弓形虫排泄—分泌抗原（ESA）鼻内免疫小鼠诱导的免疫应答的观察

发布时间：2018-05-16 16:41

本文选题：弓形虫 + ESA　；参考：《山西医科大学》2006年硕士论文

【摘要】： 目的本研究首先观察弓形虫感染小鼠腹水中分离的排泄-分泌抗原(excreted/secreted antigens ESA)鼻内免疫小鼠后对不同黏膜部位和系统的免疫效应,又观察了Vero细胞株体外培养弓形虫并提取ESA鼻内免疫小鼠后对不同黏膜部位和系统的免疫效应以及诱导免疫应答持续时间的观察。探讨不同途径获得的弓形虫ESA鼻内免疫诱导的免疫应答机制及免疫效果,为弓形虫鼻内复合疫苗研制奠定理论基础。方法本研究分三部分:第一部分观察弓形虫感染小鼠腹水中分离的排泄-分泌抗原(ESA)鼻内免疫小鼠后对不同黏膜部位和系统的免疫效应。实验组分别用从感染弓形虫小鼠后24hESA组、48h ESA组、72h ESA组提取的ESA 20μg/只鼻内免疫小鼠2次,间隔2周;对照组用20μl/只PBS滴鼻。观察小鼠健康和死亡情况,记录体重。在末次免疫后第3w处死小鼠,称取脾脏重量;计数派伊尔氏(Peyer’s patches, PP)个数;分离PP淋巴细胞、脾淋巴细胞、小肠上皮内淋巴细胞(intraepithelial lymphocyte, IEL)及肠系膜淋巴结(mesenteric lymph node, MLN)细胞并计数。眼眶采血和收集直肠内粪便,ELISA检测粪内sIgA水平及血清内特异性IgG。第二部分观察Vero细胞株体外培养弓形虫并提取ESA鼻内免疫小鼠后对不同黏膜部位和系统的免疫效应。实验组分别用5d、7d、9d、11d、14d提取的ESA 20μg/只鼻内免疫小鼠2次,间隔2周;对照组用20μl/只无攻虫的细胞培养上清液滴鼻。观察小鼠健康和死亡情况,记录体重。在末次免疫后第3w处死小鼠,称取脾脏重量;计数PP个数;分离PP、脾淋巴细胞、肠上皮内淋巴细胞及肠系膜淋巴结细胞并计数。在二次免疫后第3周处死小鼠,收集粪便和眼眶采血,ELISA检测粪内sIgA水平及血清内特异性IgG。第三部分本研究观察了Vero细胞株体外培养弓形虫并提取的ESA鼻内免疫BALB/c小鼠后不同黏膜部位和系统的免疫效应及其持续时间。实验组以免疫原性好的ESA(20μg/只)为抗原,鼻内免疫,对照组用未接种弓形虫的细胞培养上清液20μl/只滴鼻。滴鼻2次(间隔2周)后分别于第1、2、3、4、5、6、7周处死小鼠。ELISA法测定血清IgG、IgA;粪便sIgA及肠液sIgA;称取脾脏重量;计数PP个数;分离PP、脾、肠上皮内淋巴细胞(intestinal intraepithelial lymphocytes, IEL)并计数。结果弓形虫感染小鼠腹水中分离的排泄-分泌抗原(ESA)鼻内免疫小鼠后72h ESA组小鼠健康状况差,体重逐渐降低,死亡4只;其它组小鼠体重仍呈增高趋势。实验各组小鼠IEL数、脾淋巴细胞数、MLN淋巴细胞数均高于对照组(P0.05),有较高的增殖活性。其中72h ESA组和48h ESA组的IEL数、脾淋巴细胞数、MLN细胞数显著高于24h ESA组(P0.01)。弓形虫ESA免疫后第3周实验各组小鼠的血清IgG水平、粪便sIgA水平均高于对照组(P0.05),其中72h ESA组和48h ESA组的抗体水平显著高于第24h ESA组(P0.01)。
[Abstract]:Objective to investigate the immunological effects of excreted-secreted antigens isolated from ascites of mice infected with Toxoplasma gondii (Toxoplasma gondii) on different mucosal sites and systems after nasal immunization with Toxoplasma gondii (Toxoplasma gondii) infected mice. The immune effects of Toxoplasma gondii (Toxoplasma gondii) on different mucosal sites and systems and the duration of induction of immune response were observed in mice immunized with ESA after nasal immunization with Toxoplasma gondii in vitro. To study the immune response mechanism and immune effect induced by different ways of intranasal immunization of Toxoplasma gondii ESA, and to lay a theoretical foundation for the development of Toxoplasma gondii intranasal compound vaccine. Methods the present study was divided into three parts: the first part was to observe the immune effects on different mucosal sites and systems of mice immunized with excretion-secretory antigen (ESA) isolated from ascites infected by Toxoplasma gondii (Toxoplasma gondii). Mice in the experimental group were immunized with ESA 20 渭 g / mouse after infection with Toxoplasma gondii (Toxoplasma gondii) for 48 h, ESA group (72 h) and control group (20 渭 l / PBS). The health and death of mice were observed and their body weight was recorded. At the third week after the last immunization, the mice were killed, the weight of spleen was taken, the number of Peyers patcheses (PPs) was counted, PP lymphocytes, spleen lymphocytes, intraepithelial lymphocytes (IELs) and mesenteric lymph node, MLN) cells of mesenteric lymph nodes were isolated and counted. SIgA levels in feces and serum specific IgGs were detected by Elisa in orbital blood collection and rectum stool collection. The second part was to observe the immune effects of Toxoplasma gondii (Toxoplasma gondii) cultured in vitro on different mucosal sites and systems in mice immunized with ESA. The mice in the experimental group were immunized with ESA 20 渭 g / mouse for 2 weeks, respectively, and the control group was infused with 20 渭 l / a cell culture supernatant. The health and death of mice were observed and their body weight was recorded. The mice were killed at the third week after the last immunization, the weight of spleen was taken, the number of PP was counted, and PPP-spleen lymphocytes, intestinal intraepithelial lymphocytes and mesenteric lymph node cells were isolated and counted. The mice were killed at the third week after the second immunization. The fecal and orbital blood samples were collected to detect the level of sIgA in feces and the specific IgGs in serum by enzyme-linked immunosorbent assay (Elisa). In the third part, the immune effects and duration of different mucosal sites and systems of Vero cell line cultured in vitro and extracted ESA from BALB/c mice were observed. The experimental group was immunized with ESA(20 渭 g / mouse, and the control group was inoculated with 20 渭 l of the culture supernatant of Toxoplasma gondii cells. The mice were killed at 7 weeks after nasal drip twice (interval 2 weeks). Elisa method was used to determine the serum IgGG IgA, the fecal sIgA and intestinal Siga, the weight of spleen, the number of PP, the separation of PPp, spleen and intestinal intraepithelial lymphocytes, and count. Results the mice inoculated with Toxoplasma gondii in ascites with excretion-secretory antigen (ESAA) were in poor health condition, their body weight gradually decreased, and 4 mice died, while the other groups showed an increasing trend of body weight. The number of IEL and spleen lymphocytes in each group was higher than that in control group (P 0.05), and it had higher proliferative activity. The number of IEL and spleen lymphocytes in 72 h ESA group and 48 h ESA group were significantly higher than that in 24 h ESA group. The levels of serum IgG and fecal sIgA were significantly higher in Toxoplasma gondii ESA group than those in control group at 3 weeks after ESA immunization. The antibody levels in 72 h ESA group and 48 h ESA group were significantly higher than those in 24 h ESA group.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

【引证文献】