平喘颗粒通过NOTCH信号通路调节哮喘大鼠树突细胞免疫功能的实验研究

发布时间：2018-01-04 06:10

本文关键词：平喘颗粒通过NOTCH信号通路调节哮喘大鼠树突细胞免疫功能的实验研究　出处：《黑龙江中医药大学》2017年博士论文　论文类型：学位论文

【摘要】：目的:观察平喘颗粒对哮喘大鼠树突细胞免疫功能的影响,探讨平喘颗粒治疗哮喘的机制方法:1.40只Wistar大鼠被随机分为空白组、模型组、地塞米松组、平喘颗粒组。除空白组外,其余各组用卵蛋白建立哮喘大鼠模型。地塞米松组给予地塞米松(0.45mg/kg)灌胃,平喘颗粒组大鼠予平喘颗粒(5.4g/kg)灌胃,空白组、模型组给予生理盐水(5ml/kg)灌胃。观察平喘颗粒对哮喘大鼠肺组织、肺DCs迁移的影响。流式细胞术检测哮喘大鼠肺 DCs CD80、CD86、MHCII表达变化。Realtime-PCR 检测肺 DCs Notch1、Jag-1 mRNA 的表达水平。Western blot 法检测肺 DCs EGR1、PKC蛋白表达水平。初步探讨平喘颗粒对哮喘大鼠DCs免疫功能调节作用。2.无菌获得大鼠骨髓树突细胞,加入rmGM-CSF(10ng/ml)、rmlL-4(10ng/ml)联合培养7天,获得DCs。将获得的DCs分别给予不同浓度LPS、DAPT刺激,确定最佳LPS、DAPT浓度。将获得的DCs随机分为空白组、LPS组、DAPT+LPS组、平喘颗粒+LPS组、空白血清+LPS组。用流式细胞术检测DCs CD80、CD86、MHCII表达变化。RealtimePCR检测 DCs Notchl、Jag-1 mRNA 的表达水平。Western blot 法检测 DCs EGR1、PKC蛋白表达水平。初步探讨平喘颗粒对DCs免疫功能调节作用。探讨平喘颗粒在LPS诱导DCs成熟过程中对Notch信号通路的调节作用。结果:1.平喘颗粒可以降低哮喘大鼠肺组织炎性浸润,改善肺脏病理状态,降低DCs向肺组织的迁移程度。每视野肺组织平均DCs浸润个数为32.00±6.16个,与模型组比较有明显差异(P0.05),与地塞米松组比较无明显差别(P0.05)。2.平喘颗粒可以降低哮喘大鼠肺DCs表型分子的表达,CD80、CD86、MHCII的表达率分别为 62.04±4.57%、54.66±4.40%和57.64±4.26%。与模型组比较有显著差异(P0.05)。3.平喘颗粒可以降低哮喘大鼠肺DCs Notch1、Jag-1mRNA表达,与模型组比较有显著差异(P0.05)。4.平喘颗粒能降低PKC蛋白表达,促进EGR1蛋白表达,两者表达量与模型组比价有显著差异(P0.05)。5.1.5μg/mlLPS刺激12h,是DCs成熟的最佳刺激条件,与2.5μg/ml LPS在刺激DCs成熟度和细胞抑制率方面没有差异(P0.05)。6.DAPT的最佳抑制浓度是20umol/L,与其他浓度比较有较显著差异(P0.05)。7.平喘颗粒血清能降低LPS诱导的DCs CD80、CD86、MHCII表达,阳性率分别为 62.26±4.44%、67.69±5.33%和 63.77±4.58%,与 LPS 组比较有显著差异(P0.05)。8.平喘颗粒血清能降低LPS诱导的DCsNotch1、Jag-1mRNA表达,与LPS组比较有显著差异(P0.05)。9.平喘颗粒血清能降低LPS诱导的PKC蛋白表达,减轻LPS对EGR1蛋白表达的抑制,两种蛋白在表达程度与LPS组比较有显著差异(P0.05)。结论:1.平喘颗粒能改善哮喘大鼠肺组织病理改变程度。2.平喘颗粒能抑制哮喘发病中DCs成熟。3.平喘颗粒能降低哮喘发病中DCs Notch1、Jag-1mRNA表达。4.平喘颗粒通过抑制DCs Notch1 mRNA表达,调节哮喘大鼠DCs免疫功能。
[Abstract]:Objective: to observe the effect of Pingchuan granule on the immune function of dendritic cells in asthmatic rats and to explore the mechanism of Pingchuan granule in the treatment of asthma. One hundred and forty Wistar rats were randomly divided into blank group and model group. Dexamethasone group, Pingchuan granule group. With the exception of blank group, the other groups were used to establish asthmatic rat model with ovalbumin. The dexamethasone group was given dexamethasone 0.45 mg / kg intragastrically. The rats in the Pingchuan granule group were given 5. 4 g / kg Pingchuan granule by stomach, while in the blank group, the model group were given 5 ml / kg normal saline. The lung tissue of asthmatic rats was observed by observing the effect of Pingchuan granule on the lung tissue of asthmatic rats. Effect of lung DCs migration. Lung DCs CD80 CD86 was detected by flow cytometry in asthmatic rats. The changes of MHCII expression. Realtime-PCR were used to detect lung DCs Notch1. The expression level of Jag-1 mRNA. Western blot method was used to detect DCs EGR1 in lung. Expression level of PKC protein. To investigate the effect of Pingchuan granule on the immune function of DCs in asthmatic rats. 2. To obtain rat bone marrow dendritic cells from aseptic rats and add rmGM-CSF10 ng / ml). After 7 days of co-culture of rmlL-4ng / ml, the obtained DCs was stimulated with