Pg-LPS对脂肪细胞内质网应激反应标志物表达的影响

发布时间：2018-06-13 17:15

本文选题：牙周炎 + 脂肪细胞　；参考：《郑州大学》2017年硕士论文

【摘要】：目的模拟牙周炎致病因子对脂肪细胞致炎的过程,在体外培养3T3-L1前脂肪细胞、诱导其向脂肪细胞分化,以牙龈卟啉单胞菌脂多糖Pg-LPS作用于脂肪细胞,观察Pg-LPS对脂肪内质网应激反应标志物GRP78、XBP1s的mRNA表达水平的影响,研究Pg-LPS对脂肪细胞内质网应激状态的影响,从而为进一步揭示牙周炎、脂肪组织炎症,与胰岛素抵抗和糖尿病发生发展的关系奠定理论基础,和为临床上治疗、预防提供理论依据。方法1、诱导3T3-L1前脂肪细胞向脂肪细胞分化,油红o染色鉴定细胞分化状态;2、将脂肪细胞分为空白对照组,LPS处理组两组;空白对照组:加入等体积0.8%的牛血清白蛋白的无血清DEME培养液,7个时间点0,30min,1h,2h、4h、8h、12h取样;LPS处理组:又分为4组,分别加入含有50ng/ml、100ng/ml、500ng/ml、1000ng/ml 4个作用浓度的Pg-LPS和0.8%的牛血清白蛋白的无血清DEME培养液,分别处理细胞7个时间点0,30min,1h,2h、4h、6h、12h取样;real-time PCR检测内质网应激反应标志物GRP78、XBP1s的mRNA表达水平;结果1、诱导分化14d后,细胞在镜下均可看到明显脂滴,3T3-l1前脂肪细胞基本分化为脂肪细胞;2、加Pg-LPS 12小时后,对照组除个别死亡细胞漂浮未见明显细胞凋亡情况,50ng/ml、100ng/ml、500ng/ml、1000ng/ml实验组与对照组相比,实验组各组均出现小面积细胞的凋亡细胞漂浮和团块集聚的情况;3、0-12h,对照组GRP78 mRNA表达水平均维持在较为恒定的基线水平;而实验组GRP78表达水平各浓度实验组均在加Pg-LPS干预早期,均出现比基线水平明显的抬高,而到达峰值后回落至靠近基线水平上下:50ng/ml组在作用30min后表达水平逐步抬高,在1h时表达量达到峰值,于靠近2h时间点回落至接近基线水平;100ng/ml组在0-1h间GRP78 mRNA相对表达量逐步出现抬高,1h-2h明显上抬达到峰值后于4h回落至基线水平附近。500ng/ml组在30min达到峰值后逐步回落,在接近2h时间点回落至基线水平。1000ng/ml组在0-1h间GRP78mRNA相对表达量逐步出现抬高,1h上抬达到峰值后于2h回落至基线水平附近。4、0-12h,对照组XBP1s mRNA表达水平均维持在较为恒定的基线水平;而实验组GRP78表达水平各浓度组均在加Pg-LPS干预早期,出现比基线水平明显的抬高而到达峰值后回落至靠近基线水平:50ng/ml组在作用30min后表达水平逐步抬高,在1h时表达量达到峰值,于靠近2H时间点回落至基线水平以下;100ng/ml组在0-1h间XBP1s mRNA相对表达量逐步出现抬高,1h-2h明显上抬达到峰值后于4h回落至基线水平附近。500ng/ml组在30min达到峰值后逐步回落,在接近1h时间点回落至基线水平。1000ng/ml组在0-1h间XBP1s mRNA相对表达量逐步出现抬高,1h上抬达到峰值后于2h回落至基线水平附近。结论:1、Pg-LPS可上调脂肪细胞内质网应激反应水平,显著升高脂肪细胞GRP78、XBP1s mRNA表达水平;2、GRP78、XBP1s mRNA表达水平和Pg-LPS作用的浓度和时间不存在相关性;3、Pg-LPS可能通过内质网应激,启动脂肪细胞的凋亡机制;
[Abstract]:Objective to simulate the process of periodontitis pathogenicity factor (PDF) on adipocytes, culture 3T3-L1 preadipocytes in vitro, induce them to differentiate into adipocytes, and treat adipocytes with lipopolysaccharide (Pg-LPS) from porphyromonas gingivalis. To observe the effect of Pg-LPS on the expression of GRP78XBP1s mRNA in adipose endoplasmic reticulum stress, and to study the effect of Pg-LPS on endoplasmic reticulum stress in adipocytes, so as to further reveal periodontitis and adipose tissue inflammation. The relationship between insulin resistance and diabetes mellitus provides theoretical basis for clinical treatment and prevention. Methods 1. 3T3-L1 preadipocytes were induced to differentiate into adipocytes. The differentiation status of the cells was identified by oil red o staining. The adipocytes were divided into two groups: blank control group and LPS treated group. Blank control group: the control group was divided into 4 groups: the control group was treated with LPS for 12 hours by adding equal volume 0.8% bovine serum albumin (BSA) serum-free DEME culture medium. Four concentrations of Pg-LPS and 0.8% bovine serum albumin (BSA) were added to Pg-LPS and 0.8% bovine serum albumin (BSA) respectively. The mRNA expression level of GRP78 XBP1s was detected by real-time PCR. Results 1. After 14 days of differentiation, the adipocytes could be seen to differentiate into adipocytes before 3T3-l1. After 12 