本文選題：玉屏風(fēng)多糖 + 紅外質(zhì)譜　； 參考：《山東農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：具有刺激免疫應(yīng)答的藥物—免疫增強(qiáng)劑的應(yīng)用,在生產(chǎn)中對動(dòng)物傳染性疾病具有提高免疫功能,并由此增強(qiáng)抵抗能力的重要作用。然而,現(xiàn)在最常用的免疫增強(qiáng)劑大多是化學(xué)合成的,在藥物殘留方面具有潛在的隱患。如今藥物的安全性引起了公眾的關(guān)注,從而使得中藥顯示出其優(yōu)越性。許多研究表明,多糖是中藥的一個(gè)重要組成部分,是存在于天然植物,微生物(細(xì)菌和真菌)和海藻類生物體內(nèi)的一類復(fù)雜的天然高分子化合物。許多藥理和臨床研究表明,多糖具有增強(qiáng)免疫,抗病毒,抗腫瘤,抗氧化,抗衰老等生物活性。本研究從玉屏風(fēng)散整體出發(fā)追蹤其多糖類活性成分,然后與方中的單味藥白術(shù)多糖作比較,通過體內(nèi)外實(shí)驗(yàn)探究玉屏風(fēng)多糖的免疫增強(qiáng)活性。首先用一步醇沉和分級醇沉法提取玉屏風(fēng)總多糖和白術(shù)總多糖及各分級多糖,通過體外對雞外周血淋巴細(xì)胞增殖的影響,篩選出3個(gè)效果較好的活性部位;然后從體外對雞淋巴細(xì)胞周期分布和淋巴細(xì)胞亞群CD4+/CD8+比例的影響,體內(nèi)對雞新城疫疫苗免疫應(yīng)答的影響,比較了3個(gè)多糖的增強(qiáng)免疫活性。實(shí)驗(yàn)分為以下五個(gè)部分:采用一步醇沉和分步醇沉法提取粗的玉屏風(fēng)總多糖(YPF-PStc)和白術(shù)總多糖(RAMPStc),3個(gè)分級白術(shù)多糖RAMPS60c、RAMPS70c和RAMPS80c。YPF-PStc和RAMPStc通過陰離子交換劑DEAE sephadex A-25分離純化,從中分離出純化的玉屏風(fēng)多糖(YPF-PStp)和白術(shù)多糖(RAMPStp),分別用苯酚-硫酸法和考馬斯亮蘭法測定多糖和蛋白含量,紅外質(zhì)譜法鑒定多糖的結(jié)構(gòu)。結(jié)果顯示,YPF-PStc提取率為13.56%,RAMPStc提取率為13.36%,分級白術(shù)多糖的提取率以RAMPS60c最高、達(dá)14.79%;YPF-PStp提取率為16.87%。RAMPStp的糖含量最高、達(dá)97.14%,比RAMPStc提高了9.93%,YPF-PStp的糖含量為85.6%,比YPF-PStc提高了18.25%;玉屏風(fēng)和白術(shù)各多糖的蛋白含量都較低。結(jié)果表明,柱層析純化后可以提高多糖的糖含量,多糖都具有糖的典型特征吸收峰。將YPF-PStc、YPF-PStp、RAMPStc、RAMPStp和RAMPS60c、RAMPS70c、RAMPS80c加入到雞外周血淋巴細(xì)胞培養(yǎng)體系中,用MTT法測定其安全濃度;然后將安全濃度下的7種多糖,分別單獨(dú)或協(xié)同PHA加入到培養(yǎng)的雞外周血淋巴細(xì)胞中,培養(yǎng)48 h后,測定淋巴細(xì)胞增殖(細(xì)胞A570值和最高淋巴細(xì)胞增殖率)的變化。結(jié)果表明,多糖單獨(dú)刺激時(shí)YPF-PStc和RAMPStp在15.625~62.5μg m L-1、YPF-PStp在7.813~62.5μg mL-1、RAMPS70c在31.25μg m L-1時(shí)能顯著促進(jìn)淋巴細(xì)胞增殖,YPF-PStp在31.25μg m L-1時(shí)的細(xì)胞增殖率最高,為31.148%;多糖與pha共同刺激時(shí),ypf-pstc在15.625~62.5μgml-1、ypf-pstp在15.625~31.25μgml-1、ramps60c在0.781~1.563μgml-1、rampstp在3.125μgml-1能顯著促進(jìn)淋巴細(xì)胞增殖,ramps60c在7.813μgml-1時(shí)的細(xì)胞增殖率最高,為29.897%。綜合評價(jià),篩選出ypf-pstp、rampstp和ramps60c可能是增強(qiáng)免疫活性的較好部位。多糖ypf-pstp、rampstp和ramps60c在31.25μgml-1(細(xì)胞增殖試驗(yàn)篩選出的最佳濃度)時(shí)刺激淋巴細(xì)胞,分別在24h、48h和72h收集細(xì)胞,經(jīng)過處理,在流式細(xì)胞儀上檢測各個(gè)時(shí)間點(diǎn)的周期分布情況。結(jié)果顯示,在72h之內(nèi)未經(jīng)任何處理的細(xì)胞大部分處于g0/g1期,其變化不明顯。與空白組比較,pha組位于g0/g1期細(xì)胞的百分比明顯降低(p0.05),處于s期和g2/m期的細(xì)胞百分比明顯增多(p0.05)。在不同時(shí)間點(diǎn),ypf-pstp、rampstp和ramps60c與pha組相比,都可以明顯降低g0/g1期細(xì)胞的百分比(p0.05),ypf-pstp和rampstp在作用于淋巴細(xì)胞48h時(shí)明顯升高s期細(xì)胞的百分比,其spf值、pi值均最大,效果最明顯。分別在24h、48h和72h收集細(xì)胞,pbs洗滌2次,流式細(xì)胞儀檢測cd4+、cd8+t淋巴細(xì)胞亞群的變化。結(jié)果顯示,在所有時(shí)間點(diǎn),試驗(yàn)組的cd4+淋巴細(xì)胞百分率均顯著高于pha對照組(p0.05),ypf-pstp和rampstp處理細(xì)胞48h后,cd4+淋巴細(xì)胞百分率顯著高于ramps60c對照組(p0.05)。多糖處理細(xì)胞48h、72h后,cd8+淋巴細(xì)胞百分率和cd4+/cd8+比值均顯著高于pha對照組(p0.05),ypf-pstp和rampstp處理細(xì)胞48h后,cd8+淋巴細(xì)胞百分率顯著高于ramps60c對照組(p0.05),表明ypf-pstp和rampstp可以明顯提高cd4+、cd8+淋巴細(xì)胞百分率及cd4+/cd8+比值。為了比較上述3個(gè)多糖的增強(qiáng)免疫作用,測定了3個(gè)多糖對雛雞新城疫疫苗免疫效果的影響。將健康1日齡海蘭褐雛公雞210只,隨機(jī)分為6組,除空白對照組外,14日齡雛雞均用新城疫iv系苗滴鼻點(diǎn)眼免疫,28日齡二免。在首次免疫的同時(shí),分別肌肉注射6mgml-1的ypf-pstp、rampstp和ramps60c,口服10mgkg-1的鹽酸左旋咪唑,無佐劑對照組注射等量生理鹽水。分別于首次免疫后第7、14、21、28d翼靜脈采血檢測血清hi抗體效價(jià),心臟采血測定外周血t淋巴細(xì)胞增殖、淋巴細(xì)胞周期和cd4+、cd8+t淋巴細(xì)胞亞群的變化,處死雛雞后稱取體重及胸腺、脾臟和法氏囊的重量,計(jì)算免疫器官指數(shù)。結(jié)果表明,ypf-pstp和rampstp在大多時(shí)間點(diǎn)能顯著提高血清抗體效價(jià)、促進(jìn)t淋巴細(xì)胞增殖、促進(jìn)淋巴細(xì)胞進(jìn)入s期和g2/m期,提高cd4+、cd8+t淋巴細(xì)胞亞群的百分率,提高雛雞免疫器官指數(shù)。其中YPF-PStp免疫增強(qiáng)活性最強(qiáng)。
