Li Duo, Yuan Song-Tao, Xie Xin-Yi, Shen Han, Liu Qing-Huai, Yao Yong
Department of Ophthalmology, the Affiliated Wuxi People's Hospital of Nanjing Medical University, Wuxi 214000, Jiangsu Province, China.
Department of Ophthalmology, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210000, Jiangsu Province, China.
Int J Ophthalmol. 2022 May 18;15(5):711-720. doi: 10.18240/ijo.2022.05.04. eCollection 2022.
To explore the functions of Chordin-like 2, which is encoded by , in the process of retinal pigmented epithelium (RPE) differentiation and damage repair.
The fetal RPE cells (fRPE) was obtained from aborted fetus which obeyed medical ethics. Real-time quantitative polymerase chain reaction was used to measure expression quantity of and other functional genes expression. Knocking down and overexpression was used to analyze the functions about Chordin-like 2. Enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion of bone morphogenetic proteins 4 (BMP4). Flow cytometry was used to analyze cell cycle. Cell morphology was observed by phase contrast microscope (PCM).
In normal RPE cells, was firstly upregulated and followed a downregulation but eventually, it was expressed higher than the cells which undergone epithelial-mesenchymal transition (EMT). After knocking down , the secretion of BMP4 was decreased, RPE-related genes () were downregulated while EMT-related genes () were upregulated. However, the expression of these related genes after overexpression of had contrary results. Chordin-like 2 also regulated the cell cycle by regulating BMP pathway. When was knocked down, more fRPE cells stayed in S phase of cell cycle, while adding BMP4 reduced the proportion of the cells in S phase. However, overexpression of increased more BMP4 secretion, this effect decreased the number of cells in S phase, but exogenous BMP inhibitor also could change this effect. At last, in the process of RPE cells differentiation, adding BMP4 at early stage could intervene normal RPE differentiation. Compared with BMP4, inhibiting BMP pathway had no significant negative effect at early stage, but suppressed differentiation at late stage.
BMP pathway can be activated in a correct temporal order, otherwise, the cells have incorrect differentiation orientation. And Chordin-like 2 plays a role in dynamic regulation of BMP pathway and it also regulates the differentiation of RPE cells. Therefore, this research enlightens a new direction to inhibit EMT and promote cell redifferentiation after injury.
探讨由[具体基因名称未给出]编码的类脊索蛋白2在视网膜色素上皮（RPE）分化及损伤修复过程中的作用。
从符合医学伦理的流产胎儿获取胎儿RPE细胞（fRPE）。采用实时定量聚合酶链反应检测[具体基因名称未给出]及其他功能基因的表达量。运用敲低和过表达技术分析类脊索蛋白2的功能。采用酶联免疫吸附测定（ELISA）检测骨形态发生蛋白4（BMP4）的分泌。通过流式细胞术分析细胞周期。利用相差显微镜（PCM）观察细胞形态。
在正常RPE细胞中，[具体基因名称未给出]先上调后下调，但最终其表达高于经历上皮-间质转化（EMT）的细胞。敲低[具体基因名称未给出]后，BMP4分泌减少，RPE相关基因（[相关基因名称未给出]）下调，而EMT相关基因（[相关基因名称未给出]）上调。然而，[具体基因名称未给出]过表达后这些相关基因的表达结果相反。类脊索蛋白2还通过调节BMP通路调控细胞周期。敲低[具体基因名称未给出]时，更多fRPE细胞停留在细胞周期的S期，而添加BMP4可降低S期细胞比例。然而，[具体基因名称未给出]过表达增加了BMP4分泌，此效应减少了S期细胞数量，但外源性BMP抑制剂也可改变此效应。最后，在RPE细胞分化过程中，早期添加BMP4可干预正常RPE分化。与BMP4相比，抑制BMP通路在早期无显著负面影响，但在后期抑制分化。
BMP通路可按正确的时间顺序被激活，否则细胞会有错误的分化方向。类脊索蛋白2在BMP通路的动态调节中起作用，且它还调节RPE细胞的分化。因此，本研究为抑制EMT及促进损伤后细胞再分化开辟了新方向。
