玉屏风多糖的制备及体内外增强免疫活性研究

发布时间：2018-06-13 17:57

本文选题：玉屏风多糖 + 红外质谱　；参考：《山东农业大学》2015年硕士论文

【摘要】：具有刺激免疫应答的药物—免疫增强剂的应用,在生产中对动物传染性疾病具有提高免疫功能,并由此增强抵抗能力的重要作用。然而,现在最常用的免疫增强剂大多是化学合成的,在药物残留方面具有潜在的隐患。如今药物的安全性引起了公众的关注,从而使得中药显示出其优越性。许多研究表明,多糖是中药的一个重要组成部分,是存在于天然植物,微生物(细菌和真菌)和海藻类生物体内的一类复杂的天然高分子化合物。许多药理和临床研究表明,多糖具有增强免疫,抗病毒,抗肿瘤,抗氧化,抗衰老等生物活性。本研究从玉屏风散整体出发追踪其多糖类活性成分,然后与方中的单味药白术多糖作比较,通过体内外实验探究玉屏风多糖的免疫增强活性。首先用一步醇沉和分级醇沉法提取玉屏风总多糖和白术总多糖及各分级多糖,通过体外对鸡外周血淋巴细胞增殖的影响,筛选出3个效果较好的活性部位;然后从体外对鸡淋巴细胞周期分布和淋巴细胞亚群CD4+/CD8+比例的影响,体内对鸡新城疫疫苗免疫应答的影响,比较了3个多糖的增强免疫活性。实验分为以下五个部分:采用一步醇沉和分步醇沉法提取粗的玉屏风总多糖(YPF-PStc)和白术总多糖(RAMPStc),3个分级白术多糖RAMPS60c、RAMPS70c和RAMPS80c。YPF-PStc和RAMPStc通过阴离子交换剂DEAE sephadex A-25分离纯化,从中分离出纯化的玉屏风多糖(YPF-PStp)和白术多糖(RAMPStp),分别用苯酚-硫酸法和考马斯亮兰法测定多糖和蛋白含量,红外质谱法鉴定多糖的结构。结果显示,YPF-PStc提取率为13.56%,RAMPStc提取率为13.36%,分级白术多糖的提取率以RAMPS60c最高、达14.79%;YPF-PStp提取率为16.87%。RAMPStp的糖含量最高、达97.14%,比RAMPStc提高了9.93%,YPF-PStp的糖含量为85.6%,比YPF-PStc提高了18.25%;玉屏风和白术各多糖的蛋白含量都较低。结果表明,柱层析纯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。将YPF-PStc、YPF-PStp、RAMPStc、RAMPStp和RAMPS60c、RAMPS70c、RAMPS80c加入到鸡外周血淋巴细胞培养体系中,用MTT法测定其安全浓度;然后将安全浓度下的7种多糖,分别单独或协同PHA加入到培养的鸡外周血淋巴细胞中,培养48 h后,测定淋巴细胞增殖(细胞A570值和最高淋巴细胞增殖率)的变化。结果表明,多糖单独刺激时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时能显著促进淋巴细胞增殖,YPF-PStp在31.25μg m L-1时的细胞增殖率最高,为31.148%;多糖与pha共同刺激时,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能显著促进淋巴细胞增殖,ramps60c在7.813μgml-1时的细胞增殖率最高,为29.897%。综合评价,筛选出ypf-pstp、rampstp和ramps60c可能是增强免疫活性的较好部位。多糖ypf-pstp、rampstp和ramps60c在31.25μgml-1(细胞增殖试验筛选出的最佳浓度)时刺激淋巴细胞,分别在24h、48h和72h收集细胞,经过处理,在流式细胞仪上检测各个时间点的周期分布情况。结果显示,在72h之内未经任何处理的细胞大部分处于g0/g1期,其变化不明显。与空白组比较,pha组位于g0/g1期细胞的百分比明显降低(p0.05),处于s期和g2/m期的细胞百分比明显增多(p0.05)。在不同时间点,ypf-pstp、rampstp和ramps60c与pha组相比,都可以明显降低g0/g1期细胞的百分比(p0.05),ypf-pstp和rampstp在作用于淋巴细胞48h时明显升高s期细胞的百分比,其spf值、pi值均最大,效果最明显。分别在24h、48h和72h收集细胞,pbs洗涤2次,流式细胞仪检测cd4+、cd8+t淋巴细胞亚群的变化。结果显示,在所有时间点,试验组的cd4+淋巴细胞百分率均显著高于pha对照组(p0.05),ypf-pstp和rampstp处理细胞48h后,cd4+淋巴细胞百分率显著高于ramps60c对照组(p0.05)。多糖处理细胞48h、72h后,cd8+淋巴细胞百分率和cd4+/cd8+比值均显著高于pha对照组(p0.05),ypf-pstp和rampstp处理细胞48h后,cd8+淋巴细胞百分率显著高于ramps60c对照组(p0.05),表明ypf-pstp和rampstp可以明显提高cd4+、cd8+淋巴细胞百分率及cd4+/cd8+比值。为了比较上述3个多糖的增强免疫作用,测定了3个多糖对雏鸡新城疫疫苗免疫效果的影响。将健康1日龄海兰褐雏公鸡210只,随机分为6组,除空白对照组外,14日龄雏鸡均用新城疫iv系苗滴鼻点眼免疫,28日龄二免。在首次免疫的同时,分别肌肉注射6mgml-1的ypf-pstp、rampstp和ramps60c,口服10mgkg-1的盐酸左旋咪唑,无佐剂对照组注射等量生理盐水。分别于首次免疫后第7、14、21、28d翼静脉采血检测血清hi抗体效价,心脏采血测定外周血t淋巴细胞增殖、淋巴细胞周期和cd4+、cd8+t淋巴细胞亚群的变化,处死雏鸡后称取体重及胸腺、脾脏和法氏囊的重量,计算免疫器官指数。结果表明,ypf-pstp和rampstp在大多时间点能显著提高血清抗体效价、促进t淋巴细胞增殖、促进淋巴细胞进入s期和g2/m期,提高cd4+、cd8+t淋巴细胞亚群的百分率,提高雏鸡免疫器官指数。其中YPF-PStp免疫增强活性最强。
[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.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S853.7

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