|
| TET2在帕金森病细胞模型中的表达及机制研究 |
| The expression and mechanism of TET2 in the pathogenesis of Parkinson's Disease |
| 投稿时间：2019-03-14 修订日期：2019-03-18 |
| DOI： |
| 中文关键词: 帕金森病 TET2 DNA羟甲基化 中脑多巴胺能神经元 |
| 英文关键词: Parkinson's disease TET2 DNA hydroxymethylation Midbrain dopaminergic neuron |
| 基金项目:HIF-1α-ATP13A2信号通路对帕金森病多巴胺能神经元损伤的保护作用和机制研究 |
|
| 摘要点击次数: 486 |
| 全文下载次数: 0 |
| 中文摘要: |
| 目的：帕金森病（PD）是中老年人第二大常见的神经变性疾病，与中脑多巴胺能神经元损伤密切相关。表观遗传学调控可能在PD的发生发展中起着重要的作用。其中，DNA甲基化是最常研究的表观遗传修饰方式。最新报道显示，TET酶可以催化DNA羟甲基化进而逆转DNA甲基化。然而，TET酶在PD发病机制中的作用目前尚不知晓。本研究在PD体外细胞模型中寻找新型表观遗传标志物，探索PD的发病机制。
方法：本次研究使用的细胞为神经母细胞瘤细胞系SH-SY5Y。首先，我们用CCK-8检测细胞活力，选取合适浓度的MPP+构建PD细胞损伤模型。再用PBS和MPP+分别处理SH-SY5Y细胞，用RT-qPCR检测了几个甲基化酶DNMT1, DNMT3A, DNMT3B与去甲基化酶TET1, TET2, TET3的mRNA的表达水平，并用蛋白印迹检测TET2蛋白水平，免疫荧光检测了TET2蛋白定位。进一步用慢病毒转染SH-SY5Y细胞敲低TET2后，检测细胞增殖。
结果：本研究发现，MPP+对SH-SY5Y细胞增殖的抑制具有时间与浓度依赖性，我们最终选择2.5 mM MPP+作为后续的细胞处理浓度。与对照组相比，MPP+处理细胞TET2的mRNA及蛋白水平表达均增加，且蛋白进入细胞核增加；同时发现，敲低TET2表达可以延缓MPP+对SH-SY5Y细胞增殖的抑制作用。
结论：在当前的研究中，我们报道了TET2蛋白可能是PD新型的表观遗传学标志物，提示我们将来也许可以使用TET2抑制剂来治疗PD，因此本研究有可能为PD提供新的治疗方向和靶点。 |
| 英文摘要: |
| Objective: Parkinson’s disease (PD) is the second most common neurodegenerative disorder, and it is associated with midbrain dopaminergic neuron (mDA) loss. Epigenetic alterations have been hypothesized to play a key role in the pathogenesis of PD. Epigenetic modifications, particularly DNA methylation, have attracted significant research attention. The ten-eleven translocation (TET) family of 5-mC hydroxylases, could oxidize DNA 5-mC to 5-hmC toward a complete removal of the methylated cytosine and consequent DNA demethylation. This study mainly investigated the novel epigenetic markers and explored the pathogenesis of PD in the cellular model.
Methods: We used the neuroblastoma cell line SH-SY5Y in this study. To find the optimal concentration of MPP+ for SH-SY5Y cells, we used CCK-8 to detect and select an optimum concentration. SH-SY5Y cells were treated with PBS and MPP+ respectively, then the mRNA expression levels of several methylases and demethylases, such as DNMT1, DNMT3A, DNMT3B, TET1, TET2 and TET3 were detected by RT-qPCR, and level of TET2 protein was detected by western blot. Finally, the protein localization of TET2 was detected by immunofluorescence. Furthermore, we knockdown TET2 with a lentivirus to detect cell proliferation.
Results: With the increase of time period orconcentration, the cell inhibition rates increased, we finally select a concentration at which the cell inhibition rate was about 50 %, which turned out to be 2.5 mM. We observed that TET2 was significantly upregulated and recruited to the nuclear bodies of SH-SY5Y cells after MPP+ treatment. And the CCK-8 assays showed that MPP+ inhibited the proliferation of SH-SY5Y cell lines, while downregulation of TET2 promoted proliferation.
Conclusions: In the present study, we reported the functions of TET2 proteins in the pathogenesis of PD as novel epigenetic markers, suggesting that we could use TET2 inhibitors to treat PD in the future, so this study may provide us a new direction and target for PD treatment. |
|
View Fulltext
查看/发表评论 下载PDF阅读器 |
| 关闭 |
|
|
|
