Mab21L2基因在人晶状体上皮细胞分化中的功能分析

发布时间：2018-01-11 20:09

  本文关键词：Mab21L2基因在人晶状体上皮细胞分化中的功能分析　出处：《湖南师范大学》2011年硕士论文　论文类型：学位论文

  更多相关文章： Mab21L2 pERK1/2 分化

【摘要】：以前的研究表明Mab21L2在眼睛尤其是晶状体发育过程中起重要作用[1,2],但是Mab21L2调控晶状体发育的分子机制目前尚不清楚.体外培养的晶状体上皮细胞,被广泛运用为研究晶状体发育和分化的模型。HLE细胞系是向体外培养的人晶状体上皮细胞中转入SV40大T抗原而得到的转化细胞系,我们利用这一细胞系研究Mab21L2在晶状体分化中的可能功能。纯化的Mab21L2表达质粒和它的载体被分别转入HLE细胞中,用400μg/ml的G418筛选获得稳定的克隆,然后用RT-PCR和Western Blot鉴定高表达Mab21L2的克隆。形态观察发现,高表达Mab21L2的克隆与载体转染的克隆相比,细胞形态有明显变化,细胞变长,有明显纤维化表型。为了进一步探索Mab21L2在晶状体分化中的功能,我们用75ng/ml和100ng/ml的FGF-basic处理Mab21L2和载体转染的稳定高表达克隆,在同样的浓度处理时,高表达Mab21L2的克隆大概只需2-3天,纤维化已经十分明显,但是转入了载体的对照克隆却需要5天才出现较明显纤维化,很明显过表达Mab21L2可以加速FGF-basic诱导的HLE细胞分化。FGF-basic处理细胞后,提取细胞总蛋白,Western Blot检测结果表明Mab21L2和载体转染的克隆中,pERK1/2的表达模式十分不同。在没有FGF-basic处理时,高表达Mab21L2的克隆中pERK1/2的表达被抑制,明显低于对照克隆中pERK1/2的表达；但是当用FGF-basic处理细胞1或2天后,高表达Mab21L2的克隆中pERK1/2的表达极大的增强,因而远高于对照克隆中的pERK1/2;但到处理4天后,高表达Mab21L2的克隆中pERK1/2表达急剧下降,又一次低于对照克隆中pERK1/2的表达。此外,表达系和对照系中,PP2A调节亚基B56γ和MEK2的表达模式也有明显差异,并且MEK2与pERK1/2的表达水平正相关,B56γ与pERK1/2的表达水平负相关。这些结果提示Mab21L2促进HLE细胞分化的分子机制是通过调节B56γ和MEK2的表达,从磷酸化和去磷酸化两方面,精确调控pERK1/2表达水平来实现的。在FGF-basic诱导HLE分化的不同阶段,Mab21L2既可正性也可负性调节pERK1/2表达水平。
[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 纬 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 纬 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.
【学位授予单位】：湖南师范大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

【参考文献】

相关期刊论文 前1条

1 蔡琪,李晓玫;丝裂原活化蛋白激酶信号转导通路研究进展[J];肾脏病与透析肾移植杂志;1999年04期

，

本文编号：1411020