本文選題：Brachyury + 胚胎干細(xì)胞��； 參考：《安徽醫(yī)科大學(xué)》2017年碩士論文

【摘要】：小鼠胚胎干細(xì)胞(mESCs)來(lái)源于胚胎著床前的多能干細(xì)胞,能夠在體外持續(xù)培養(yǎng)并保持自我更新的能力,同時(shí)又具有多向分化的潛能,可以分化成機(jī)體的各種細(xì)胞類型。目前己經(jīng)在發(fā)育分化模型的構(gòu)建中起到不可或缺的作用,同時(shí)廣泛的用于再生醫(yī)學(xué)的研究及細(xì)胞學(xué)移植的研究。經(jīng)典的Wnt/β-catenin信號(hào)通路在胚胎的發(fā)育以及多種干細(xì)胞的分化中起著關(guān)鍵的作用。研究表明,Wnt/β-catenin信號(hào)通路在胚胎時(shí)期心臟的發(fā)育以及血管形成等過(guò)程中起到了不可或缺的作用,在胚胎干細(xì)胞分化過(guò)程的特定的時(shí)間段激活該信號(hào)通路可誘導(dǎo)胚胎干細(xì)胞定向分化成心肌細(xì)胞。Brachyury(T)基因是Wnt/β-catenin信號(hào)通路的下游靶基因,也是中胚層的標(biāo)志基因,在胚胎發(fā)育中胚層形成的過(guò)程中高度表達(dá)。為了研究T基因在mESCs分化過(guò)程中的作用,我們通過(guò)PB(PiggyBac)質(zhì)粒過(guò)表達(dá)該基因,將構(gòu)建好過(guò)表達(dá)質(zhì)粒后轉(zhuǎn)染到mESCs中,采用LIF/serum培養(yǎng)基培養(yǎng),并用細(xì)胞計(jì)數(shù)的方法檢測(cè)過(guò)表達(dá)T基因后細(xì)胞的增殖情況,并用堿性磷酸酶染色(AP染色)方法檢測(cè)mESCs的分化情況。分化實(shí)驗(yàn)是采用單個(gè)細(xì)胞懸浮培養(yǎng)的方法首先形成擬胚體(EBs),形成的EBs可形成三個(gè)胚層,其中中胚層可進(jìn)一步分化形成心肌細(xì)胞。研究報(bào)道,單獨(dú)使用GSK3β抑制劑BIO可以誘導(dǎo)胚胎干細(xì)胞分化成心肌細(xì)胞。我們研究表明在mESCs分化早期階段加入GSK3β抑制劑CHIR99021(CHIR)可以促進(jìn)胚胎干細(xì)胞向心肌細(xì)胞分化,實(shí)驗(yàn)時(shí)我們加入CHIR作為陽(yáng)性對(duì)照組。研究結(jié)果表明,過(guò)表達(dá)T基因后細(xì)胞的增殖慢于對(duì)照組,且細(xì)胞的分化傾向增加且更易于分化。分化實(shí)驗(yàn)的qRT-PCR結(jié)果表明,三組隨著分化時(shí)間的增加心肌標(biāo)志基因cTnT、Cx43、Nkx2.5、和α-MHC的表達(dá)量增加,其中過(guò)表達(dá)T基因的T組表達(dá)量高于陰性對(duì)照PB組,而低于陽(yáng)性對(duì)照CH組。Western blot結(jié)果顯示第10天T組的Cx43和α-MHC蛋白的表達(dá)量高于PB組而低于CH組,第15天的蛋白表達(dá)量趨勢(shì)與第10天一樣,但各組相應(yīng)的蛋白表達(dá)量高于第10天的表達(dá)量。免疫熒光染色結(jié)果與Western blot結(jié)果趨勢(shì)一樣即三組中α-MHC和Cx43蛋白免疫熒光均陽(yáng)性,T組熒光強(qiáng)于PB組,CH組熒光強(qiáng)于T組。因此,過(guò)表達(dá)T基因可促進(jìn)mESCs向心肌細(xì)胞分化,分化效率弱于GSK3β抑制劑CHIR的誘導(dǎo)效率。為了進(jìn)一步研究T基因的促進(jìn)mESCs心肌細(xì)胞的分化作用,我們采用慢病毒pLKO.1質(zhì)粒系統(tǒng)敲低該基因,將重組質(zhì)粒用相同的方法轉(zhuǎn)染到mESCs中,采用LIF/serum培養(yǎng)基培養(yǎng),用懸浮培養(yǎng)方法形成EBs。用qRT-PCR檢測(cè)心肌標(biāo)志基因的表達(dá)情況,用Western blot檢測(cè)心肌標(biāo)志蛋白。實(shí)驗(yàn)結(jié)果表明三組隨著分化時(shí)間的增加心肌標(biāo)志基因cTnT、Cx43、Nkx2.5和α-MHC的表達(dá)量增加,其中敲低組的表達(dá)量低于對(duì)照組。Western blot結(jié)果顯示第10天敲低組的Cx43和α-MHC蛋白的表達(dá)量低于對(duì)照組,第15天的蛋白表達(dá)量趨勢(shì)與第10天一樣,但各組相應(yīng)的蛋白表達(dá)量高于第10天的表達(dá)量。因此,敲低T基因后心肌細(xì)胞分化效率降低。我們研究表明,T基因可以促進(jìn)小鼠胚胎干細(xì)胞向心肌細(xì)胞分化,但促分化的效率低于GSK3抑制劑CHIR的誘導(dǎo)效率。
[Abstract]:Mouse embryonic stem cells (mESCs) originate from the pluripotent stem cells of the embryo, which can continue to be cultured in vitro and maintain their ability to renew themselves. At the same time, they have the potential to differentiate into various types of cells. At present, it has played an indispensable role in the construction of the developmental differentiation model and is widely used. The classical Wnt/ beta -catenin signaling pathway plays a key role in the development of the embryo and the differentiation of a variety of stem cells. The study shows that the Wnt/ beta -catenin signaling pathway plays an indispensable role in the development of the embryonic heart and the formation of blood vessels. The specific time period of the differentiation process of fetal stem cells activates the signal pathway to induce the embryonic stem cells to differentiate into.Brachyury (T) gene as the downstream target gene of the Wnt/ beta -catenin signaling pathway, and also a marker gene of the mesoderm. It is highly expressed in the process of embryonic development of the mesoderm. In order to study the T gene in mESCs In the process of differentiation, we expressed the gene through the PB (PiggyBac) plasmid, then transfected the plasmid and transfected into mESCs, cultured the LIF/serum medium, and detected the proliferation of the cells after the expression of T gene with the cell count method. The differentiation of mESCs was detected by