【作者】 卢秀红；

【导师】 张为宇；

【作者基本信息】 广西大学 ， 预防兽医学， 2008， 硕士

【摘要】 大片吸虫病(Fasciolosis)是一种常见的水牛寄生虫病,了解水牛免疫细胞的各种特性,是防御水牛大片吸虫病的基础。研究一:为选择适宜的实验动物,采用酶联免疫吸附实验(ELISA)筛选大片吸虫阴性的9、15和24月龄的水牛各5头。对所有实验动物的外周血细胞进行总数计数和分类计数,同时分离出不同月龄(9、15和24月龄)健康水牛外周血单个核细胞(PBMC),与不同浓度的非特异刺激因子植物血凝素(PHA)和刀豆素A(ConA)(0.125、0.25、0.5、1、2、3、4μg/孔)进行培养,采用四甲基偶氮唑盐微量酶反应比色法(MTT)测定增殖能力,计算刺激指数(SI)。白细胞总数计数和分类计数实验结果表明,9、15和24月龄组外周血细胞总数分别是17.1×10~9个/ml、16.4×10~9个/ml和16.5×10~9个/ml;其中淋巴细胞分别为9.7×10~9个/ml、9.1×10~9个/ml和7.9×10~9个/ml;嗜酸性粒细胞分别为0.3×10~9个/ml、0.1×10~9个/ml和0.3×10~9个/ml。白细胞分类计数和总数计数在不同月龄水牛间的差异不显著。细胞增殖实验结果表明:9、15和24月龄组在PHA刺激下(0.125～3.000μg/孔)SI随PHA浓度的升高而增高,分别为1.3～3.3、1.0～3.0和1.5～3.6。各月龄组水牛细胞在ConA刺激下(0.125～4μg/孔)的SI随ConA浓度的升高而增高,分别为1.0～1.5、1.1～1.6和1.0～1.6。PHA或ConA刺激下的SI在不同月龄水牛间差异也不显著。这提示9、15、24月龄水牛均适宜用作进一步的实验。PHA刺激下的SI和ConA刺激下的SI在同一月龄水牛组内差异显著(P＜0.05),且PHA刺激下的SI比ConA刺激下的SI高。研究二:为了了解非特异性刺激原(ConA、PHA、LPS)和FgESP对水牛PBMC表达IFN-γ和IL-10的水平的影响,及FgESP对各刺激因子的调节作用。从水牛胆囊中收集大片吸虫成虫并制备大片吸虫分泌排泄产物(FgESP)。应用FgESP和非特异性刺激原(ConA、PHA、LPS)刺激15月龄水牛外周血单个核细胞,实验设有ConA、PHA、LPS、FgESP、FgESP+LPS、FgESP+ConA和FgESP+PHA 7个实验组,并设有阴性对照。采用ELISA方法检测各实验组及阴性对照组在各刺激原刺激6、12、24和48小时(h)下PBMC表达IFN-γ和IL-10的浓度,比较各组实验在不同时间段IFN-γ和IL-10的水平。结果显示ConA、PHA和LPS三者均于6h刺激水牛PBMC产生IFN-γ。PHA刺激细胞产生IFN-γ出现的峰值最早(6h,83.41 pg/ml),ConA刺激细胞产生IFN-γ出现的峰值最晚(24h,87.76 pg/ml)。ConA、PHA和LPS三者在各时间段刺激细胞产生的IL-10都较少,与阴性对照比较差异不显著。在ConA、PHA和LPS三组中,ConA刺激PBMC产生的IFN-γ最强(24h峰值,87.76 pg/ml),LPS刺激PBMC产生的IFN-γ最弱(6h峰值,47.5 pg/ml)。ESP单独刺激PBMC检测到的IFN-γ和IL-10都较少,与阴性比较差异都不显著。FgESP+ConA、PHA+FgESP和FgESP+LPS三组实验分别与ConA、PHA和LPS三组实验相比,的IFN-γ区别都不明显。FgESP+ConA和PHA+FgESP两组实验分别与ConA和PHA实验组相比,IL-10都明显升高(P＜0.05),升高的最高指数都在24h,分别为2.91和2.92;FgESP+LPS实验组与LPS比较,IL-10区别不明显。实验结果表明健康水牛PBMC对ConA和PHA抗原比较敏感,且在ConA刺激下产生IFN-γ量较大,这为今后大片吸虫疫苗设计奠定了基础;在FgESP的调节作用下,ConA和PHA刺激PBMC产生的IL-10升高明显,且升高指数都在24h。FgESP对PBMC这一免疫调节特征为今后的大片吸虫防治打下基础。更多还原

