乌司他丁下调LPS诱导的小鼠巨噬细胞MiR-21表达及初步调控机制研究

发布时间：2018-05-05 19:42

本文选题：脓毒症 + 微小RNA-21　；参考：《重庆医科大学》2017年硕士论文

【摘要】：目的:探讨乌司他丁在脂多糖诱导的RAW264.7细胞的保护作用及对mi R-21表达影响。方法:(1)用不同浓度(100,250,500 ng/m L)LPS刺激RAW264.7细胞,RT-PCR法检测mi R-21的表达、肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)m RNA、白介素-6(interleukin-6,IL-6)m RNA的表达;(2)采用500ng/m L LPS刺激RAW264.7细胞6h、12h、24h,采用RT-PCR检测TNF-αm RNA;(3)四甲基偶氮唑(Methyl thiazolyl tetrazolium,MTT)法检测不同浓度(10,100,1000U/m L)UTI对RAW264.7细胞活性的影响;设置正常组、模型组(LPS 500ng/m L剌激组)、实验组(LPS 500ng/m L+UTI 1000U/m L组)和阴性实验对照组(UTI 1000U/m L),观察RAW264.7细胞生长状态;(4)用不同浓度(10,100,1000 U/m L)UTI预处理2h后,LPS刺激小鼠RAW264.7细胞诱导炎症模型,RT-PCR检测肿瘤坏死因子TNF-αm RNA、IL-6 m RNA、mi R-21的表达,Western Blot法检测UTI(1000 U/m L)时PTEN蛋白表达水平。(5)设置空白组、模型组(LPS 500 ng/m L剌激组)、实验组(LPS+mi R-21模拟剂、LPS+mi R-21抑制剂组):RT-PCR法检测mi R-21的表达,Western Blot法检测PTEN蛋白表达水平。结果:1.UTI对细胞的保护作用1 UTI对RAW264.7细胞的毒性作用:UTI浓度小于1000U/m L时对RAW264.7细胞无毒性作用,各组细胞OD值比较差异无统计学意义(P=0.117);2.不同浓度(10,100,1000U/m L)UTI能有效降低TNF-αm RNA表达及IL-6 m RNA表达(P10.05,P20.05)。2.不同LPS浓度下TNF-αm RNA、IL-6 m RNA及mi R-21的表达情况与正常组相比,在LPS(100,250,500ng/m L)刺激RAW264.7细胞后,mi R-21、TNF-αm RNA及IL-6 m RNA的分泌量与基础分泌量相比均明显升高(P0.05),并随LPS浓度升高而增加(P0.05)。3.UTI对mi R-21表达的影响不同浓度(10,100,1000U/m L)UTI能有效降低mi R-21的表达,差异有统计学意义(P0.05)。4.mi R-21对PTEN蛋白表达的影响与空白组相比较,在LPS组中,mi R-21表达上调,PTEN蛋白表达下调;在加入.mi R-21抑制剂组中,mi R-21表达明显下降,PTEN蛋白表达明显升高;mi R-21模拟剂组中,mi R-21表达明显升高,PTEN蛋白表达明显下降(P0.05)。5.UTI对PTEN蛋白表达的影响基于上述UTI下调mi R-21表达水平的实验结果,进一步观察UTI对mi R-21靶基因PTEN蛋白的影响。实验结果发现,与空白组比较,PTEN蛋白表达水平在LPS组中明显下降,在UTI组中表达水平明显升高,差异具有统计学意义(P0.05)。结论:1.UTI可降低LPS诱导的RAW264.7细胞中TNF-α、IL-6 m RNA的表达。2.LPS可诱导RAW264.7细胞mi R-21的高表达,且呈浓度依赖性;mi R-21高表达可抑制PTEN蛋白的表达。3.UTI可显著下调mi R-21的表达,并上调靶基因PTEN蛋白表达水平。综上所述,UTI可抑制LPS诱导RAW264.7细胞的炎症反应,对细胞起着保护作用,其机制可能与UTI引起mi R-21表达下调有关。
[Abstract]:Aim: to investigate the protective effect of ulinastatin on lipopolysaccharide-induced RAW264.7 cells and its effect on the expression of miR-21. Methods the expression of miR-21 was detected by RT-PCR in RAW264.7 cells stimulated with different concentrations of 100250500 ng/m L)LPS. TNF- 伪 -Tumor necrosis factor- 伪 -TNF- 伪 -mRNAs, interleukin-6h6 interleukin-6tr (IL-6m RNA expression) were used to stimulate RAW264.7 cells for 6 h and 12 h to 24 h by 500ng/m L LPS. TNF- 伪 m RNA-3) tetrazolium L)UTI (MTT) assay was used to detect the effect of different concentrations of 10 100 渭 m L)UTI on RAW264.7 cell activity. In normal group, TNF- 伪 m RNA-3) was used to detect the effect of TNF- 伪 necrosis factor- 伪 on the activity of RAW264.7 cells with different concentrations of TNF- 伪 necrosis factor- 伪 (TNF- 伪 necrosis factor- 伪 -TNF- 伪 -TNF- 伪 -TNF- 伪 -TNF- 伪). The model group was stimulated by 500ng/m L, the experimental group was treated with 500ng/m L UTI 1000U/m L) and the negative control group was used to observe the growth state of RAW264.7 cells. The RAW264.7 cells were pretreated with different concentrations of 10100U / m L)UTI for 2 hours. The model of RAW264.7 cells was induced by RT-PCR after pretreatment with