alkaline phosphatase staining (AP staining). The differentiation experiment is to form a single cell suspension culture method first to form the embryoid body (EBs), and the formation of EBs can form three germ layers, in which the mesoderm can further differentiate into cardiomyocytes. It is reported that the use of GSK3 beta inhibitor BIO alone can induce embryonic stem cells to differentiate into cardiac myocytes. Our study showed that in the early differentiation of mESCs. The addition of GSK3 beta inhibitor CHIR99021 (CHIR) could promote the differentiation of embryonic stem cells into cardiomyocytes. We added CHIR as a positive control group at the time of experiment. The results showed that the proliferation of cells after the overexpression of T gene was slower than that of the control group, and the differentiation tendency of the cells increased and the differentiation was more easily differentiated. The qRT-PCR results of the differentiation experiment showed that the three groups were three groups. The expression of myocardial marker gene cTnT, Cx43, Nkx2.5, and alpha -MHC increased with the time of differentiation, and the expression of T in group T gene was higher than that of negative control PB group, and the result of.Western blot in CH group was lower than that of the positive control CH group. The expression of Cx43 and alpha protein in the tenth day T group was higher than that in the group but was lower than that of the group and fifteenth days of protein expression. The quantity trend was the same as that of tenth days, but the expression of corresponding protein in each group was higher than that of tenth days. The results of immunofluorescence staining were the same as that of Western blot. The immunofluorescence of alpha -MHC and Cx43 protein in the three groups was positive, the fluorescence of T group was stronger than that of the PB group, and the fluorescence of CH group was stronger than that of the T group. Therefore, the overexpression of T gene could promote the differentiation of mESCs into the cardiomyocytes. The differentiation efficiency is weaker than the induction efficiency of GSK3 beta inhibitor CHIR. In order to further study the role of T gene in promoting the differentiation of mESCs cardiomyocytes, we use the lentivirus pLKO.1 plasmid system to knock down the gene and transfect the recombinant plasmid into mESCs with the same method, using LIF/serum culture medium to form EBs. using qRT- to form qRT-. The expression of myocardial marker gene was detected by PCR, and myocardial marker protein was detected by Western blot. The experimental results showed that the expression of myocardial marker gene cTnT, Cx43, Nkx2.5 and alpha -MHC in the three groups increased with the time of differentiation, and the expression of the knockout group was lower than the control group.Western blot results showed that the Cx43 and alpha -MHC eggs of the tenth day knockout group were in the lower group. The expression of white was lower than that of the control group. The expression of protein in fifteenth days was the same as that of tenth days, but the expression of protein in each group was higher than that of tenth days. Therefore, the efficiency of cardiomyocyte differentiation decreased after knocking down the T gene. Our study showed that the T gene could promote the differentiation of mouse embryonic stem cells to the cardiomyocytes, but the efficiency of promoting differentiation. The induction efficiency is lower than the GSK3 inhibitor CHIR.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R54

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