(xì)胞中的調(diào)控作用利用構(gòu)建的shRNA干擾載體和EpCAM超表達(dá)載體,觀察其對(duì)PEF細(xì)胞誘導(dǎo)重編程過(guò)程的影響。敲低EpCAM表達(dá)會(huì)降低iPS誘導(dǎo)中的AP(alklin phosphatase,堿性磷酸酶)陽(yáng)性克隆形成率,而超表達(dá)EpCAM則能夠顯著提高AP陽(yáng)性率。在建系的豬DOX-iPS細(xì)胞中敲低EpCAM,導(dǎo)致內(nèi)源OCT4、SOX2、LIN28、SALL4和ESRRB的表達(dá)顯著下調(diào),而超表達(dá)EpCAM則導(dǎo)致這些基因的顯著上調(diào)。另外,敲低內(nèi)源EpCAM的表達(dá),DOX-iPS細(xì)胞克隆團(tuán)的形態(tài)會(huì)出現(xiàn)松散狀,表明EpCAM敲低導(dǎo)致的內(nèi)源多能基因下調(diào),進(jìn)而造成iPS細(xì)胞出現(xiàn)分化狀態(tài)。3.豬EpCAM相關(guān)信號(hào)通路本研究發(fā)現(xiàn),豬EpCAM在細(xì)胞膜上經(jīng)蛋白酶水解,能夠產(chǎn)生一個(gè)胞內(nèi)多肽EpICD。通過(guò)利用小分子抑制劑抑制蛋白酶TACE和PS-2活性,可以降低EpCAM的裂解,減少EpICD的產(chǎn)生。通過(guò)RT-PCR檢測(cè)發(fā)現(xiàn),在豬不同組織細(xì)胞中TACE和PS-2的m RNA水平存在差異,表明在不同細(xì)胞中,EpCAM可能存在不同的裂解效率,從而產(chǎn)生不同豐度的EpICD來(lái)調(diào)控其下游基因。通過(guò)在DOX-iPS細(xì)胞中添加TACE和PS-2的抑制劑,會(huì)使EpCAM靶基因OCT4,SOX2,LIN28和ESRRB等的表達(dá)量下調(diào),說(shuō)明EpCAM的轉(zhuǎn)錄調(diào)控作用是由其裂解產(chǎn)生的EpICD實(shí)現(xiàn)的。將超表達(dá)EpICD的載體轉(zhuǎn)染DOX-iPS細(xì)胞,可以顯著促進(jìn)iPS細(xì)胞中EpCAM下游靶基因的表達(dá)水平。在對(duì)PEF細(xì)胞進(jìn)行誘導(dǎo)重編程時(shí),超表達(dá)EpICD可以提高形成克隆的AP陽(yáng)性率。但是,在非iPS細(xì)胞培養(yǎng)條件下,在PEF中超表達(dá)EpICD無(wú)法激活EpCAM下游靶基因,而在培養(yǎng)體系中添加GSK3抑制劑CHIR99021,則EpCAM靶基因啟動(dòng)子可以被激活。原因是GSK3對(duì)beta-CATENIN的降解具有促進(jìn)作用,抑制GSK3的表達(dá)可以提高beta-CATENIN的水平。因此,EpCAM依賴beta-CATENIN信號(hào)通路來(lái)發(fā)揮對(duì)下游靶基因的轉(zhuǎn)錄調(diào)控作用。
[Abstract]:Pig is not only an important economic animal, but also an ideal medical model animal. Obtaining porcine pluripotent stem cells is of great significance to animal breeding and regenerative medicine. However, there is no report on the establishment of the pig ESCs line at present. It has been reported that there are still many problems in the established pig iPS cells (including previous reports in the laboratory). Sex related genes, which discover the differences in the signaling pathways between pigs and humans, can help us understand the molecular regulatory mechanism of porcine pluripotent stem cells. EpCAM (epithelial cell adhesion molecule) is highly expressed in domestic and foreign established pig iPSCs cells. In humans and rats, EpCAM can also be used as a marker to indicate pluripotent activity. It is used for identification and screening of iPS and other pluripotent cells. In addition to being a pluripotent marker, the intracellular hydrolysate of EpCAM, EpICD, can enter the nucleus and regulate the expression of some genes. In this study, the porcine EpCAM gene and protein were used as the research object. By detecting the expression of EpCAM in pig and established pig iPS cells, the expression of EpCAM was determined. The relationship between EpCAM and porcine pluripotent activity was established. The regulation relationship between EpCAM and porcine pluripotent gene was established by knocking down and overexpressing EpCAM in iPS cells. By knocking down or overexpressing EpCAM in the process of reprogramming the pig somatic cells to iPS, it was found that EpCAM and its cracking product EpICD play an important role in the process of reprogramming of cell reprogramming. After further study on the regulation and signaling pathway of EpCAM protein, the mode of action of EpCAM in porcine pluripotent cells and the transcriptional spectrum of.1.EpCAM gene and its expression and regulation are clarified, and the changes in the expression of EpCAM during the detection of pig tissues, iPS cells, early embryonic development and cell reprogramming are tried to establish EpCA. The relationship between M and porcine pluripotent activity; the expression regulation of porcine EpCAM gene in iPS cells was attempted by building a porcine EpCAM promoter report vector. The results showed that the expression of EpCAM could be detected in all the tissues of the