[Abstract]:The application of a drug that stimulates the immune response - the application of immune enhancers, plays an important role in improving immune function and enhancing resistance in production of animal infectious diseases. However, most of the most commonly used Immunoenhancers are chemically synthesized and have potential potential for drug residues. Now the safety of drugs is safe. Many studies show that polysaccharide is an important component of traditional Chinese medicine and is a complex natural polymer compound existing in natural plants, microorganisms (bacteria and fungi) and seaweed organisms. Many pharmacological and clinical studies have shown that polysaccharides are enhanced. Immunization, antivirus, anti-tumor, antioxidation, antiaging biological activity. This study traced the polysaccharide active components from the Yuping wind dispersion, and compared with the single flavour polysaccharide of Chinese Rhizoma Atractylodes macrocephala in the prescription, and explored the immune enhancement activity of Yuping wind polysaccharides in vivo and in vitro. First, one step alcohol precipitation and fractionated alcohol precipitation were used to extract the general Yuping wind. The total polysaccharide and polysaccharide of Atractylodes macrocephala and various fractionated polysaccharides were used to screen 3 active sites through the effect on the proliferation of peripheral blood lymphocytes in chicken in vitro, and then the effects on the lymphocyte cycle distribution and the proportion of CD4+/CD8+ in lymphocyte subsets in vitro, and in vivo effects on the immune response of chicken new city vaccine were compared, and 3 polysaccharides were compared. The experiment was divided into five parts: the crude polysaccharide (YPF-PStc) and total polysaccharide (RAMPStc) of Yuping wind were extracted with one step alcohol precipitation and stepwise alcohol precipitation, and 3 classified polysaccharide RAMPS60c, RAMPS70c and RAMPS80c.YPF-PStc and RAMPStc were separated and purified by the anion exchange agent DEAE Sephadex A-25, from which pure and pure were isolated and purified. The polysaccharide and protein content of Yuping wind polysaccharide (YPF-PStp) and Atractylodes macrocephala (RAMPStp) were determined by the phenol sulfuric acid method and Coomassie bright blue method respectively. The structure of polysaccharide was identified by infrared mass spectrometry. The results showed that the extraction rate of YPF-PStc was 13.56%, the extraction rate of RAMPStc was 13.36%, and the extraction rate of Polysaccharide from the grade Atractylodes macrocephala was the highest, 14.79%; YPF-PStp The sugar content of 16.87%.RAMPStp was the highest, 97.14%, 9.93% higher than that of RAMPStc, the sugar content of YPF-PStp was 85.6%, and 18.25% higher than that of YPF-PStc; the protein content of each polysaccharide in Yuping wind and Atractylodes macrocephala were low. The results showed that the column chromatography could improve the sugar content of polysaccharide, and the polysaccharide had the typical characteristic absorption peak of sugar. YPF -PStc, YPF-PStp, RAMPStc, RAMPStp and RAMPS60c, RAMPS70c, RAMPS80c were added to the peripheral blood lymphocyte culture system of chicken, and the safety concentration was determined by MTT method. Then 7 kinds of polysaccharides under the safe concentration were added to the cultured chicken peripheral blood cells separately or in collaboration with PHA, and the lymphocyte proliferation was determined after 48 h, and the cell A570 was determined. The results showed that YPF-PStc and RAMPStp in 15.625~62.5 mu g m L-1, YPF-PStp in 7.813~62.5 u g mL-1, RAMPS70c at 31.25 micron G can promote lymphocyte proliferation when the