【Abstract】 Fasciolosis is a common disease of domestic water buffalos(Babalus bubalis).Better understanding of the characteristics of water buffalos immunocyte is to control this disease.In the first part of our study,We used enzyme linked immunosorbent assay (ELISA)to selecte 15 non-infected buffalos of 9,15 and 24 months.White blood cell(WBC)counts and differential WBC counts were calculated for each animal. Simultaneously,we isolated the PBMC of buffalos in differential age groups. These PBMC were stimulated with PHA and ConA at 0.125,0.25,0.5,1,2,3 and 4μg/well,respectively.MTT was applied to determine the PBMC proliferation and the stimulation index(SI)was calculated.The results showed that WBC counts of 9,15 and 24 months were 17.1×10~9 entries/ml,16.4×10~9 entries/ml and 16.5×10~9 entries/ml,respectively.Lymphocyte counts were 9.7×10~9 entries/ml,9.1×10~9 entries/ml and 7.9×10~9 entries/ml.Eosinophile granulocyte count of every group were 0.3×10~9 entries/ml,0.1×10~9 entries/ml and 0.3×10~9 entries/ml,respectively.Unremarkable changes were observed in WBC counts and differential counts.PBMC proliferation showed that the SI correlated positively with PHA and ConA concentration 0.125-4.000μg/well.In 9,15 and 24 months age groups,The SI of PHA-stimula ted PBMC were 1.3-3.3, 1.0-3.0 and 1.5-3.6,respectively.The SI of ConA-stimulated PBMC was 1.0-1.5, 1.1-1.6 and 1.0-1.6,respectively.The differences of SI in all age groups were insignificant.Further-research was facilated through above experiments.In the second part of our study,to know the modulation of IFN-γ,and IL-10 prouduction by F.gigantica excretory secretory products(FgESP)in water buffalos PBMC.FgESP was made from F.gigantica adults collected from gallbladders of buffalos.It was used as a stimulator along non-specific stimulators(ConA,PHA and LPS)to stimulate PBMC.We established FgESP, LPS,LPS+FgESP,ConA,ConA+FgESP,PHA and PHA+FgESP seven experimental groups and one control.The IFN-γ,and IL-10 production were estimated by ELISA at 4 time points(6,12,24 and 48h).The results showed that IFN-γlevels increased significantly at 6h in group of ConA,PHA and LPS. IFN-γproduction in PHA group was early and displayed a pesk at 6h(83.41 pg/mL).IFN-γproduction in ConA group was late displayed a pesk at 24h (87.76 pg/mL).Little IL-10 production In these groups was estimated.In groups of PHA,ConA and LPS,IFN-γlevels in the group of ConA was the highest (87.76 pg/mL)at 24h.IFN-γlevel in LPS group was the lowest at 6h(47.5 pg/mL).Little IL-10 and little IFN-γproduction was estimated in FgESP group in any time point.Through comparing ConA+FgESP,PHA+FgESP with ConA, PHA and conrol,we found the trends of little dropping on IFN-γand hight rising on IL-10(P＜0.05).The max upregulating index in ConA+FgESP and PHA+FgESP groups were 2.91 and 2.92 at 24h.The differents of IFN-γand IL-10 between groups FgESP+LPS and LPS was not significant.In this part of our study suggested that water buffalos PBMC was highly susceptible to ConA, it facilitated foundation of vaccine design in future.FgESP can induce regulation in immunity-escaped of Fasciola gigantica.更多还原