线粒体活性氧与糖尿病性白内障的关系及茶多酚干预作用的实验研究

发布时间：2018-04-20 10:41

本文选题：晶状体上皮细胞 + 茶多酚　；参考：《山东中医药大学》2010年博士论文

【摘要】：目的：通过体内外实验探讨高糖及茶多酚干预对大鼠晶状体上皮细胞的影响,阐明茶多酚对糖尿病性白内障的预计疗效及作用机制。方法：体外培养大鼠晶状体上皮细胞,培养基中葡萄糖浓度分别设定为5mmol/l、30mmol/l两种浓度,在30mmol/l葡萄糖浓度培养基中分别加入茶多酚配成0.1μmol/l和0.3μmol/l茶多酚浓度的培养基进行细胞培养。用MMT检测晶状体上皮细胞的活性,流式细胞仪检测LEC细胞死亡率和线粒体膜电位,线粒体活性氧的变化,以及使用免疫细胞化学和免疫荧光法检测内质网表达量的变化。利用统计学分析高糖及茶多酚干预对大鼠晶状体上皮细胞的影响。体内试验通过STZ尾静脉注射诱导建立糖尿病大鼠模型,分为正常组、STZ诱导糖尿病组(STZ-DM)和茶多酚干预糖尿病组(TP-DM)三组,通过HE染色、电镜观察大鼠晶状体的超微结构,生化法测定各组大鼠晶状体中GSH、GR及CAT生化指标的活性,RT-PCR法检测各组大鼠晶状体中UCPs、MnSODmRNA的表达量变化,探讨茶多酚对STZ诱导糖尿病大鼠晶状体上皮细胞干预的影响。结果：在高葡萄糖浓度条件下培养的上皮细胞逐渐丧失原有特征,形态表现多样,渐则细胞肿胀,出现空泡和颗粒,细胞数量明显减少,传代后容易老化,死亡率CDR(%)明显升高,线粒体膜电位MMP(FI)降低,线粒体活性氧增多,内质网的阳性率明显降低；而茶多酚干预组细胞则较好的保持了上皮细胞的形态,细胞之间紧密相靠,排列规则,较少出现细胞肿胀、空泡和颗粒等,传代未见老化,细胞死亡率降低,线粒体膜电位升高,线粒体活性氧的产生明显减少,并且内质网的阳性率明显升高。低浓度与高浓度的茶多酚干预效果差别不明显。体外实验表明在建模8周时,STZ-DM组76.2%的糖尿病大鼠晶状体表现为Ⅲ级混浊,而TP-DM组糖尿病大鼠晶状体混浊发展不明显,30%的糖尿病大鼠晶状体表现为Ⅱ级混浊。第12周时STZ-DM组糖尿病大鼠晶状体完全混浊,而TP-DM组61.1%糖尿病大鼠晶状体表现Ⅲ级混浊。扫描电镜观察大鼠的晶状体的超微结构,发现STZ-DM组大鼠的晶状体纤维严重受损,而TP-DM组受损明显减轻；通过HE染色观察大鼠的晶状体细胞形态,发现STZ-DM组的大鼠晶状体纤维细胞内有大量未被降解的细胞器,无细胞器区域缩小。而TP-DM组晶状体纤维细胞未被降解的细胞器明显减少,无细胞器区域显著扩大。通过对大鼠晶状体中CAT的活性和GSH的含量进行测量,发现STZ-DM大鼠晶状体中CAT的活性和GSH的含量均明显下降,而TP-DM组晶状体中保持了CAT的活性和GSH的含量；应用RT-PCR的方法对大鼠晶状体中UCPs、MnSOD表达的变化进行检测,发现STZ-DM大鼠晶状体中UCP2及MnSOD mRNA表达量逐渐减少,而TP-DM组大鼠晶状体中的UCP2及MnSOD mRNA表达增加且维持在较高水平。结论：大鼠晶状体上皮细胞在高糖条件下培养时细胞凋亡的各项特征明显加强；而应用茶多酚干预后,以上细胞凋亡现象对比高糖组均有明显减轻。表明茶多酚可延缓和阻止晶状体上皮细胞受损伤,对高糖条件下晶状体上皮细胞的凋亡具有抑制作用,对防治糖尿病性白内障有可预计的积极疗效。体内实验表明茶多酚可以显著减轻糖尿病性白内障大鼠晶状体的超微结构的损伤,维持晶状体内正常的细胞器降解,可以保持CAT的活性和GSH的含量,维持晶状体正常的氧化还原状态,甚至逆转了抗氧化酶类活性的降低；茶多酚可以上调糖尿病大鼠晶状体中的UCP2及MnSOD mRNA表达并使其维持在较高水平,进而影响晶状体中活性氧的清除,阻止或者延缓晶状体的混浊,显著延缓糖尿病性白内障的发生。
[Abstract]:Objective : To study the effects of high glucose and tea polyphenols on lens epithelial cells in rats by in vivo and in vivo experiments , and clarify the expected curative effect and mechanism of tea polyphenols on diabetic cataract .

Methods : The rat lens epithelial cells were cultured in vitro . The concentration of glucose in the culture medium was set to 5 mmol / l and 30 mmol / l . The effects of high glucose and tea polyphenols on the rat lens epithelial cells were determined by HE staining and electron microscope .

Results : The epithelial cells cultured under the condition of high glucose concentration gradually lose their original characteristics , the morphology is varied , the cells are swollen , vacuoles and particles appear , the number of cells is obviously reduced , the mortality CDR ( % ) is obviously increased , the mitochondrial membrane potential MMP ( FI ) is decreased , the mitochondrial active oxygen is increased , and the positive rate of the endoplasmic reticulum is obviously reduced ;
The results showed that the lens performance of diabetic rats was not significantly higher than that in TP - DM group .
The activity of CAT and the content of GSH in the lens were measured by HE staining , while the activity of CAT and the content of GSH were observed in the lens of TP - DM group .
The changes of UCP and MnSOD in the lens of rats were detected by RT - PCR , and the expression of UCP2 and MnSOD mRNA in the lens was gradually decreased , while the expression of UCP2 and MnSOD mRNA in the lens of TP - DM rats was increased and maintained at a higher level .

Conclusion : The characteristics of apoptosis in rat lens epithelial cells were significantly enhanced when cultured in high glucose condition .
The results showed that tea polyphenols can delay and prevent lens epithelial cells from being damaged , and can inhibit the apoptosis of lens epithelial cells under the condition of high glucose .
The tea polyphenol can raise the expression of UCP2 and MnSOD mRNA in the lens of diabetic rats and maintain it at a high level , thereby affecting the removal of active oxygen in the lens , preventing or delaying the opacity of the lens , and obviously delaying the occurrence of diabetic cataract .

【学位授予单位】：山东中医药大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R587.1;R776.1

【参考文献】