结核分枝杆菌H37Ra和有机硒联合免疫效果的研究

发布时间：2018-03-28 04:18

本文选题：结核分枝杆菌　切入点：H37Ra　出处：《重庆医科大学》2007年硕士论文

【摘要】： 目的:1.探讨结核分枝杆菌H37Ra与有机硒联合免疫小鼠后,细菌在小鼠体内定植的情况,以及诱导机体产生的特异性细胞免疫功能。2.构建可表达结核分枝杆菌分泌蛋白Ag85B的靶细胞,并研究结核分枝杆菌H37Ra与有机硒联合免疫小鼠后的特异性细胞毒作用及其免疫机制。方法:1.将C57BL/6小鼠分为6组(每组12只),即⑴H37Ra+Se组、⑵BCG+Se组、⑶H37Ra组、⑷BCG组、⑸Se组和⑹PBS对照组。小鼠连续灌服有机硒1周后,采用皮内注射方式接种H37Ra或BCG,然后继续喂硒4周。免疫4周和8周后,检测小鼠脏器指数及结核杆菌在脏器内的定植情况。2.分离各组小鼠脾淋巴细胞,体外用PPD刺激,MTT法检测脾淋巴细胞的刺激指数(Stimulation Index,SI);ELISA法检测培养上清液中IFN-γ、IL-2的产量。3.构建可表达结核分枝杆菌分泌蛋白Ag85B的靶细胞,MTT法检测免疫小鼠脾淋巴细胞的杀伤活性。4.免疫8周后,RT-PCR法检测脾淋巴细胞中穿孔素、颗粒酶B mRNA的表达。结果:1.免疫4周或8周后,H37Ra+Se组的脾指数、胸腺指数和脾肺荷菌量略高于BCG+Se组、H37Ra组,但差异无显著性(P0.05)。2. H37Ra+Se组的SI、IL-2和IFN-γ均显著高于H37Ra组(P0.05),但与BCG+Se组相比,IFN-γ有显著性差异,其他指标无显著性差异(P0.05);随着时间的延长,各项指标均下降。3.转染阳性细胞中有结核分枝杆菌分泌蛋白Ag85B的表达,H37Ra+Se组脾淋巴细胞的杀伤活性显著高于H37Ra组(P0.05),略高于BCG+Se组(P0.05)。4. H37Ra+Se组小鼠穿孔素及颗粒酶B mRNA的表达量均显著高于PBS组(P0.05),略高于BCG+Se组(P0.05);H37Ra+Se组与H37Ra组比较,穿孔素mRNA的表达量有显著性差异(P0.05),而颗粒酶B mRNA的表达量,前者略多于后者,但无统计学意义(P0.05)。结论:1. H37Ra和有机硒联合免疫小鼠后结核菌能在小鼠脾脏、肺脏定植至少60天。2. H37Ra和有机硒联合免疫小鼠后诱导机体产生的细胞免疫效果显著优于H37Ra或BCG单独免疫,略优于BCG和有机硒联合免疫。3.构建表达结核分枝杆菌分泌蛋白Ag85B的靶细胞及MTT法检测特异性杀伤活性,为研究结核疫苗免疫的细胞毒作用提供了较好的研究手段。4. H37Ra和有机硒联合免疫小鼠后可以诱导脾淋巴细胞的杀伤活性,其杀伤机制可能与细胞中穿孔素、颗粒酶B mRNA表达增加有关。
[Abstract]:Objective 1. To investigate the colonization of mycobacterium tuberculosis (H37Ra) combined with organic selenium in mice and the specific cellular immune function induced by mycobacterium tuberculosis. 2. To construct the target cells expressing the secreting protein Ag85B of Mycobacterium tuberculosis. The specific cytotoxic effect and immune mechanism of mice immunized with mycobacterium tuberculosis H37Ra and organic selenium were studied. Methods: 1. C57BL/6 mice were divided into 6 groups (12 rats in each group, I. e., 1H37Ra se group, 2 BCG se group, 3 BCG group, 4 BCG group, 5 se group and 6PBS control group). After one week of continuous administration of organic selenium, the mice were given organic selenium for 1 week. The mice were inoculated with H37Ra or BCG by intradermal injection, and then given selenium for 4 weeks. After immunization for 4 weeks and 8 weeks, the viscera index and the colonization of Mycobacterium tuberculosis in the organs were detected. The stimulation index of splenic lymphocytes was detected by PPD stimulation assay in vitro. Elisa was used to detect the production of IL-2 in culture supernatant. The target cells expressing Ag85B secreted by Mycobacterium tuberculosis were constructed to detect the spleen lymphocytes of immunized mice. After 8 weeks of immunization, the perforin in splenic lymphocytes was detected by RT-PCR. Expression of granzyme B mRNA. Results 1. The spleen index, thymus index and the amount of splenic and pulmonary bacilli in H37Ra se group were slightly higher than those in BCG se group, but there was no significant difference between H37Ra se group and H37Ra group, but there was significant difference between H37Ra se group and BCG se group, but there was a significant difference between H37Ra se group and H37Ra group, but there was a significant difference between H37Ra se group and BCG se group, but there was no significant difference between H37Ra se group and BCG se group, but there was no significant difference between H37Ra se group and H37Ra group (P 0.05), but there was no significant difference between H37Ra se group and BCG se group, but there was no significant difference between H37Ra se group and H37Ra group. There was no significant difference in other indexes (P 0.05). The activity of splenic lymphocytes in H37Ra se group was significantly higher than that in H37Ra group, and slightly higher than that in BCG se group. 4. Perforin and granzyme B in H37Ra se group. The expression of mRNA was significantly higher than that of PBS group (P 0.05) and slightly higher than that of BCG se group (P 0.05) and H37Ra se group compared with H37Ra group. The expression of perforin mRNA was significantly different (P 0.05), while the expression of granzyme B mRNA was slightly higher in the former than in the latter, but there was no significant difference between the two groups. Conclusion H37Ra combined with organic selenium can immunize mice with tuberculous bacillus at least 60 days after inoculation of mice spleen and lung. The cellular immunity of mice immunized with H37Ra and organic selenium is better than that of H37Ra or BCG alone. The target cells expressing the secreting protein Ag85B of Mycobacterium tuberculosis were constructed and the specific killing activity was detected by MTT method. 4. H37Ra and organoselenium combined immunized mice can induce the killing activity of spleen lymphocytes, which may be related to the cytotoxicity of perforin in cells. The increased expression of granzyme B mRNA was related to the expression of granzyme
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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