Sep15基因沉默對(duì)高糖誘導(dǎo)的人晶狀體上皮細(xì)胞中整合素表達(dá)的影響
發(fā)布時(shí)間：2018-09-06 09:36
【摘要】：白內(nèi)障是主要的致盲性眼疾。據(jù)報(bào)道,大約有高于43%的盲人患者是由白內(nèi)障引起,嚴(yán)重地影響人們生活質(zhì)量�，F(xiàn)在由于生活條件的提高,糖尿病白內(nèi)障的發(fā)病率越來越高,受到人們的關(guān)注。而晶狀體上皮細(xì)胞正常的增殖、遷移、分化是保證晶狀體正常發(fā)育的基礎(chǔ)。一些證據(jù)已經(jīng)表明必需微量元素硒(Se)對(duì)眼起重要的保護(hù)作用,其中內(nèi)質(zhì)網(wǎng)硒蛋白Sep15在維持眼晶狀體透明中起著關(guān)鍵作用,但是Sep15在其中的機(jī)理仍不清楚。因此研究Sep15對(duì)白內(nèi)障的防治作用及機(jī)理有著非常重要的科學(xué)意義。整合素α6在晶狀體組織形態(tài)、增殖、分化中發(fā)揮著重要作用。本文采用人眼晶狀體上皮細(xì)胞(h LECs)SRΑ01/04細(xì)胞系為研究對(duì)象,采用MTT、Western-Blot、RT-PCR手段探討Sep15基因沉默對(duì)高糖誘導(dǎo)的h LECs中整合素表達(dá)的影響,主要結(jié)果如下:(1)采用MTT法測(cè)定不同濃度的葡萄糖對(duì)細(xì)胞存活率的影響,結(jié)果表明當(dāng)葡萄糖濃度為45mM時(shí),對(duì)細(xì)胞的損傷程度最大,可明顯誘發(fā)內(nèi)質(zhì)網(wǎng)應(yīng)激。然而當(dāng)葡萄糖濃度為25mM時(shí),細(xì)胞的存活率最高,整合素α6的表達(dá)最高,表明葡萄糖濃度為25mM是h LECs細(xì)胞的最佳生長(zhǎng)環(huán)境。在此基礎(chǔ)上,用40ng/m L bFGF促進(jìn)hLE細(xì)胞的分化,bFGF作用時(shí)間的長(zhǎng)短對(duì)整合素mRNΑ和protein表達(dá)不同。RT-PCR和WB結(jié)果分別顯示bFGF作用時(shí)間為6h,整合素α6 mRNΑ表達(dá)最高;bFGF作用時(shí)間為24h,整合素α6 protein表達(dá)最高。當(dāng)用bFGF和Glucose共同作用細(xì)胞時(shí),當(dāng)葡萄糖的濃度為25mM時(shí),細(xì)胞的存活率最高,整合素α6 mRNΑ和protein表達(dá)水平都為最高;當(dāng)葡萄糖濃度為45mM時(shí)反而抑制整合素的表達(dá)。可見高濃度的葡萄糖嚴(yán)重?fù)p傷細(xì)胞,影響整合素α6的表達(dá)。(2)研究了Sep15基因沉默對(duì)高糖誘導(dǎo)的hLECs中整合素表達(dá)的影響,結(jié)果表明Sep15基因沉默更加劇了高糖對(duì)hLECs分化過程中對(duì)整合素的影響作用。說明Sep15對(duì)晶狀體上皮細(xì)胞的分化具有相應(yīng)的保護(hù)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 偽 6 plays an important role in lens morphology, proliferation and differentiation. Human lens epithelial cell line (h LECs) SR 偽 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 偽 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 偽 6 mRN 偽 was 24 h, and the expression of integrin 偽 6 protein was the highest. When bFGF and Glucose were used together, when glucose concentration was 25mM, cell survival rate was the highest, integrin 偽 6 mRN 偽 -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 偽 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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R776.1
本文編號(hào)：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 偽 6 plays an important role in lens morphology, proliferation and differentiation. Human lens epithelial cell line (h LECs) SR 偽 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 偽 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 偽 6 mRN 偽 was 24 h, and the expression of integrin 偽 6 protein was the highest. When bFGF and Glucose were used together, when glucose concentration was 25mM, cell survival rate was the highest, integrin 偽 6 mRN 偽 -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 偽 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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R776.1
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