日本血吸虫中间宿主的遗传学及免疫学研究

发布时间：2018-07-20 15:19

【摘要】： 目的研究日本血吸虫(Schistosome japonicum)中间宿主湖北钉螺(Oncomelania hupensis)生物学特性、血淋巴细胞大量获取的方法学、血淋巴细胞的形态学、免疫学、遗传学特性,以进一步揭示日本血吸虫攻击并感染钉螺的侵入机制及其自身保护性机制,研究钉螺分类、遗传特性及筛选抗性株钉螺,为血吸虫病流行病学调查研究、血吸虫病防治策略的制订提供理论依据。方法参照外周淋巴器官中淋巴细胞的悬浮收集法,获取钉螺血淋巴细胞,Giemsa染色后观察其形态。血淋巴细胞经结晶紫染色后分别计数悬浮法、传统压片法及针刺法获取的血淋巴细胞数,并对其进行方差分析和Dunnett-t检验。取冻融的血淋巴细胞上清进行免疫沉淀、抑菌、吞噬杀菌实验。十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析血淋巴细胞蛋白组份相对分子质量(Mr)。凝胶层析纯化分离钉螺血淋巴细胞蛋白。RMBI1640培养基培养钉螺血淋巴细胞。将秋水仙素作用后的钉螺血淋巴细胞进行低渗、固定、气干法制片和染色,显微镜下阅片,进行染色体核型分析。结果(1)钉螺血淋巴细胞分为圆形有丝状伪足、嗜酸性圆形无丝状伪足、嗜碱性圆形无丝状伪足和梭形细胞4种形态,平均直径依次约为10.93、6.13、6.08及11.06μm,分别约占细胞总数的50%、30%、5%及15%。每只钉螺悬浮法、传统压片法及针刺法获取的血淋巴细胞均数分别为1.50、0.66及0.03×104/ml。 (2)悬浮法与传统压片法、针刺法总体均数差异有统计学意义(F =281.47, P0.01)。进一步Dunnett-t检验,悬浮法与压片法、悬浮法与针刺法的总体均数差异有统计学意义(t1=15.67,t2=24.50,两组P0.01)。 (3)冻融的血淋巴细胞上清,与日本血吸虫虫卵可溶性抗原(SEA)反应出现絮状沉淀。 (4)抑菌试验血淋巴细胞上清对金黄色葡萄球菌和大肠埃希菌出现明显的抑菌圈。 (5)血淋巴细胞对白色念珠菌的吞噬率和杀菌率分别为86%、46%。 (6)血淋巴细胞蛋白组份相对分子质量约为Mr 112 300、107 100、97 200、73 500、60 000及12 000。 (7)凝胶层析分离纯化钉螺血淋巴细胞蛋白得到两个主峰。 (8)RMBI1640培养钉螺血淋巴细胞因取材污染未能培养成功。 (9)湖北钉螺的染色体数2n=34,核型公式为14m+8Sm+8St+2t+性染色体。结论悬浮法可获得大量钉螺血淋巴细胞,其具有沉淀SEA、抑制金黄色葡萄球菌和大肠埃希菌生长及吞噬杀灭白色念珠菌等免疫功能。SDS-PAGE显示钉螺血淋巴细胞蛋白Mr约为112 300, 107 100, 972 00, 73 500, 600 00, 12 000。凝胶层析发现钉螺血淋巴细胞蛋白有两个主峰。用钉螺血淋巴细胞气干法制备染色体标本,方法简便,增加有丝分裂中期核型,图像清晰,染色体伸展,形态良好,着丝粒位置明显,可读性好,便于进行核型分析。对钉螺血淋巴细胞的研究,将为研究钉螺与日本血吸虫之间的共同抗原、相容性及其抗性机制、筛选抗性株钉螺、钉螺对灭螺药物的敏感性及耐药性机制等提供参考资料。
[Abstract]:Objective to study the biological characteristics of Hubei Oncomelania Snail (Oncomelania hupensis), the intermediate host of Schistosoma japonicum (Schistosome japonicum), the method of obtaining a large number of blood lymphocytes, morphological, immunological and genetic characteristics of the blood lymphocytes, in order to further reveal the invasion mechanism of the Japanese blood sucking worm and the invasion mechanism of Oncomelania snails and their protective machines. To study the classification of Oncomelania snails, the genetic characteristics and the screening of Oncomelania snails, which provide a theoretical basis for the epidemiological investigation of schistosomiasis and the formulation of schistosomiasis control strategies.
Methods the blood lymphocytes of Oncomelania snails were collected from the peripheral lymphoid organs to obtain the blood lymphocytes of Oncomelania snails, and the morphology was observed after Giemsa staining. The number of blood lymphocytes was counted by the suspension method, the traditional compression method and the acupuncture method were counted, and the blood lymphocytes were analyzed by variance analysis and Dunnett-t test. Lymphocyte supernatant was immunoprecipitation, bacteriostasis, phagocytosis and bactericidal experiment. The relative molecular mass (Mr) of the blood lymphocyte protein components was analyzed by twelve alkyl sulfonate polyacrylamide gel electrophoresis (SDS-PAGE). The blood lymphocyte of Oncomelania snails was cultured and isolated by gel chromatography and purified. After colchicine, colchicine was used. The Oncomelania blood lymphocytes were subjected to low osmotic, fixed, air dried tablets and staining.
