Sep15基因沉默对高糖诱导的人晶状体上皮细胞中整合素表达的影响
发布时间：2018-09-06 09:36
【摘要】：白内障是主要的致盲性眼疾。据报道,大约有高于43%的盲人患者是由白内障引起,严重地影响人们生活质量。现在由于生活条件的提高,糖尿病白内障的发病率越来越高,受到人们的关注。而晶状体上皮细胞正常的增殖、迁移、分化是保证晶状体正常发育的基础。一些证据已经表明必需微量元素硒(Se)对眼起重要的保护作用,其中内质网硒蛋白Sep15在维持眼晶状体透明中起着关键作用,但是Sep15在其中的机理仍不清楚。因此研究Sep15对白内障的防治作用及机理有着非常重要的科学意义。整合素α6在晶状体组织形态、增殖、分化中发挥着重要作用。本文采用人眼晶状体上皮细胞(h LECs)SRΑ01/04细胞系为研究对象,采用MTT、Western-Blot、RT-PCR手段探讨Sep15基因沉默对高糖诱导的h LECs中整合素表达的影响,主要结果如下:(1)采用MTT法测定不同浓度的葡萄糖对细胞存活率的影响,结果表明当葡萄糖浓度为45mM时,对细胞的损伤程度最大,可明显诱发内质网应激。然而当葡萄糖浓度为25mM时,细胞的存活率最高,整合素α6的表达最高,表明葡萄糖浓度为25mM是h LECs细胞的最佳生长环境。在此基础上,用40ng/m L bFGF促进hLE细胞的分化,bFGF作用时间的长短对整合素mRNΑ和protein表达不同。RT-PCR和WB结果分别显示bFGF作用时间为6h,整合素α6 mRNΑ表达最高;bFGF作用时间为24h,整合素α6 protein表达最高。当用bFGF和Glucose共同作用细胞时,当葡萄糖的浓度为25mM时,细胞的存活率最高,整合素α6 mRNΑ和protein表达水平都为最高;当葡萄糖浓度为45mM时反而抑制整合素的表达。可见高浓度的葡萄糖严重损伤细胞,影响整合素α6的表达。(2)研究了Sep15基因沉默对高糖诱导的hLECs中整合素表达的影响,结果表明Sep15基因沉默更加剧了高糖对hLECs分化过程中对整合素的影响作用。说明Sep15对晶状体上皮细胞的分化具有相应的保护作用。
[Abstract]:Cataract is the leading cause of blindness. It is reported that more than 43% of blind patients are caused by cataracts, seriously affecting people's quality of life. Due to the improvement of living conditions, the incidence of diabetic cataract is increasing. The normal proliferation, migration and differentiation of lens epithelial cells are the basis to ensure the normal development of lens. Some evidences have shown that selenium (Se) plays an important role in protecting eyes. Endoplasmic reticulum selenium protein (Sep15) plays a key role in maintaining lens transparency, but the mechanism of Sep15 is still unclear. Therefore, it is of great scientific significance to study the role and mechanism of Sep15 in the prevention and treatment of cataract. Integrin 伪 6 plays an important role in lens morphology, proliferation and differentiation. Human lens epithelial cell line (h LECs) SR 伪 01 / 04 was used to investigate the effect of Sep15 gene silencing on the expression of integrin in h LECs induced by high glucose by MTT,Western-Blot,RT-PCR. The main results were as follows: (1) the effects of glucose concentration on cell survival were measured by MTT method. The results showed that the damage to cells was the greatest when glucose concentration was 45mM, which could induce endoplasmic reticulum stress. However, when glucose concentration was 25mM, the survival rate of cells was the highest, and the expression of integrin 伪 6 was the highest, indicating that glucose concentration of 25mM was the best growth environment for h LECs cells. On this basis, The effects of 40ng/m L bFGF on the expression of integrin mRN and protein in hLE cells were different. RT-PCR and WB showed that the time of bFGF treatment was 6 h, the highest expression of integrin 伪 6 mRN 伪 was 24 h, and the expression of integrin 伪 6 protein was the highest. When bFGF and Glucose were used together, when glucose concentration was 25mM, cell survival rate was the highest, integrin 伪 6 mRN 伪 -alpha and protein expression was the highest, but when glucose concentration was 45mM, integrin expression was inhibited. It can be seen that high concentration of glucose seriously damages the cells and affects the expression of integrin 伪 6. (2) the effect of Sep15 gene silencing on the expression of integrin in hLECs induced by high glucose was studied. The results showed that Sep15 gene silencing increased the effect of high glucose on integrin in the differentiation of hLECs. These results suggest that Sep15 has a protective effect on the differentiation of lens epithelial cells.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R776.1
本文编号：2225938
[Abstract]:Cataract is the leading cause of blindness. It is reported that more than 43% of blind patients are caused by cataracts, seriously affecting people's quality of life. Due to the improvement of living conditions, the incidence of diabetic cataract is increasing. The normal proliferation, migration and differentiation of lens epithelial cells are the basis to ensure the normal development of lens. Some evidences have shown that selenium (Se) plays an important role in protecting eyes. Endoplasmic reticulum selenium protein (Sep15) plays a key role in maintaining lens transparency, but the mechanism of Sep15 is still unclear. Therefore, it is of great scientific significance to study the role and mechanism of Sep15 in the prevention and treatment of cataract. Integrin 伪 6 plays an important role in lens morphology, proliferation and differentiation. Human lens epithelial cell line (h LECs) SR 伪 01 / 04 was used to investigate the effect of Sep15 gene silencing on the expression of integrin in h LECs induced by high glucose by MTT,Western-Blot,RT-PCR. The main results were as follows: (1) the effects of glucose concentration on cell survival were measured by MTT method. The results showed that the damage to cells was the greatest when glucose concentration was 45mM, which could induce endoplasmic reticulum stress. However, when glucose concentration was 25mM, the survival rate of cells was the highest, and the expression of integrin 伪 6 was the highest, indicating that glucose concentration of 25mM was the best growth environment for h LECs cells. On this basis, The effects of 40ng/m L bFGF on the expression of integrin mRN and protein in hLE cells were different. RT-PCR and WB showed that the time of bFGF treatment was 6 h, the highest expression of integrin 伪 6 mRN 伪 was 24 h, and the expression of integrin 伪 6 protein was the highest. When bFGF and Glucose were used together, when glucose concentration was 25mM, cell survival rate was the highest, integrin 伪 6 mRN 伪 -alpha and protein expression was the highest, but when glucose concentration was 45mM, integrin expression was inhibited. It can be seen that high concentration of glucose seriously damages the cells and affects the expression of integrin 伪 6. (2) the effect of Sep15 gene silencing on the expression of integrin in hLECs induced by high glucose was studied. The results showed that Sep15 gene silencing increased the effect of high glucose on integrin in the differentiation of hLECs. These results suggest that Sep15 has a protective effect on the differentiation of lens epithelial cells.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R776.1
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