different concentrations of 10100U / m L)UTI for 2 hours. The expression of TNF- 伪 m RNA-IL-6 m RNAi R-21 was detected. Western Blot assay was used to detect the expression level of PTEN protein in UTI(1000 UP / mL. (5) blank group was set up. The expression of miR-21 in the model group was detected by ng/m RT-PCR and the expression level of PTEN protein was detected by Western Blot method in the LPS-mi R-21 mimic of the experimental group and the LPS-mi R-21 inhibitor group. Results 1. The cytotoxic effect of UTI on RAW264.7 cells. When the concentration of UTI was lower than that of 1000U/m L, there was no toxic effect on RAW264.7 cells. There was no significant difference in OD value between the two groups. The expression of TNF- 伪 m RNA and the expression of IL-6 m RNA were significantly decreased by different concentrations of 10100U ~ 1000Um L)UTI. The expression of IL-6 m RNA and miR-21 in TNF- 伪 m RNAs at different concentrations of LPS was significantly higher than that in normal controls. After stimulation of RAW264.7 cells with LPS(100250500ng/m L, the secretion of Mi R-21 TNF- 伪 m RNA and IL-6 m RNA increased significantly compared with the basal secretion, and increased with the increase of LPS concentration. 3. The effect of UTI on the expression of miR-21 was observed. The difference was statistically significant (P 0.05) .4.mi R-21 had a significant effect on the expression of PTEN protein compared with the control group. In the LPS group, the expression of PTEN R-21 up-regulated the expression of PTEN protein. The expression of PTEN protein decreased significantly in the group treated with .mi R-21 inhibitor; the expression of PTEN protein increased significantly in the mimic group of miR-21; the expression of PTEN protein decreased significantly in the group of mi-R-21 mimics; the effect of P0.05. 5.UTI on the expression of PTEN protein was based on the down-regulation of miR-21 protein by the above-mentioned UTI. Expression level of the experimental results, To investigate the effect of UTI on PTEN protein of miR-21 target gene. The results showed that compared with the blank group, the expression level of PTEN protein in the LPS group was significantly decreased, and the expression level in the UTI group was significantly increased. The difference was statistically significant (P 0.05). Conclusion: 1. UTI can decrease the expression of TNF- 伪 IL-6 m RNA in RAW264.7 cells induced by LPS. 2.LPs can induce the high expression of miR-21 in RAW264.7 cells, and the high expression of PTEN protein in RAW264.7 cells can be inhibited in a dose-dependent manner. 3. UTI can significantly down-regulate the expression of miR-21. The expression level of target gene PTEN protein was upregulated. In conclusion, LPS can inhibit the inflammatory response of RAW264.7 cells induced by UTI, which may be related to the down-regulation of miR-21 expression induced by UTI.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R459.7

【参考文献】