本文選題：絨山羊 + 毛乳頭細(xì)胞��； 參考：《內(nèi)蒙古大學(xué)》2017年碩士論文

【摘要】：羊絨在絨山羊毛囊中發(fā)育形成,由毛乳頭細(xì)胞通過(guò)多種信號(hào)通路來(lái)調(diào)控其生長(zhǎng)和發(fā)育,因此毛乳頭細(xì)胞也被稱為是調(diào)控毛囊發(fā)育和周期的控制中心。內(nèi)蒙古絨山羊毛囊具有兩種類型,包括初級(jí)毛囊和次級(jí)毛囊。這兩種毛囊分別會(huì)產(chǎn)生羊毛和羊絨兩種不同類型的毛纖維。調(diào)控這兩種毛囊的生長(zhǎng)和發(fā)育是多種信號(hào)因子相互作用的結(jié)果。本實(shí)驗(yàn)以阿爾巴斯絨山羊兩種毛乳頭細(xì)胞為模型,研究與絨毛生長(zhǎng)相關(guān)的Lef1和Msx2基因?qū)γ轭^細(xì)胞的增殖作用。1、過(guò)表達(dá)和干擾載體的構(gòu)建通過(guò)基因片段克隆、酶切和連接等方法,成功構(gòu)建了 4種載體,分別是Lef1基因過(guò)表達(dá)載體、Lef1干擾載體、Msx2基因過(guò)表達(dá)載體和Msx2基因干擾載體。2、阿爾巴斯絨山羊初級(jí)和次級(jí)毛乳頭細(xì)胞的培養(yǎng)及鑒定通過(guò)常規(guī)細(xì)胞培養(yǎng)法,本實(shí)驗(yàn)成功培養(yǎng)了阿爾巴斯絨山羊的初級(jí)和次級(jí)毛乳頭細(xì)胞,并利用熒光免疫法檢測(cè)毛乳頭細(xì)胞的特異性標(biāo)記因子α-SMA和CD133,鑒定本實(shí)驗(yàn)所培養(yǎng)的細(xì)胞確實(shí)為毛乳頭細(xì)胞。3、Lef1基因的過(guò)表達(dá)和干擾細(xì)胞株的轉(zhuǎn)染及檢測(cè)通過(guò)脂質(zhì)體轉(zhuǎn)染方法將Lef1基因的過(guò)表達(dá)載體和干擾載體成功轉(zhuǎn)入阿爾巴斯絨山羊的初級(jí)和次級(jí)毛乳頭細(xì)胞中,大量培養(yǎng)后,得到Lef1基因的4種細(xì)胞株,分別為L(zhǎng)ef1基因過(guò)表達(dá)的初級(jí)和次級(jí)毛乳頭細(xì)胞株,以及Lef1基因干擾的初級(jí)次級(jí)毛乳頭細(xì)胞株。Real-time PCR和Western Blot檢測(cè)Lef1基因及相關(guān)基因β-catenin、C-myc和cyclinD1的表達(dá)量發(fā)現(xiàn):以未轉(zhuǎn)染任何質(zhì)粒的細(xì)胞作為實(shí)驗(yàn)對(duì)照組,在初級(jí)毛乳頭細(xì)胞中Lef1過(guò)表達(dá)和干擾組的表達(dá)量分別是對(duì)照的9.25和0.2倍。而在次級(jí)毛乳頭細(xì)胞中,Lef1過(guò)表達(dá)和干擾組的表達(dá)量分別是對(duì)照的10.53和0.21倍。實(shí)驗(yàn)結(jié)果表明本實(shí)驗(yàn)成功建立了 Lef1基因的四種細(xì)胞株。在實(shí)驗(yàn)中檢測(cè)各個(gè)樣本中Lef1、β-catenin、C-myc和cyclinD1基因的表達(dá)情況發(fā)現(xiàn):兩種毛乳頭細(xì)胞中Lef1基因的表達(dá)變化與β-catenin、C-myc和cyclinD1基因的表達(dá)變化趨勢(shì)一致。實(shí)驗(yàn)結(jié)果表明:在兩種毛乳頭細(xì)胞中Lef1基因與β-catenin、C-myc和cyclinD1基因的表達(dá)呈正相關(guān)性。