LPS和热应激对猪颗粒细胞的影响及HSP70参与调控机制的研究

发布时间：2018-03-30 11:25

本文选题：颗粒细胞　切入点：LPS　出处：《山东农业大学》2016年硕士论文

【摘要】：在众多影响动物繁殖的环境因素中,内毒素(Endotoxin)感染和热应激(Heat stress)是两个非常重要的方面。内毒素化学本质是脂多糖(Lipopolysaccharide,LPS)。LPS和热应激都能够通过多种路径对动物机体造成不良影响,本研究着重探讨二者对动物繁殖产生的不良影响。已有研究结果证明,LPS或热应激作用于活体动物时,均能显著抑制生殖内分泌轴,抑制激素分泌、发情、排卵,降低受胎率和活胎率等。卵泡颗粒细胞(Granulosa Cell)是一种高度分化的体细胞,与卵母细胞关系密切,在动物繁殖活动中发挥重要作用,而关于LPS和热应激对颗粒细胞的直接影响及作用机理仍缺乏深入了解。因此,本研究以离体培养的猪颗粒细胞为模型,旨在探讨LPS和热应激对颗粒细胞形态、增殖、凋亡和激素分泌功能的影响,并对作用路径进行初步研究。经过前期预实验确定LPS实验处理时间为48 h,热应激处理实验为3 h。利用扫描电子显微镜分别放大1000×、5000×、10000×和15000×观察LPS或热应激对细胞形态产生的影响。在LPS对颗粒细胞增殖、凋亡和雌激素分泌影响的研究中,利用CCK8测定细胞增殖情况;利用Annexin V-FITC/PI细胞凋亡检测试剂盒借助流式细胞仪检测细胞凋亡情况;利用ELISA法测定培养液中E2含量,利用Western Blot检测细胞MAPKs蛋白含量,包括ERK、Phospho ERK、JNK、Phospho JNK、p38、Phospho p38 MAPK;利用qRT-PCR检测与细胞增殖周期相关的基因FN1、IGF2、IGFBP2、Cyclin D1、Cyclin D2和P27kip的表达。针对LPS和热应激对颗粒细胞功能的影响检测了基因P450arom、FSHR的表达。同时检测了HSP70基因的表达,研究发现,LPS和热应激都能够激活颗粒细胞HSP70基因,使这两个环境不良因素对动物繁殖影响产生交汇点,因此利用小分子物质HSP70抑制剂(VER 155008)和HSP70激活剂(STA-4783)针对HSP70作相应处理,随后分别检测三个基因的表达和HSP70蛋白的表达。并在HSP70过表达的基础上利用Western Blot和细胞免疫荧光染色实验对Phospho Smad3进行了定量和定位。结果显示LPS处理后,细胞饱满,有较多微绒毛发和伪足,细胞状态和活力均优于对照组。1000 ng?mL-1浓度LPS即可显著促进细胞增殖、抑制细胞凋亡、提高活细胞百分率(P0.05),而500 ng?mL-1 LPS即可促进与细胞生长增殖、周期相关基因FN1(P0.01),IGF2、IGFBP2(P0.05),Cyclin D1、Cyclin D2(P0.01)的表达,抑制阻碍细胞周期的基因P27kip(P0.01)的表达。细胞MAPKs蛋白含量中,各总蛋白含量不变,各磷酸化蛋白含量增加。热应激处理使细胞固缩,微绒毛及伪足消失,细胞状态比对照组差。LPS和热应激均可强烈抑制芳香化酶P450arom和FSHR基因的表达(P0.01),LPS刺激还能够显著抑制E2的分泌(P0.01)。LPS和热应激均可在转录和反应水平上显著地上调HSP70基因的相对表达量(P0.01)。经HSP70抑制剂(VER155008)处理后,HSP70基因相对表达量代偿性上调,蛋白表达量被抑制。P450arom和FSHR基因的相对表达量显著回调(P0.01)。在应用HSP70激活剂(STA-4783)后,HSP70基因被极显著激活(P0.01),基因相对表达量增加超过50倍,蛋白表达量显著上调。P450arom和FSHR基因的表达与前者相反,出现极显著下调。HSP70过表达3 h和48 h均能明显抑制Smad3磷酸化和向核转移,核内荧光变暗,且Phospho Smad3蛋白量显著降低。以上结果表明,LPS可以促进颗粒细胞增殖并抑制细胞凋亡,LPS和热应激抑制颗粒细胞功能。二者对颗粒细胞激素分泌功能的影响是通过HSP70实现的,HSP70阻碍Phospho Smad3的核转移是其发挥作用的路径之一。
[Abstract]:There are many factors that influence the animal breeding environment, endotoxin (Endotoxin) infection and heat stress (Heat stress) are two important aspects. The chemical nature of endotoxin is lipopolysaccharide (Lipopolysaccharide, LPS).LPS and heat stress can cause adverse effects on the animal body through various approaches, this research focuses on the adverse effects of the two on the animal breeding. The research results show that LPS or heat stress in a living animal, could significantly inhibit the reproductive endocrine axis, inhibiting hormone secretion, estrus, ovulation, reducing the pregnancy rate and live birth rate. Granulosa cells (Granulosa Cell) is a kind of highly differentiated somatic cells, and oocyte closely, play an important role in animal breeding activities, and on the direct effect of LPS and heat stress on granulosa cells and the mechanism is still a lack of understanding. Therefore, this study in vitro Cultured porcine granulosa cells as a model to investigate LPS and heat stress on the proliferation of granule cell morphology, apoptosis, and hormone secretion, and preliminary study on effect of path. After pre experiment to determine the LPS treatment time was 48 h, 3 h. heat stress experiment by scanning electron microscopy respectively amplified 1000 *, 5000 *, 10000 * and 15000 * LPS observation or heat stress effects on cell morphology. In LPS on the proliferation of granulosa cells, apoptosis and estrogen secretion, cell proliferation was measured by CCK8; the cell apoptosis was detected by flow