Brachyury在小鼠胚胎干细胞分化为心肌细胞中的作用

发布时间：2018-05-01 02:01

本文选题：Brachyury + 胚胎干细胞　；参考：《安徽医科大学》2017年硕士论文

【摘要】：小鼠胚胎干细胞(mESCs)来源于胚胎着床前的多能干细胞,能够在体外持续培养并保持自我更新的能力,同时又具有多向分化的潜能,可以分化成机体的各种细胞类型。目前己经在发育分化模型的构建中起到不可或缺的作用,同时广泛的用于再生医学的研究及细胞学移植的研究。经典的Wnt/β-catenin信号通路在胚胎的发育以及多种干细胞的分化中起着关键的作用。研究表明,Wnt/β-catenin信号通路在胚胎时期心脏的发育以及血管形成等过程中起到了不可或缺的作用,在胚胎干细胞分化过程的特定的时间段激活该信号通路可诱导胚胎干细胞定向分化成心肌细胞。Brachyury(T)基因是Wnt/β-catenin信号通路的下游靶基因,也是中胚层的标志基因,在胚胎发育中胚层形成的过程中高度表达。为了研究T基因在mESCs分化过程中的作用,我们通过PB(PiggyBac)质粒过表达该基因,将构建好过表达质粒后转染到mESCs中,采用LIF/serum培养基培养,并用细胞计数的方法检测过表达T基因后细胞的增殖情况,并用碱性磷酸酶染色(AP染色)方法检测mESCs的分化情况。分化实验是采用单个细胞悬浮培养的方法首先形成拟胚体(EBs),形成的EBs可形成三个胚层,其中中胚层可进一步分化形成心肌细胞。研究报道,单独使用GSK3β抑制剂BIO可以诱导胚胎干细胞分化成心肌细胞。我们研究表明在mESCs分化早期阶段加入GSK3β抑制剂CHIR99021(CHIR)可以促进胚胎干细胞向心肌细胞分化,实验时我们加入CHIR作为阳性对照组。研究结果表明,过表达T基因后细胞的增殖慢于对照组,且细胞的分化倾向增加且更易于分化。分化实验的qRT-PCR结果表明,三组随着分化时间的增加心肌标志基因cTnT、Cx43、Nkx2.5、和α-MHC的表达量增加,其中过表达T基因的T组表达量高于阴性对照PB组,而低于阳性对照CH组。Western blot结果显示第10天T组的Cx43和α-MHC蛋白的表达量高于PB组而低于CH组,第15天的蛋白表达量趋势与第10天一样,但各组相应的蛋白表达量高于第10天的表达量。免疫荧光染色结果与Western blot结果趋势一样即三组中α-MHC和Cx43蛋白免疫荧光均阳性,T组荧光强于PB组,CH组荧光强于T组。因此,过表达T基因可促进mESCs向心肌细胞分化,分化效率弱于GSK3β抑制剂CHIR的诱导效率。为了进一步研究T基因的促进mESCs心肌细胞的分化作用,我们采用慢病毒pLKO.1质粒系统敲低该基因,将重组质粒用相同的方法转染到mESCs中,采用LIF/serum培养基培养,用悬浮培养方法形成EBs。用qRT-PCR检测心肌标志基因的表达情况,用Western blot检测心肌标志蛋白。实验结果表明三组随着分化时间的增加心肌标志基因cTnT、Cx43、Nkx2.5和α-MHC的表达量增加,其中敲低组的表达量低于对照组。Western blot结果显示第10天敲低组的Cx43和α-MHC蛋白的表达量低于对照组,第15天的蛋白表达量趋势与第10天一样,但各组相应的蛋白表达量高于第10天的表达量。因此,敲低T基因后心肌细胞分化效率降低。我们研究表明,T基因可以促进小鼠胚胎干细胞向心肌细胞分化,但促分化的效率低于GSK3抑制剂CHIR的诱导效率。
[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.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54

【相似文献】