同時(shí)也比較了 Lef1基因的四種細(xì)胞株中相關(guān)基因的表達(dá)情況,結(jié)果發(fā)現(xiàn):Lef1、β-catenin、cyclinD1在次級(jí)毛乳頭細(xì)胞中的表達(dá)量分別是初級(jí)毛乳頭細(xì)胞的1.28、2.19、1.16倍。而C-myc在次級(jí)毛乳頭細(xì)胞中表達(dá)量是初級(jí)毛乳頭細(xì)胞的0.37倍。實(shí)驗(yàn)結(jié)果表明:與絨毛生長(zhǎng)的相關(guān)的Lef1、β-catenin、cyclinD1和C-myc基因在兩種毛乳頭細(xì)胞中的表達(dá)量是不同的。4.Msx2基因的過(guò)表達(dá)和干擾細(xì)胞株的轉(zhuǎn)染及檢測(cè)通過(guò)脂質(zhì)體轉(zhuǎn)染方法將Msx2基因的過(guò)表達(dá)載體和干擾載體成功轉(zhuǎn)入阿爾巴斯絨山羊的初級(jí)和次級(jí)毛乳頭細(xì)胞中,大量培養(yǎng)后,得到Msx2基因的4種細(xì)胞株,分別為Msx2基因過(guò)表達(dá)的初級(jí)和次級(jí)毛乳頭細(xì)胞株,以及Msx2基因干擾的初級(jí)和次級(jí)毛乳頭細(xì)胞株。Real-time PCR和Western Blot檢測(cè)Msx2基因及相關(guān)基因Lef1、BMP-2的表達(dá)量發(fā)現(xiàn):以未轉(zhuǎn)染任何質(zhì)粒的細(xì)胞作為實(shí)驗(yàn)對(duì)照組,在初級(jí)毛乳頭細(xì)胞中Msx2過(guò)表達(dá)和干擾組的表達(dá)量分別是對(duì)照的11.85和0.31倍。在次級(jí)毛乳頭細(xì)胞中,Msx2過(guò)表達(dá)和干擾組的表達(dá)量分別是對(duì)照的10.59和0.45倍。實(shí)驗(yàn)結(jié)果表明本實(shí)驗(yàn)成功建立了 Msx2基因的四種細(xì)胞株。在實(shí)驗(yàn)中檢測(cè)各個(gè)樣本中Msx2、Lef1和BMP-2基因的表達(dá)情況發(fā)現(xiàn):在兩種毛乳頭細(xì)胞中Msx2基因的表達(dá)與Lef1和BMP-2基因的表達(dá)變化趨勢(shì)一致。實(shí)驗(yàn)結(jié)果表明:在毛乳頭細(xì)胞中Msx2基因的表達(dá)與Lef1和BMP-2基因的表達(dá)呈正相關(guān)性。通過(guò)對(duì)各細(xì)胞株生長(zhǎng)情況的觀察,發(fā)現(xiàn)Lef1和Msx2基因?qū)γ轭^細(xì)胞的增殖能力起到正調(diào)控作用,Lef1和Msx2基因的干擾使得毛乳頭細(xì)胞的增殖能力下降。上述實(shí)驗(yàn)研究表明:在毛乳頭細(xì)胞中Lef1基因與β-catenin、C-myc和cyclin D1基因呈正相關(guān)性的調(diào)控,Msx2基因與Lef1和BMP-2基因呈正相關(guān)性的調(diào)控,Lef1和Msx2基因?qū)γ轭^細(xì)胞的增殖具有正調(diào)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.
【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S827

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