polysaccharide was stimulated alone, and the cell proliferation rate was the highest at 31.25 mu, 31.148%. At the time, ypf-pstc at 15.625~62.5 gml-1, ypf-pstp in 15.625~31.25, gml-1, ramps60c in 0.781~1.563 u gml-1, rampstp at 3.125 mu gml-1 can significantly promote lymphocyte proliferation, and the proliferation rate of ramps60c at 7.813 Mu is the highest. Sites. Polysaccharides ypf-pstp, rampstp and ramps60c stimulated lymphocytes at 31.25 mu gml-1 (the optimum concentration screened by cell proliferation test). Cells were collected in 24h, 48h and 72h respectively. After processing, the periodic distribution of each time point was detected by flow cytometry. The results showed that most of the cells were not treated in 72h. In the g0/g1 phase, the change was not obvious. Compared with the blank group, the percentage of PHA cells at g0/g1 stage decreased significantly (P0.05), and the percentage of cells in S and g2/m stages increased significantly (P0.05). At different time points, ypf-pstp, rampstp and ramps60c were significantly lower than those of PHA group. TP significantly increased the percentage of s cells in the action of lymphocyte 48h. The SPF value and pI value were the most significant. The cells were collected in 24h, 48h and 72h, respectively, and PBS washed 2 times, and the flow cytometry was used to detect cd4+ and cd8+t lymphocyte subgroups. The results showed that the percentage of cd4+ lymphocytes in the test group was significantly higher at all time points. In the PHA control group (P0.05), the percentage of cd4+ lymphocyte in the cells treated with ypf-pstp and rampstp was significantly higher than that of the ramps60c control group (P0.05). The percentage of cd8+ lymphocytes and the cd4+/cd8+ ratio were significantly higher than those in the ramps60c control group (P0.05). Higher than the ramps60c control group (P0.05), the results showed that ypf-pstp and rampstp could significantly increase the percentage of cd4+, cd8+ lymphocyte and cd4+/cd8+ ratio. In order to compare the enhanced immunity of the 3 polysaccharides, the effects of 3 polysaccharides on the immune effect of the new chicks were measured. The 1 day old chick chicks were divided into 6 groups randomly. In the blank control group, 14 day old chicks were immunized with NDV IV vaccine and 28 days of age two. At the same time, the first immunization was given to the ypf-pstp, rampstp and ramps60c of 6mgml-1, the oral 10mgkg-1 hydrochloric acid levoimidazole, and the non adjuvant control group with equal amount of normal saline. Respectively, the first 7,14,21,28d wing vein after the first immunization. The serum HI antibody titer was measured by blood sampling, the proliferation of T lymphocyte in peripheral blood, the lymphocyte cycle and the changes of cd4+ and cd8+t lymphocyte subgroups were measured by heart blood sampling. The weight of body weight and thymus, spleen and bursa of the chicks were weighed and the immune organ index was calculated. The results showed that ypf-pstp and rampstp could significantly increase the serum level at most time points. The antibody titer promoted the proliferation of T lymphocytes, promoted the entry of lymphocytes into S and g2/m stages, increased the percentage of cd4+, cd8+t lymphocyte subsets, and increased the immune organ index of chicks, among which the YPF-PStp enhanced activity was the strongest.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S853.7

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本文編號：2014910