Sep15对晶状体细胞分化紊乱的抑制机理和防障作用

Based on the pathogenesis of cataract and the fact of cataract formation in Sep 15 knockout mice, this project will take the differentiation process of lens epithelial cells into fiber cells as a point of penetration, rat primary lens epithelial cell and human lens epithelial cell line SRA01/04 as research objects, research the influences of Sep 15 gene silencing, endoplasmic reticulum stress, oxidative stress, nitration stress and high sugar on the differentiation of lens epithelial cells to fiber cell using fluorescent quantitative PCR, western blot, flow cytometric, CD spectrum, mass spectrum and so on, especially explore the effects of these factors on the gene expression of signal molecules and growth factors which play important roles in regulation of cell differentiation, analyze the effects of oxidation and nitration modification of lens proteins resulted from those stresses on the molecular structure of lens crystallins, compare their differences before and after Sep15 gene silence, then discuss the function and mechanism of Sep15 in the differentiation of lens epithelial cells into fiber cells on the level of cell and molecule, as well as the role in preventing cataracts according to these results. In addition, an intervention role of Ebselen as a mimic compound of selenoproteins on the interferences of reticulum stress, oxidative stress, nitration stress and high sugar on the differentiation of lens epithelial cells into fiber cell will be detected in order to examine the possibility of selenoprotein mimic compound as preventive medicine for preventing cataracts. From above, this project is of great scientific significance and potential application value to provide some clues for understanding the function and mechanism of Sep15 in cataract prevention.

本项目基于白内障病理和硒蛋白Sep15基因敲除导致鼠患白内障等结果，以晶状体上皮细胞为对象，以其分化这一关键环节为切入点，采用定量PCR、western blot、流式细胞仪、CD谱和质谱等手段，研究Sep15基因沉默、内质网应激、氧化应激、氮化应激和高糖等因素对晶状体上皮细胞向纤维细胞分化的影响，重点研究这些因素对在细胞分化中起重要调节作用的信号分子和生长因子基因表达的影响，分析这些因素引起的氧化和硝化修饰导致晶状体球蛋白分子结构的变化，比较Sep15基因沉默前后引起细胞凋亡率差异，从细胞和分子水平探讨Sep15在晶状体细胞分化紊乱中的作用和机理，及对白内障发生的预防作用；还采用硒蛋白的模拟物Ebselen对Sep15基因沉默、氧化应激、氮化应激和高糖影响细胞分化进行干预，以探索其预防白内障的可能性。通过这些研究为认识硒在白内障预防中的作用及机理提供线索，具有重要的科学意义和应用价值。

Sep15（15kD硒蛋白）是一个内质网蛋白，其基因敲除鼠的下一代出生1.5个月就出现晶状体浑浊，但其发病机理不清楚。本项目以人晶状体上皮（hLE）细胞分化这一关键环节为切入点，采用RNA干扰、定量PCR、蛋白质印迹、流式细胞仪等手段，研究了Sep15对内质网应激（ERS）试剂Tm（依霉素）和高糖等因素导致hLE细胞分化紊乱的调节作用，比较了Sep15基因敲低前后这些因素诱发细胞凋亡率的差异，从细胞和分子水平探讨了Sep15在hLE细胞分化紊乱中的作用和机理；并研究了Ebselen和硒（亚硒酸钠）的补充对D-半乳糖型白内障的干预作用，以及补充硒上调hLE细胞中硒蛋白对ERS干扰细胞分化的修复作用。结果发现，Sep15基因敲低能一定程度上抑制bFGF诱导hLE细胞β-crystallin的合成，加剧Tm引起的分化抑制。其机理与Sep15基因敲低加剧Tm诱发的线粒体损伤有关，但Sep15并不是通过抑制ERS来调节hLE细胞分化的紊乱，而是通过对hLE细胞分化信号通路的调节来缓解ERS导致的氧化应激、进而减少细胞凋亡来调节hLE细胞的分化。补硒可明显上调Sep15，SelR和SelS蛋白表达来缓解Tm诱发的hLE细胞ERS，但Sep15蛋白对硒的补充更为敏感，提示Sep15在晶状体上皮细胞中可能发挥主要的作用。此外，SelR可以通过抑制ONOO‾导致F-actin蛋白的硝化和ERK的失活，保护hLE细胞对ONOO‾诱导的F-actin的损伤发挥重要作用。动物实验的结果发现给半乳糖型糖尿病大鼠补硒或Ebselen可以延缓半乳糖性白内障的形成，而这种延缓作用也与硒蛋白的上调、抑制半乳糖诱发的氧化应激和抑制半乳糖激活醛糖还原酶的活性、保护hLE细胞和纤维细胞的损伤有关。这些结果为理解Sep15在眼晶状体中的作用机理以及硒在白内障预防中的应用提供了有益的证据和线索，具有重要的科学意义和潜在的应用价值。