本文關(guān)鍵詞：Mab21L2基因在人晶狀體上皮細(xì)胞分化中的功能分析　出處：《湖南師范大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： Mab21L2 pERK1/2 分化

【摘要】：以前的研究表明Mab21L2在眼睛尤其是晶狀體發(fā)育過程中起重要作用[1,2],但是Mab21L2調(diào)控晶狀體發(fā)育的分子機(jī)制目前尚不清楚.體外培養(yǎng)的晶狀體上皮細(xì)胞,被廣泛運(yùn)用為研究晶狀體發(fā)育和分化的模型。HLE細(xì)胞系是向體外培養(yǎng)的人晶狀體上皮細(xì)胞中轉(zhuǎn)入SV40大T抗原而得到的轉(zhuǎn)化細(xì)胞系,我們利用這一細(xì)胞系研究Mab21L2在晶狀體分化中的可能功能。純化的Mab21L2表達(dá)質(zhì)粒和它的載體被分別轉(zhuǎn)入HLE細(xì)胞中,用400μg/ml的G418篩選獲得穩(wěn)定的克隆,然后用RT-PCR和Western Blot鑒定高表達(dá)Mab21L2的克隆。形態(tài)觀察發(fā)現(xiàn),高表達(dá)Mab21L2的克隆與載體轉(zhuǎn)染的克隆相比,細(xì)胞形態(tài)有明顯變化,細(xì)胞變長,有明顯纖維化表型。為了進(jìn)一步探索Mab21L2在晶狀體分化中的功能,我們用75ng/ml和100ng/ml的FGF-basic處理Mab21L2和載體轉(zhuǎn)染的穩(wěn)定高表達(dá)克隆,在同樣的濃度處理時(shí),高表達(dá)Mab21L2的克隆大概只需2-3天,纖維化已經(jīng)十分明顯,但是轉(zhuǎn)入了載體的對(duì)照克隆卻需要5天才出現(xiàn)較明顯纖維化,很明顯過表達(dá)Mab21L2可以加速FGF-basic誘導(dǎo)的HLE細(xì)胞分化。FGF-basic處理細(xì)胞后,提取細(xì)胞總蛋白,Western Blot檢測結(jié)果表明Mab21L2和載體轉(zhuǎn)染的克隆中,pERK1/2的表達(dá)模式十分不同。在沒有FGF-basic處理時(shí),高表達(dá)Mab21L2的克隆中pERK1/2的表達(dá)被抑制,明顯低于對(duì)照克隆中pERK1/2的表達(dá)；但是當(dāng)用FGF-basic處理細(xì)胞1或2天后,高表達(dá)Mab21L2的克隆中pERK1/2的表達(dá)極大的增強(qiáng),因而遠(yuǎn)高于對(duì)照克隆中的pERK1/2;但到處理4天后,高表達(dá)Mab21L2的克隆中pERK1/2表達(dá)急劇下降,又一次低于對(duì)照克隆中pERK1/2的表達(dá)。此外,表達(dá)系和對(duì)照系中,PP2A調(diào)節(jié)亞基B56γ和MEK2的表達(dá)模式也有明顯差異,并且MEK2與pERK1/2的表達(dá)水平正相關(guān),B56γ與pERK1/2的表達(dá)水平負(fù)相關(guān)。這些結(jié)果提示Mab21L2促進(jìn)HLE細(xì)胞分化的分子機(jī)制是通過調(diào)節(jié)B56γ和MEK2的表達(dá),從磷酸化和去磷酸化兩方面,精確調(diào)控pERK1/2表達(dá)水平來實(shí)現(xiàn)的。在FGF-basic誘導(dǎo)HLE分化的不同階段,Mab21L2既可正性也可負(fù)性調(diào)節(jié)pERK1/2表達(dá)水平。
[Abstract]:Previous studies have shown that Mab21L2 plays an important role in the development of the eyes, especially the lens. [However, the molecular mechanism of Mab21L2 regulating lens development is not clear. Lens epithelial cells cultured in vitro. HLE cell line is a transformed cell line derived from SV40 large T antigen transferred into human lens epithelial cells in vitro. We used this cell line to study the possible function of Mab21L2 in lens differentiation. The purified Mab21L2 expression plasmid and its vector were transferred into HLE cells respectively. The stable clones were screened by G418 of 400 渭 g / ml, and then the high expression Mab21L2 clones were identified by RT-PCR and Western Blot. Compared with the vector transfected clones, the high expression Mab21L2 clones showed obvious changes in cell morphology and cell length. In order to further explore the function of Mab21L2 in lens differentiation. We treated Mab21L2 with 75ng / ml and 100ng / ml FGF-basic and stably overexpressed clones transfected with vector, at the same concentration. The highly expressed Mab21L2 clones took only 2-3 days and the fibrosis was already very obvious, but it took 5 days for the control clones transferred to the vector to show more obvious fibrosis. Overexpression of Mab21L2 could accelerate the differentiation of HLE cells induced by FGF-basic. FGF-basic treatment could extract the total protein. The results of Western Blot analysis showed that the expression pattern of pERK1 / 2 in Mab21L2 and vector transfected clones was very different, without FGF-basic treatment. The expression of pERK1/2 in the clone with high expression of Mab21L2 was inhibited, which was significantly lower than that in the control clone. However, when treated with FGF-basic for 1 or 2 days, the expression of pERK1/2 in highly expressed Mab21L2 clones was significantly enhanced. Therefore, it was much higher than pERK1 / 2 in the control clone; However, after 4 days of treatment, the expression of pERK1/2 in the clones with high expression of Mab21L2 decreased sharply, again lower than that in the control clones. In addition, the expression of pERK1/2 in the expressed lines and the control lines was also decreased. The expression patterns of PP2A regulatory subunit B56 緯 and MEK2 were also significantly different, and the expression levels of MEK2 and pERK1/2 were positively correlated. These results suggest that the molecular mechanism of Mab21L2 promoting the differentiation of HLE cells is by regulating the expression of B56 緯 and MEK2. From two aspects of phosphorylation and dephosphorylation, the expression level of pERK1/2 was precisely regulated. At different stages of HLE differentiation induced by FGF-basic. Mab21L2 can regulate pERK1/2 expression both positively and negatively.
【學(xué)位授予單位】：湖南師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R346

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 蔡琪,李曉玫;絲裂原活化蛋白激酶信號(hào)轉(zhuǎn)導(dǎo)通路研究進(jìn)展[J];腎臟病與透析腎移植雜志;1999年04期

，

本文編號(hào)：1411020