Results (1) the blood lymphocytes of Oncomelania snails were divided into 4 types, round and round, eosinophilic, non filamentous, basophilic, and spindle cells, with an average diameter of about 10.93,6.13,6.08 and 11.06 mu m, respectively, accounting for 50%, 30%, 5% and 15% of the total cells, respectively. The mean blood lymphocyte counts were 1.50,0.66 and 0.03 x 104/ml. respectively.
(2) there was significant difference in the total average number between the suspension method and the traditional compression method (F =281.47, P0.01). Further Dunnett-t test, suspension method and compression method, the total mean difference between suspension method and acupuncture method was statistically significant (t1=15.67, t2=24.50, and two groups of P0.01).
(3) the supernatant of frozen thawed lymphocytes showed flocculus deposition in response to soluble antigen (SEA) of Schistosoma japonicum eggs.
(4) bacteriostatic test showed that the supernatant of blood lymphocytes had obvious bacteriostatic circle against Staphylococcus aureus and Escherichia coli.
(5) the phagocytosis rate and bactericidal rate of blood lymphocytes to Candida albicans were 86%, 46%. respectively.
(6) the relative molecular mass of the components of the blood lymphocyte protein is about Mr 112300107 100,97 200,73 500,60 000 and 12000.
(7) separation and purification of hemocytes from Oncomelania hupensis by gel chromatography revealed two main peaks.
(8) RMBI1640 cultured snail blood lymphocytes were not successfully cultured because of contamination.
(9) the chromosome number of Oncomelania hupensis in Hubei is 2n=34, and the karyotype formula is 14m+8Sm+8St+2t+ sex chromosome.
Conclusion a large number of Oncomelania Snail blood lymphocytes can be obtained by suspension method, which can precipitate SEA, inhibit the growth of Staphylococcus aureus and Escherichia coli, and phagocytosis and kill Candida albicans..SDS-PAGE shows that the blood lymphocyte protein Mr of Oncomelania snails is about 112300, 107100, 97200, 73500, 60000, 12000. gel chromatography to detect the hemolymph of Oncomelania snails The cell protein has two main peaks. The preparation of chromosome specimens with Oncomelania Oncomelania blood lymphocyte gas drying method is simple and convenient to increase the metaphase karyotype of the mitosis. The image is clear, the chromosomes extend, the morphology is good, the location of the centromere is obvious, the readability is good, and the karyotype analysis is easy to carry out. The common antigens, compatibility and resistance mechanism among them, screening Oncomelania hupensis, Oncomelania hupensis, sensitivity and drug resistance mechanism of snail were provided.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R383

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