pig, and the expression of EpCAM was higher in the abundant organ tissues of the epithelial cells. In the porcine iPS cells and pigs that were built, the porcine iPS cells and pigs were formed. In the comparison of vascular cells, the expression level of EpCAM in all iPS cells was higher than that of PEF in the progenitor cells, indicating that EpCAM was activated during reprogramming; the expression pattern of EpCAM was positively correlated with the key pluripotent genes, but negatively correlated with the gene of the germ layer differentiation; the expression of EpCAM also showed a certain correlation with the related genes of MET. The multipotent gene expression analysis in the embryo shows that the expression pattern of EpCAM in the early porcine embryos is similar to that of the key pluripotent gene expression patterns..OCT4 and SOX2 proteins have a downregulation effect on the EpCAM promoter, while KLF4, c-MYC, NANOG, LIN28, SALL4 and ESRRB are up to regulate.2.EpCAM in the EpCAM promoter. The effect of shRNA interference carrier and EpCAM overexpression vector on the regulation of cell reprogramming and iPS cells was used to observe the effect on the reprogramming process of PEF cells. Low EpCAM expression could reduce the formation rate of AP (alklin phosphatase, alkaline phosphatase) positive clones in iPS induced, and the overexpression EpCAM could be significantly raised. The high AP positive rate. The expression of OCT4, SOX2, LIN28, SALL4 and ESRRB decreased significantly in the OCT4, SOX2, LIN28, SALL4 and ESRRB in the built pig cells, and the overexpression of EpCAM resulted in the significant up-regulation of these genes. Moreover, the expression of low endogenous EpCAM and the form of DOX-iPS cell clones appeared loosely, indicating the endogenous multiple energy caused by low EpCAM knockout. The gene down-regulation leads to the emergence of iPS cell differentiation state.3. EpCAM related signaling pathway. It is found that porcine EpCAM can produce an intracellular polypeptide EpICD. by protease hydrolysis on the cell membrane, which can reduce the fragmentation of EpCAM and reduce the production of EpICD by using small molecule inhibitors to reduce the activity of EpCAM and reduce the production of EpICD. Through RT-PCR, it can reduce the production of EpICD. The detection results showed that the m RNA levels of TACE and PS-2 in different tissues of pigs were different, indicating that in different cells, EpCAM may have different cracking efficiency, resulting in the production of different abundance EpICD to regulate its downstream genes. By adding TACE and PS-2 inhibitor in DOX-iPS cells, EpCAM target genes will be OCT4, SOX2, etc. The expression of EpCAM is downregulated, indicating that the transcriptional regulation of EpICD is realized by its lysis. Transfection of the vector of overexpression EpICD to DOX-iPS cells can significantly promote the expression level of the target gene of the downstream EpCAM in iPS cells. In the reprogramming of PEF cells, the overexpression of EpICD can increase the AP positive rate of the formation of the clone. However, under the condition of non iPS cell culture, the overexpression of EpICD in PEF can not activate the target gene of the downstream EpCAM, and the GSK3 inhibitor CHIR99021 is added to the culture system. The promoter of the EpCAM target gene can be activated. The reason is that GSK3 can promote the degradation of beta-CATENIN, and the expression of inhibition GSK3 can increase the beta-CATENIN level. EpCAM relies on beta-CATENIN signaling pathway to regulate the transcription of downstream target genes.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S828

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