黄芪多糖影响雏鸡免疫器官TLR4信号转导通路的研究

发布时间：2018-07-07 22:20

本文选题：APS + TLR4信号转导通路　；参考：《东北农业大学》2015年硕士论文

【摘要】：为了探索黄芪多糖(Astragalus Polysaccharides,APS)在雏鸡免疫器官中对TLR4信号转导通路的影响,本试验首先采用水提醇沉法提取黄芪粗多糖,Sevag法除蛋白得到精多糖APS,以葡萄糖为标准,苯酚-硫酸法测得该提取物糖含量为84.7%。之后,将140只7日龄雏鸡随机分为四组,即APS高剂量组(I组)、APS中剂量组(II组)、APS低剂量组(III组)和空白对照组(C组)。I、II、III组雏鸡于7日龄以灌服方式分别给予80 mg·m L-1、40 mg·mL-1、20 mg·mL-1APS液0.2mL,1次/天,连续5天,C组雏鸡以相同方式给予相同剂量生理盐水。14日龄时采取滴口方式进行鸡传染性法氏囊病活疫苗免疫。在首次给药后1、3、5、7天(即雏鸡8、10、12、14日龄)及疫苗免疫后7、14、21天(即雏鸡21、28、35日龄),每组随机抽取5只雏鸡,取其胸腺、脾脏、法氏囊,采用实时荧光定量PCR技术及免疫酶组织化学技术对各组雏鸡胸腺、脾脏、法氏囊中的ch TLR4 m RNA与蛋白表达、ch TLR4信号转导通路衔接蛋白分子(ch My D88,ch TRIF)、转录因子(ch NF-κB,ch IRF3)及其诱导产物(ch IFN-β,ch TNF-α)m RNA的表达进行检测。结果显示,在各检测时间点,APS试验组雏鸡ch TLR4 m RNA表达量高于对照组或与对照组相当。其中胸腺组织中ch TLR4 m RNA表达量在雏鸡8日龄时APS高、中剂量组极显著高于对照组(p0.01),接下来时高时低,之后与对照组持平,免疫后第三周其表达量又显著高于对照组(p0.01),低剂量组ch TLR4 m RNA表达量也较对照组有所提高,但较高、中剂量组延迟。脾脏中chTLR4 mRNA表达量水平在APS高剂量组上调突出,从整个检测时间段来看,高剂量组和中剂量组APS显著上调了ch TLR4 mRNA表达,低剂量组效果不如高、中剂量组明显。法氏囊中ch TLR4 m RNA表达量在连续灌服APS后逐渐显著高于对照组(p0.01或p0.05),免疫后也保持着相对较高水平的表达,并且低剂量APS组对ch TLR4 m RNA表达量的上调作用要优于高、中剂量组。在各组织中,ch TLR4蛋白表达的变化趋势与ch TLR4 mRNA的表达变化基本一致。雏鸡胸腺、脾脏、法氏囊中ch My D88,ch TRIF,ch NF-κB,ch IRF3 mRNA表达量随着ch TLR4 mRNA表达上调,也明显上调;细胞因子ch IFN-β,ch TNF-αmRNA表达量与空白对照组有一定的差异,在免疫前,试验组各免疫器官中ch IFN-β、ch TNF-αm RNA的表达量和对照组相比显著增高(p0.01或p0.05)或者持平,免疫后试验组ch IFN-β,ch TNF-αm RNA表达水平显著上调,整个试验阶段各个检测点与对照组相比较,结果有所不同。本研究表明,APS可激活雏鸡胸腺、脾脏、法氏囊中ch TLR4信号转导通路My D88依赖途径和My D88非依赖途径,优化雏鸡免疫状态,进一步从雏鸡体内受体模式及信号转导的角度阐明APS调节雏鸡免疫功能的分子机制,为APS在提高雏鸡抗病能力的作用研究及APS在家禽养殖方面的科学应用提供科学依据。
[Abstract]:In order to explore the effect of Astragalus polysaccharides (APS) on the signal transduction pathway of TLR4 in the immune organs of chicks, we first extracted Astragalus polysaccharides Sevag from Astragalus membranaceus by water extraction and alcohol precipitation method to obtain spermatoglycan APSs, which was based on glucose as the standard. The sugar content of the extract was determined to be 84.7 by phenol-sulfuric acid method. After that, 140 7-day-old chicks were randomly divided into four groups: the high dose APS group (group I) and the control group (group C): low dose group (group III) and control group (group C). The chickens in the control group were given 80 mg mL ~ (-1) mg ml ~ (-1) and 20 mg / m ~ (-1) APS solution 0.2mL ~ (-1) a day respectively at the age of 7 days. For 5 consecutive days, chickens in group C were immunized with the same dose of physiological saline at the age of 14 days with the same dose of live vaccine of infectious bursal disease (IBD). Five chicks were randomly selected from each group for thymus, spleen and bursa of Fabricius on 1 ~ 3 ~ 5 ~ 5 ~ 5 ~ 7 days after first administration (that is, 810 ~ 12 ~ (12) ~ 14 ~ 14 ~ 14 ~ (th) days) and 7 ~ 14 ~ 21 days after vaccination (i.e., 21 ~ 2 835 days of age of a chicks), and 5 chicks were randomly selected from each group to obtain thymus, spleen and bursa of Fabricius. The thymus and spleen of chicks in each group were studied by real-time fluorescence quantitative PCR and immunohistochemistry. The expression of ch TLR4 mRNA and protein in bursa