Lef1和Msx2基因在阿尔巴斯绒山羊毛乳头细胞增殖中的调控作用

发布时间：2018-05-24 12:27

  本文选题：绒山羊 + 毛乳头细胞　； 参考：《内蒙古大学》2017年硕士论文

【摘要】：羊绒在绒山羊毛囊中发育形成,由毛乳头细胞通过多种信号通路来调控其生长和发育,因此毛乳头细胞也被称为是调控毛囊发育和周期的控制中心。内蒙古绒山羊毛囊具有两种类型,包括初级毛囊和次级毛囊。这两种毛囊分别会产生羊毛和羊绒两种不同类型的毛纤维。调控这两种毛囊的生长和发育是多种信号因子相互作用的结果。本实验以阿尔巴斯绒山羊两种毛乳头细胞为模型,研究与绒毛生长相关的Lef1和Msx2基因对毛乳头细胞的增殖作用。1、过表达和干扰载体的构建通过基因片段克隆、酶切和连接等方法,成功构建了 4种载体,分别是Lef1基因过表达载体、Lef1干扰载体、Msx2基因过表达载体和Msx2基因干扰载体。2、阿尔巴斯绒山羊初级和次级毛乳头细胞的培养及鉴定通过常规细胞培养法,本实验成功培养了阿尔巴斯绒山羊的初级和次级毛乳头细胞,并利用荧光免疫法检测毛乳头细胞的特异性标记因子α-SMA和CD133,鉴定本实验所培养的细胞确实为毛乳头细胞。3、Lef1基因的过表达和干扰细胞株的转染及检测通过脂质体转染方法将Lef1基因的过表达载体和干扰载体成功转入阿尔巴斯绒山羊的初级和次级毛乳头细胞中,大量培养后,得到Lef1基因的4种细胞株,分别为Lef1基因过表达的初级和次级毛乳头细胞株,以及Lef1基因干扰的初级次级毛乳头细胞株。Real-time PCR和Western Blot检测Lef1基因及相关基因β-catenin、C-myc和cyclinD1的表达量发现:以未转染任何质粒的细胞作为实验对照组,在初级毛乳头细胞中Lef1过表达和干扰组的表达量分别是对照的9.25和0.2倍。而在次级毛乳头细胞中,Lef1过表达和干扰组的表达量分别是对照的10.53和0.21倍。实验结果表明本实验成功建立了 Lef1基因的四种细胞株。在实验中检测各个样本中Lef1、β-catenin、C-myc和cyclinD1基因的表达情况发现:两种毛乳头细胞中Lef1基因的表达变化与β-catenin、C-myc和cyclinD1基因的表达变化趋势一致。实验结果表明:在两种毛乳头细胞中Lef1基因与β-catenin、C-myc和cyclinD1基因的表达呈正相关性。同时也比较了 Lef1基因的四种细胞株中相关基因的表达情况,结果发现:Lef1、β-catenin、cyclinD1在次级毛乳头细胞中的表达量分别是初级毛乳头细胞的1.28、2.19、1.16倍。而C-myc在次级毛乳头细胞中表达量是初级毛乳头细胞的0.37倍。实验结果表明:与绒毛生长的相关的Lef1、β-catenin、cyclinD1和C-myc基因在两种毛乳头细胞中的表达量是不同的。4.Msx2基因的过表达和干扰细胞株的转染及检测通过脂质体转染方法将Msx2基因的过表达载体和干扰载体成功转入阿尔巴斯绒山羊的初级和次级毛乳头细胞中,大量培养后,得到Msx2基因的4种细胞株,分别为Msx2基因过表达的初级和次级毛乳头细胞株,以及Msx2基因干扰的初级和次级毛乳头细胞株。Real-time PCR和Western Blot检测Msx2基因及相关基因Lef1、BMP-2的表达量发现:以未转染任何质粒的细胞作为实验对照组,在初级毛乳头细胞中Msx2过表达和干扰组的表达量分别是对照的11.85和0.31倍。在次级毛乳头细胞中,Msx2过表达和干扰组的表达量分别是对照的10.59和0.45倍。实验结果表明本实验成功建立了 Msx2基因的四种细胞株。在实验中检测各个样本中Msx2、Lef1和BMP-2基因的表达情况发现:在两种毛乳头细胞中Msx2基因的表达与Lef1和BMP-2基因的表达变化趋势一致。实验结果表明:在毛乳头细胞中Msx2基因的表达与Lef1和BMP-2基因的表达呈正相关性。通过对各细胞株生长情况的观察,发现Lef1和Msx2基因对毛乳头细胞的增殖能力起到正调控作用,Lef1和Msx2基因的干扰使得毛乳头细胞的增殖能力下降。上述实验研究表明:在毛乳头细胞中Lef1基因与β-catenin、C-myc和cyclin D1基因呈正相关性的调控,Msx2基因与Lef1和BMP-2基因呈正相关性的调控,Lef1和Msx2基因对毛乳头细胞的增殖具有正调控作用。
[Abstract]:Cashmere is developed in the cashmere wool sac, which regulates the growth and development of the hair papilla cells through a variety of signal pathways. Therefore, the dermal papilla cells are also known as control centers for the development and cycle of hair follicles. The wool sac of Inner Mongolia cashmere mountain has two types, including primary hair follicles and secondary follicles. The two kinds of hair follicles produce sheep respectively. The growth and development of these two kinds of wool and cashmere are the result of the interaction between the two kinds of hair follicles. In this experiment, two hair papilla cells of Alba cashmere goats were used as models to study the proliferation of Lef1 and Msx2 genes related to the growth of villi, and to overexpress and interfere with the carrier of the dermal papilla cells. 4 kinds of vectors were constructed through gene fragment cloning, enzyme cutting and connection, such as Lef1 gene overexpression vector, Lef1 interference carrier, Msx2 gene overexpression vector and Msx2 gene interference carrier.2. The primary and secondary dermal papilla cells of the EBA cashmere goat were cultured and identified by conventional cell culture method. The primary and secondary dermal papilla cells of Alba cashmere goats were cultured, and the specific marker factor alpha -SMA and CD133 were detected by fluorescence immunoassay. It was identified that the cells cultured in this experiment were.3 of the dermal papilla cells, the overexpression of Lef1 gene and the transfection of the interfering cell lines and the transfection by liposome. The overexpression vector and interference vector of the Lef1 gene were successfully transferred into the primary and secondary dermal papilla cells of the alpha cashmere goat. After a large number of cultures, 4 Lef1 genes were obtained, respectively, the primary and secondary