hours of Pg-LPS addition, the adipocytes could basically differentiate into adipocytes. There was no obvious apoptosis in the control group except individual dead cells floating. The experimental group was compared with the control group. The apoptotic cells floating and agglomeration of small area cells were observed in each group of experimental group for 30 ~ 12 hours, and the expression level of GRP78 mRNA in control group was maintained at a relatively constant baseline level, while the expression level of GRP78 in experimental group was at the early stage of Pg-LPS intervention, and the expression level of GRP78 in experimental group was at the early stage of Pg-LPS intervention. The expression level of 50 ng / ml / ml group increased gradually after the action of 30min, and reached the peak level at 1 h, and reached the peak level at 1 h. The relative expression of GRP78 mRNA in the 100ng / ml group decreased to close to the baseline level at close to 2 h. The relative expression of GRP78 mRNA gradually increased from 0 to 1 h and reached the peak value at 1h-2 h, then dropped to the baseline level at 4 h after reaching the peak level. The expression of GRP78 mRNA in the 100ng / ml group decreased gradually after the peak value of 30min reached the baseline level. The relative expression of GRP78 mRNA decreased to the baseline level at close to 2 h. The relative expression level of GRP78 mRNA in the control group was gradually elevated at 1 h and reached its peak value within 1 h, then dropped to the baseline level at 2 h, and the expression level of XBP1s mRNA in the control group was maintained at a relatively constant baseline level. In the experimental group, the expression of GRP78 increased significantly after the treatment with Pg-LPS, and then decreased to the baseline level (1: 50ng / ml). The expression level of GRP78 increased gradually after the treatment of 30min, and reached the peak level at 1 h. The relative expression of XBP1s mRNA in the 100ng / ml group decreased to the baseline level at the time point near 2H. The relative expression of XBP1s mRNA gradually increased from 0 to 1h and reached the peak value at 1h-2h, and then decreased to the baseline level at 4h after the peak value of 30min reached the peak. The relative expression of XBP1s mRNA decreased to the baseline level at close to 1h. The relative expression of XBP1s mRNA gradually increased to the peak at 1h and then decreased to the baseline level at 2h. Conclusion the stress level of endoplasmic reticulum (ER) in adipocytes can be upregulated by Pg-LPS, and the expression of GRP78 XBP1s mRNA in adipocytes is significantly increased. There is no correlation between the expression of GRP78 XBP1s mRNA and the concentration and time of Pg-LPS, which may be induced by endoplasmic reticulum stress. The mechanism of adipocyte apoptosis was initiated.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781.4

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