different concentrations of LPS-DAPT to determine the best LPS. The concentration of DAPT. The obtained DCs was randomly divided into two groups: the control group, the control group, the control group, the control group, the control group, the control group, the control group and the LPS group. In blank serum LPS group, DCs Notchl was detected by flow cytometry. The expression level of Jag-1 mRNA. Western blot method was used to detect DCs EGR1. The effect of Pingchuan granule on the immune function of DCs and on the signal pathway of Notch in the process of DCs maturation induced by LPS were studied. 1. Pingchuan granule can reduce the inflammatory infiltration of lung tissue in asthmatic rats. The average number of DCs infiltration per field of lung was 32.00 卤6.16. Compared with the model group, there was a significant difference between the model group and the dexamethasone group. Compared with the dexamethasone group, there was no significant difference between the two groups. Pingchuan granule could reduce the expression of DCs molecule in the lung of asthmatic rats. The expression rate of CD86MHCII was 62.04 卤4.57% respectively. 54.66 卤4.40% and 57.64 卤4.26.The difference was significant compared with the model group. Pingchuan granule could reduce the DCs Notch1 in the lung of asthmatic rats. Compared with the model group, the expression of Jag-1mRNA was significantly different from that of the model group. Pingchuan granule could decrease the expression of PKC protein and promote the expression of EGR1 protein. There was a significant difference between the two groups and the model group. P0.05, 5.1.5 渭 g / ml LPS was the best stimulation condition for DCs maturation for 12 h. There was no difference between 2.5 渭 g / ml LPS and 2.5 渭 g / ml LPS in stimulating DCs maturity and cell inhibition rate. The optimal inhibitory concentration of DAPT was 20umol / L. Compared with other concentrations, the serum of Pingchuan granule could decrease the expression of DCs CD80, CD86 and MHCII induced by LPS. The positive rates were 62.26 卤4.44 and 67.69 卤5.33% and 63.77 卤4.58%, respectively. Compared with LPS group, the serum of Pingchuan granule could decrease the expression of LPS induced DCsNotch1 Jag-1 mRNA. Compared with LPS group, the serum of Pingchuan granule could decrease the expression of PKC protein induced by LPS and reduce the inhibition of EGR1 protein expression by LPS. There was significant difference in the expression of the two proteins between the LPS group and the LPS group (P0.05). Conclusion 1. Pingchuan granule can improve the pathological changes of lung tissue in asthmatic rats. 2.Pingchuan granule can inhibit DCs maturation. 3. Pingchuan granule can reduce DCs in asthma. Notch1. Jag-1mRNA expression. 4. Pingchuan granule regulates the DCs immune function of asthmatic rats by inhibiting the expression of DCs Notch1 mRNA.
【学位授予单位】：黑龙江中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R285.5

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