of Fabricius was detected by detecting the expression of chTLR4 signal transduction pathway (ch my D8H TRIF), transcription factor (ch NF- 魏 BNch IRF3) and its induced product (ch IFN- 尾 chTNF- 伪) mRNA. The results showed that the expression of chTLR4 mRNA in the APS experimental group was higher than that in the control group or similar to that in the control group. The expression of chTLR4 mRNA in thymus tissue was significantly higher than that in the control group at 8 days of age (p0.01), then higher than that in the control group (p0.01), and then remained the same as that in the control group. At the third week after immunization, the expression of ch TLR4 mRNA in the low dose group was significantly higher than that in the control group (p0.01), but the expression of ch TLR4 mRNA in the low dose group was higher than that in the control group, but delayed in the middle dose group. The expression of chTLR4 mRNA in spleen was significantly up-regulated in the high dose APS group. In the whole detection period, the expression of ch TLR4 mRNA was significantly up-regulated in the high dose group and the middle dose group. The effect of the low dose group was lower than that of the middle dose group, and that of the middle dose group was obvious. The expression of ch TLR4 mRNA in bursa of Fabricius was significantly higher than that of control group (p0.01 or p0.05) after continuous administration of APS, and the expression of ch TLR4 mRNA in the low dose APS group was higher than that in the middle dose group (p0.01 or p0.05), and the up-regulation of ch TLR4 mRNA expression in the low dose APS group was better than that in the high dose group. The change trend of TLR4 protein expression in all tissues was consistent with the change of ch TLR4 mRNA expression. In chicks thymus, spleen and bursa of Fabricius, the expression of chMy D88chTRIFch NF- 魏 Bnch IRF3 mRNA was significantly up-regulated with the expression of ch TLR4 mRNA, and the expression of cytokine ch IFN- 尾 chTNF- 伪 mRNA was different from that of control group. The expression of chIFN- 尾 -nch TNF- 伪 mRNA in the immune organs of the experimental group was significantly higher than that in the control group (p0.01 or p0.05), and the expression of ch IFN- 尾 -nch TNF- 伪 mRNA was significantly up-regulated in the experimental group after immunization. The result is different. This study indicated that APS could activate chTLR4 signal transduction pathway in thymus, spleen, bursa of Fabricius and its dependent pathway, my D88 dependent pathway and my D88 independent pathway, so as to optimize the immune status of chicks. The molecular mechanism of APS regulating the immune function of chicks was further elucidated from the point of view of receptor pattern and signal transduction in chicks, which provided scientific basis for the study of APS in improving the disease resistance of chicks and the scientific application of APS in poultry breeding.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S853.7

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