本文選題：APS + TLR4信號(hào)轉(zhuǎn)導(dǎo)通路��； 參考：《東北農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：為了探索黃芪多糖(Astragalus Polysaccharides,APS)在雛雞免疫器官中對(duì)TLR4信號(hào)轉(zhuǎn)導(dǎo)通路的影響,本試驗(yàn)首先采用水提醇沉法提取黃芪粗多糖,Sevag法除蛋白得到精多糖APS,以葡萄糖為標(biāo)準(zhǔn),苯酚-硫酸法測(cè)得該提取物糖含量為84.7%。之后,將140只7日齡雛雞隨機(jī)分為四組,即APS高劑量組(I組)、APS中劑量組(II組)、APS低劑量組(III組)和空白對(duì)照組(C組)。I、II、III組雛雞于7日齡以灌服方式分別給予80 mg·m L-1、40 mg·mL-1、20 mg·mL-1APS液0.2mL,1次/天,連續(xù)5天,C組雛雞以相同方式給予相同劑量生理鹽水。14日齡時(shí)采取滴口方式進(jìn)行雞傳染性法氏囊病活疫苗免疫。在首次給藥后1、3、5、7天(即雛雞8、10、12、14日齡)及疫苗免疫后7、14、21天(即雛雞21、28、35日齡),每組隨機(jī)抽取5只雛雞,取其胸腺、脾臟、法氏囊,采用實(shí)時(shí)熒光定量PCR技術(shù)及免疫酶組織化學(xué)技術(shù)對(duì)各組雛雞胸腺、脾臟、法氏囊中的ch TLR4 m RNA與蛋白表達(dá)、ch TLR4信號(hào)轉(zhuǎn)導(dǎo)通路銜接蛋白分子(ch My D88,ch TRIF)、轉(zhuǎn)錄因子(ch NF-κB,ch IRF3)及其誘導(dǎo)產(chǎn)物(ch IFN-β,ch TNF-α)m RNA的表達(dá)進(jìn)行檢測(cè)。結(jié)果顯示,在各檢測(cè)時(shí)間點(diǎn),APS試驗(yàn)組雛雞ch TLR4 m RNA表達(dá)量高于對(duì)照組或與對(duì)照組相當(dāng)。其中胸腺組織中ch TLR4 m RNA表達(dá)量在雛雞8日齡時(shí)APS高、中劑量組極顯著高于對(duì)照組(p0.01),接下來(lái)時(shí)高時(shí)低,之后與對(duì)照組持平,免疫后第三周其表達(dá)量又顯著高于對(duì)照組(p0.01),低劑量組ch TLR4 m RNA表達(dá)量也較對(duì)照組有所提高,但較高、中劑量組延遲。脾臟中chTLR4 mRNA表達(dá)量水平在APS高劑量組上調(diào)突出,從整個(gè)檢測(cè)時(shí)間段來(lái)看,高劑量組和中劑量組APS顯著上調(diào)了ch TLR4 mRNA表達(dá),低劑量組效果不如高、中劑量組明顯。法氏囊中ch TLR4 m RNA表達(dá)量在連續(xù)灌服APS后逐漸顯著高于對(duì)照組(p0.01或p0.05),免疫后也保持著相對(duì)較高水平的表達(dá),并且低劑量APS組對(duì)ch TLR4 m RNA表達(dá)量的上調(diào)作用要優(yōu)于高、中劑量組。在各組織中,ch TLR4蛋白表達(dá)的變化趨勢(shì)與ch TLR4 mRNA的表達(dá)變化基本一致。雛雞胸腺、脾臟、法氏囊中ch My D88,ch TRIF,ch NF-κB,ch IRF3 mRNA表達(dá)量隨著ch TLR4 mRNA表達(dá)上調(diào),也明顯上調(diào);細(xì)胞因子ch IFN-β,ch TNF-αmRNA表達(dá)量與空白對(duì)照組有一定的差異,在免疫前,試驗(yàn)組各免疫器官中ch IFN-β、ch TNF-αm RNA的表達(dá)量和對(duì)照組相比顯著增高(p0.01或p0.05)或者持平,免疫后試驗(yàn)組ch IFN-β,ch TNF-αm RNA表達(dá)水平顯著上調(diào),整個(gè)試驗(yàn)階段各個(gè)檢測(cè)點(diǎn)與對(duì)照組相比較,結(jié)果有所不同。本研究表明,APS可激活雛雞胸腺、脾臟、法氏囊中ch TLR4信號(hào)轉(zhuǎn)導(dǎo)通路My D88依賴途徑和My D88非依賴途徑,優(yōu)化雛雞免疫狀態(tài),進(jìn)一步從雛雞體內(nèi)受體模式及信號(hào)轉(zhuǎn)導(dǎo)的角度闡明APS調(diào)節(jié)雛雞免疫功能的分子機(jī)制,為APS在提高雛雞抗病能力的作用研究及APS在家禽養(yǎng)殖方面的科學(xué)應(yīng)用提供科學(xué)依據(jù)。
[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- 偽) 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- 偽 mRNA was different from that of control group. The expression of chIFN- 尾 -nch TNF- 偽 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- 偽 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.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S853.7

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本文編號(hào)：2106462