cytometry using Annexin V-FITC/PI cell apoptosis detection kit; determination the content of E2 in the culture medium by ELISA method, using Western Blot to detect MAPKs protein content, including ERK, Phospho ERK, JNK, Phospho JNK, p38 Phospho, p38 MAPK; using qRT-PCR detection and cell Cell cycle related genes FN1, IGF2, IGFBP2, Cyclin, D1, D2 expression of Cyclin and P27kip. The effect of LPS and heat stress on granulosa cell function test of the P450arom gene, the expression of FSHR. At the same time to detect the expression of HSP70 gene, the study found, LPS and heat stress can activate the granulosa cell HSP70 gene so, these two factors have adverse environmental confluence on animal reproductive effects, therefore the use of small molecule inhibitors of HSP70 (VER 155008) and HSP70 activator (STA-4783) for the corresponding treatment according to the HSP70, then detected the expression of three genes and HSP70 protein expression. And the overexpression of HSP70 by Western on the basis of Blot and immunofluorescence staining experiments on Phospho Smad3 for the quantitative and positioning. The results showed that LPS treated cells full, more microvilli and pseudopodia of hair cell state and vitality, are better than the control group.1000 n G? ML-1 concentration of LPS could significantly promote cell proliferation, inhibit apoptosis, increase the percentage of viable cells (P0.05), and 500 ng? ML-1 and LPS can promote the growth and proliferation of cell cycle related genes (P0.01, FN1), IGF2, IGFBP2 (P0.05), Cyclin D1, Cyclin D2 (P0.01) expression of suppressor gene P27kip blocked the cell cycle (P0.01). The expression of MAPKs protein in the same cell, the total protein content, protein content increased acidification of various phosphorus. Heat stress to cell shrinkage, microvilli and pseudopodia disappeared, the cell state worse than the control group.LPS and heat stress can strongly inhibit the expression of aromatase P450arom and FSHR gene (P0.01), LPS stimulation can also significantly inhibit the secretion of E2 (P0.01) expression of.LPS and heat stress can be found in the transcription and reaction level significantly up-regulated the HSP70 gene (P0.01). The HSP70 inhibitor (VER155008) treatment, the relative expression of HSP70 gene The amount of compensatory up-regulated protein expression, relative expression of.P450arom was inhibited and FSHR gene significantly callback (P0.01). In the application of HSP70 activator (STA-4783), HSP70 gene was significantly activated (P0.01), relative gene expression increased more than 50 times, the protein expression was significantly up-regulated.P450arom expression and the former and the FSHR gene on the contrary, significant downregulation of.HSP70 expression of 3 h and 48 h inhibited the phosphorylation of Smad3 and nuclear transfer, nuclear fluorescence dimmed, and the Phospho Smad3 protein was significantly decreased. These results indicate that LPS can promote the proliferation of granulosa cells and inhibit cell apoptosis, and inhibition of LPS heat stress granules cell function. Effects of the two hormones on the secretion of granulosa cells is realized through HSP70, HSP70 block Phospho Smad3 nuclear transfer is one of the way to give full play to the role.

【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S828

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