茶多酚对氧化损伤奶牛乳腺上皮细胞的干预作用及机制研究

发布时间：2018-05-27 09:32

本文选题：茶多酚 + 奶牛　；参考：《内蒙古农业大学》2017年硕士论文

【摘要】：本论文分四个部分,主要研究茶多酚对氧化损伤奶牛乳腺上皮细胞的干预作用及机制。试验一采用奶牛乳腺细胞构建乳腺上皮细胞系,使其达到最佳的细胞系模型。试验二用H_2O_2处理奶牛乳腺上皮细胞,筛选出H_2O_2最佳诱导剂量和作用时间,建立氧化应激损伤模型。试验三在建立奶牛乳腺上皮细胞氧化损伤模型的基础上,添加不同浓度的茶多酚,通过细胞培养液中抗氧化指标的变化探讨茶多酚对氧化应激所致的奶牛乳腺上皮细胞损伤的影响,并筛选出最佳茶多酚添加剂量。试验四利用特殊性MAPK通路抑制剂分别将p38、JNK、ERK5和ERK1/2四条MAPK通路阻断,从而筛选出MAPK通路中对茶多酚影响Nrf2起关键作用的因子,揭示出茶多酚对奶牛乳腺抗氧化功能的影响机制。试验结果如下:(1)本试验成功建立了奶牛乳腺上皮细胞的体外培养方法,2×10~4个/mL可以作为以后的细胞接种浓度。采用免疫荧光染色方法成功鉴定了 BMECs特有骨架蛋白-角蛋白的存在,证明本试验所使用细胞为BMECs,可用于后续的试验研究。(2)当H_2O_2作用浓度为600μM,作用时间为6h,乳腺上皮细胞产生了明显的氧化损伤,但这种损伤不会造成细胞大量死亡,损伤还有可能会被修复,可作为建立乳腺上皮细胞氧化损伤模型时的适宜条件。(3)茶多酚主要通过抗氧化作用中和细胞内产生的过多的ROS,提高细胞内Nrf2的表达,减少氧化应激,起到保护乳腺上皮细胞免受H_2O_2损伤的作用。当作用浓度100μg/mL,作用时间12h时可作为茶多酚保护乳腺上皮细胞产生氧化损伤的最适条件。(4)茶多酚可提高H_2O_2损伤的乳腺上皮细胞中Nrf2通路和MAPK通路中的基因的表达,阻断剂DNCB下调了 MAPK信号通路中ERK1/2和p38的表达,说明TP对H_2O_2损伤的乳腺上皮细胞的保护作用主要是通过调节MAPK通路中ERK1/2和p38的表达来促进Nrf2的表达和Nrf2介导的HO-1的表达。综上所述,TP对H202损伤的乳腺上皮细胞具有保护作用,其机制主要是通过调节MAPK通路中ERK1/2和p38的表达来促进Nrf2和Nrf2介导的HO-1的表达,减缓细胞受到氧化应激造成的损伤,进而降低由氧化应激造成的疾病的发生率。
[Abstract]:This paper is divided into four parts to study the effect and mechanism of tea polyphenols on oxidative injury of dairy cow mammary epithelial cells. In experiment 1, the mammary epithelial cell line was constructed by using dairy mammary gland cells to make it the best cell line model. In the second experiment, H_2O_2 was used to treat dairy cow mammary epithelial cells. The optimal dose and time of H_2O_2 induction were screened out, and the oxidative stress injury model was established. On the basis of the model of oxidative injury of dairy cow mammary epithelial cells, different concentrations of tea polyphenols were added in experiment 3. The effects of tea polyphenols on the injury of dairy cow mammary epithelial cells induced by oxidative stress were studied through the changes of antioxidant indexes in cell culture medium. The optimum additive amount of tea polyphenols was screened out. In experiment 4, the specific MAPK pathway inhibitor was used to block the four MAPK pathways of p38 MAPK ERK5 and ERK1/2, respectively, so as to screen out the factors that play a key role in the influence of tea polyphenols on Nrf2 in the MAPK pathway, and to reveal the mechanism of the effects of tea polyphenols on the antioxidant function of dairy cows' mammary glands. The results are as follows: (1) in this experiment, the method of in vitro culture of dairy cow mammary epithelial cells was established successfully. 2 脳 10 ~ 4 / mL of milk cow mammary epithelial cells could be used as the subsequent cell inoculation concentration. The existence of BMECs specific skeleton protein keratin was successfully identified by immunofluorescence staining. It is proved that the cells used in this study are BMECs, which can be used for further experimental study. (2) when the concentration of H_2O_2 is 600 渭 M and the exposure time is 6 h, the epithelial cells of mammary gland produce obvious oxidative damage, but this kind of damage will not result in a large number of cell death. The injury may also be repaired, which can be used as a suitable condition for the establishment of oxidative injury model of breast epithelial cells. Tea polyphenols can increase the expression of Nrf2 and reduce oxidative stress mainly through the antioxidant action and the excessive ROSs produced in the cells. It can protect breast epithelial cells from H_2O_2 damage. When the concentration of tea polyphenols was 100 渭 g / mL and the exposure time was 12 h, tea polyphenols could be used as the best condition for protecting breast epithelial cells from oxidative damage.) Tea polyphenols could increase the expression of Nrf2 pathway and MAPK pathway gene in breast epithelial cells damaged by H_2O_2. The blocking agent DNCB down-regulated the expression of ERK1/2 and p38 in MAPK signaling pathway, suggesting that TP can promote the expression of Nrf2 and Nrf2 mediated HO-1 by regulating the expression of ERK1/2 and p38 in MAPK pathway. In conclusion, TP has protective effect on H202 damaged mammary epithelial cells. The mechanism is to promote the expression of Nrf2 and Nrf2 mediated HO-1 by regulating the expression of ERK1/2 and p38 in the MAPK pathway, and to slow down the damage caused by oxidative stress. This in turn reduces the incidence of diseases caused by oxidative stress.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S823

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