發(fā)布時間：2018-05-01 02:01

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

【摘要】：小鼠胚胎干細胞(mESCs)來源于胚胎著床前的多能干細胞,能夠在體外持續(xù)培養(yǎng)并保持自我更新的能力,同時又具有多向分化的潛能,可以分化成機體的各種細胞類型。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,同時廣泛的用于再生醫(yī)學的研究及細胞學移植的研究。經(jīng)典的Wnt/β-catenin信號通路在胚胎的發(fā)育以及多種干細胞的分化中起著關(guān)鍵的作用。研究表明,Wnt/β-catenin信號通路在胚胎時期心臟的發(fā)育以及血管形成等過程中起到了不可或缺的作用,在胚胎干細胞分化過程的特定的時間段激活該信號通路可誘導胚胎干細胞定向分化成心肌細胞。Brachyury(T)基因是Wnt/β-catenin信號通路的下游靶基因,也是中胚層的標志基因,在胚胎發(fā)育中胚層形成的過程中高度表達。為了研究T基因在mESCs分化過程中的作用,我們通過PB(PiggyBac)質(zhì)粒過表達該基因,將構(gòu)建好過表達質(zhì)粒后轉(zhuǎn)染到mESCs中,采用LIF/serum培養(yǎng)基培養(yǎng),并用細胞計數(shù)的方法檢測過表達T基因后細胞的增殖情況,并用堿性磷酸酶染色(AP染色)方法檢測mESCs的分化情況。分化實驗是采用單個細胞懸浮培養(yǎng)的方法首先形成擬胚體(EBs),形成的EBs可形成三個胚層,其中中胚層可進一步分化形成心肌細胞。研究報道,單獨使用GSK3β抑制劑BIO可以誘導胚胎干細胞分化成心肌細胞。我們研究表明在mESCs分化早期階段加入GSK3β抑制劑CHIR99021(CHIR)可以促進胚胎干細胞向心肌細胞分化,實驗時我們加入CHIR作為陽性對照組。研究結(jié)果表明,過表達T基因后細胞的增殖慢于對照組,且細胞的分化傾向增加且更易于分化。分化實驗的qRT-PCR結(jié)果表明,三組隨著分化時間的增加心肌標志基因cTnT、Cx43、Nkx2.5、和α-MHC的表達量增加,其中過表達T基因的T組表達量高于陰性對照PB組,而低于陽性對照CH組。Western blot結(jié)果顯示第10天T組的Cx43和α-MHC蛋白的表達量高于PB組而低于CH組,第15天的蛋白表達量趨勢與第10天一樣,但各組相應的蛋白表達量高于第10天的表達量。免疫熒光染色結(jié)果與Western blot結(jié)果趨勢一樣即三組中α-MHC和Cx43蛋白免疫熒光均陽性,T組熒光強于PB組,CH組熒光強于T組。因此,過表達T基因可促進mESCs向心肌細胞分化,分化效率弱于GSK3β抑制劑CHIR的誘導效率。為了進一步研究T基因的促進mESCs心肌細胞的分化作用,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檢測心肌標志基因的表達情況,用Western blot檢測心肌標志蛋白。實驗結(jié)果表明三組隨著分化時間的增加心肌標志基因cTnT、Cx43、Nkx2.5和α-MHC的表達量增加,其中敲低組的表達量低于對照組。Western blot結(jié)果顯示第10天敲低組的Cx43和α-MHC蛋白的表達量低于對照組,第15天的蛋白表達量趨勢與第10天一樣,但各組相應的蛋白表達量高于第10天的表達量。因此,敲低T基因后心肌細胞分化效率降低。我們研究表明,T基因可以促進小鼠胚胎干細胞向心肌細胞分化,但促分化的效率低于GSK3抑制劑CHIR的誘導效率。
[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.

【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R54

【相似文獻】

相關(guān)期刊論文 前10條

1 諶兵來,胡隨瑜,謝小毛,劉小平;小鼠胚胎干細胞建系新方法的初步研究[J];湖南大學學報(自然科學版);2003年05期

2 趙玲;杜曉蘭;王建民;陳林;;小鼠胚胎干細胞的培養(yǎng)[J];現(xiàn)代生物醫(yī)學進展;2007年06期

3 譚廣云;謝萬華;周焱;黃永業(yè);宋紅肖;李棟;宋娜;袁婷;段新平;逄大欣;歐陽紅生;;綠色熒光小鼠胚胎干細胞的分離與培養(yǎng)[J];現(xiàn)代生物醫(yī)學進展;2010年12期

4 阮靜;沈潔;汪錚;崔大祥;;調(diào)控量子點對小鼠胚胎干細胞活性影響的研究[J];東南大學學報(醫(yī)學版);2011年01期

5 宗卉,劉乃豐,葉銀英,劉紅林,汪河海,范必勤;小鼠胚胎干細胞的培養(yǎng)與建系[J];南京鐵道醫(yī)學院學報;1997年02期

6 姚丹丹,苑春慧;不同多羥基化合物對小鼠胚胎干細胞低溫貯存的影響[J];醫(yī)學理論與實踐;2005年04期

7 徐令;麥友剛;溫麗華;梅志勇;;小鼠胚胎干細胞體外分化為肝細胞的實驗研究[J];中山大學學報(醫(yī)學科學版);2005年S1期

8 沈岳飛;羅杰峰;莫雪安;龍桂芳;;小鼠胚胎干細胞的分離培養(yǎng)與鑒定[J];廣西醫(yī)科大學學報;2006年01期

9 黃t，

本文編號：1827214