dermal papilla cells of the Lef1 gene, and the primary secondary dermal papilla cell strain of the Lef1 gene,.Real-time PCR, and the Lef1 gene interference. Western Blot detected the expression of Lef1 gene and related gene beta -catenin, C-myc and cyclinD1: the expression of Lef1 overexpression and interference group in primary dermal papilla cells was 9.25 and 0.2 times respectively in the primary dermal papilla cells, while in primary dermal papilla cells, Lef1 overexpression and interference groups were in the primary dermal papilla cells. The expression was 10.53 and 0.21 times as high as that of the control. The experimental results showed that the Lef1 gene was successfully established in this experiment. In the experiment, the expression of Lef1, beta -catenin, C-myc and cyclinD1 gene in each sample was detected: the changes of the Lef1 gene in the two dermal papilla cells and the table of the beta -catenin, C-myc and cyclinD1 genes. The results showed that the expression of Lef1 gene in two kinds of dermal papilla cells was positively correlated with the expression of beta -catenin, C-myc and cyclinD1 genes. Meanwhile, the expression of related genes in the four cell lines of the Lef1 gene was also compared. The results showed that the expression of Lef1, beta -catenin, cyclinD1 in secondary dermal papilla cells was respectively The expression of C-myc in primary dermal papilla cells is 0.37 times that of primary dermal papilla cells. Experimental results show that the expression of Lef1, beta -catenin, cyclinD1 and C-myc genes related to the growth of villus is the overexpression and interference of different.4.Msx2 genes in the two kinds of dermal papilla cells. The transfection and detection of the Msx2 gene were successfully transfected into the primary and secondary dermal papilla cells of the alpha cashmere goat by liposome transfection. After a large number of cultures, 4 Msx2 gene cells were obtained, respectively, the primary and secondary dermal papilla cells of the Msx2 gene, and the Msx2 gene. The perturbed primary and secondary dermal papilla cells,.Real-time PCR and Western Blot, detected the Msx2 gene and related gene Lef1, and the expression of BMP-2 was found: the cells that were not transfected with any plasmid were used as the experimental control group. The expression of Msx2 overexpression and interference in primary dermal papilla cells was 11.85 and 0.31 times as high as that of the control. In the cells, the expression of Msx2 overexpression and interference group was 10.59 and 0.45 times as high as that of the control. The experimental results showed that the four cell lines of the Msx2 gene were successfully established in this experiment. In the experiment, the expression of Msx2, Lef1 and BMP-2 genes in each sample was detected. The expression of Msx2 gene in the two dermal papilla cells and the Lef1 and BMP-2 genes were found in the experiment. The results showed that the expression of Msx2 gene in the dermal papilla cells was positively correlated with the expression of Lef1 and BMP-2 genes. Through the observation of the growth of each cell line, it was found that Lef1 and Msx2 genes played a positive role in the proliferation of dermal papilla cells, and the interference of Lef1 and Msx2 genes made the dermal papilla fine. The experimental study indicated that the Lef1 gene in the dermal papilla cells has a positive correlation with the genes of beta -catenin, C-myc and cyclin D1, and the Msx2 gene has a positive correlation with the Lef1 and BMP-2 genes, and Lef1 and Msx2 genes have a positive regulation on the proliferation of dermal papilla cells.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S827

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