【摘要】： 目的研究日本血吸蟲(chóng)(Schistosome japonicum)中間宿主湖北釘螺(Oncomelania hupensis)生物學(xué)特性、血淋巴細(xì)胞大量獲取的方法學(xué)、血淋巴細(xì)胞的形態(tài)學(xué)、免疫學(xué)、遺傳學(xué)特性,以進(jìn)一步揭示日本血吸蟲(chóng)攻擊并感染釘螺的侵入機(jī)制及其自身保護(hù)性機(jī)制,研究釘螺分類、遺傳特性及篩選抗性株釘螺,為血吸蟲(chóng)病流行病學(xué)調(diào)查研究、血吸蟲(chóng)病防治策略的制訂提供理論依據(jù)。 方法參照外周淋巴器官中淋巴細(xì)胞的懸浮收集法,獲取釘螺血淋巴細(xì)胞,Giemsa染色后觀察其形態(tài)。血淋巴細(xì)胞經(jīng)結(jié)晶紫染色后分別計(jì)數(shù)懸浮法、傳統(tǒng)壓片法及針刺法獲取的血淋巴細(xì)胞數(shù),并對(duì)其進(jìn)行方差分析和Dunnett-t檢驗(yàn)。取凍融的血淋巴細(xì)胞上清進(jìn)行免疫沉淀、抑菌、吞噬殺菌實(shí)驗(yàn)。十二烷基磺酸鈉-聚丙烯酰胺凝膠電泳(SDS-PAGE)分析血淋巴細(xì)胞蛋白組份相對(duì)分子質(zhì)量(Mr)。凝膠層析純化分離釘螺血淋巴細(xì)胞蛋白。RMBI1640培養(yǎng)基培養(yǎng)釘螺血淋巴細(xì)胞。將秋水仙素作用后的釘螺血淋巴細(xì)胞進(jìn)行低滲、固定、氣干法制片和染色,顯微鏡下閱片,進(jìn)行染色體核型分析。 結(jié)果(1)釘螺血淋巴細(xì)胞分為圓形有絲狀偽足、嗜酸性圓形無(wú)絲狀偽足、嗜堿性圓形無(wú)絲狀偽足和梭形細(xì)胞4種形態(tài),平均直徑依次約為10.93、6.13、6.08及11.06μm,分別約占細(xì)胞總數(shù)的50%、30%、5%及15%。每只釘螺懸浮法、傳統(tǒng)壓片法及針刺法獲取的血淋巴細(xì)胞均數(shù)分別為1.50、0.66及0.03×104/ml。 (2)懸浮法與傳統(tǒng)壓片法、針刺法總體均數(shù)差異有統(tǒng)計(jì)學(xué)意義(F =281.47, P0.01)。進(jìn)一步Dunnett-t檢驗(yàn),懸浮法與壓片法、懸浮法與針刺法的總體均數(shù)差異有統(tǒng)計(jì)學(xué)意義(t1=15.67,t2=24.50,兩組P0.01)。 (3)凍融的血淋巴細(xì)胞上清,與日本血吸蟲(chóng)蟲(chóng)卵可溶性抗原(SEA)反應(yīng)出現(xiàn)絮狀沉淀。 (4)抑菌試驗(yàn)血淋巴細(xì)胞上清對(duì)金黃色葡萄球菌和大腸埃希菌出現(xiàn)明顯的抑菌圈。 (5)血淋巴細(xì)胞對(duì)白色念珠菌的吞噬率和殺菌率分別為86%、46%。 (6)血淋巴細(xì)胞蛋白組份相對(duì)分子質(zhì)量約為Mr 112 300、107 100、97 200、73 500、60 000及12 000。 (7)凝膠層析分離純化釘螺血淋巴細(xì)胞蛋白得到兩個(gè)主峰。 (8)RMBI1640培養(yǎng)釘螺血淋巴細(xì)胞因取材污染未能培養(yǎng)成功。 (9)湖北釘螺的染色體數(shù)2n=34,核型公式為14m+8Sm+8St+2t+性染色體。 結(jié)論懸浮法可獲得大量釘螺血淋巴細(xì)胞,其具有沉淀SEA、抑制金黃色葡萄球菌和大腸埃希菌生長(zhǎng)及吞噬殺滅白色念珠菌等免疫功能。SDS-PAGE顯示釘螺血淋巴細(xì)胞蛋白Mr約為112 300, 107 100, 972 00, 73 500, 600 00, 12 000。凝膠層析發(fā)現(xiàn)釘螺血淋巴細(xì)胞蛋白有兩個(gè)主峰。用釘螺血淋巴細(xì)胞氣干法制備染色體標(biāo)本,方法簡(jiǎn)便,增加有絲分裂中期核型,圖像清晰,染色體伸展,形態(tài)良好,著絲粒位置明顯,可讀性好,便于進(jìn)行核型分析。對(duì)釘螺血淋巴細(xì)胞的研究,將為研究釘螺與日本血吸蟲(chóng)之間的共同抗原、相容性及其抗性機(jī)制、篩選抗性株釘螺、釘螺對(duì)滅螺藥物的敏感性及耐藥性機(jī)制等提供參考資料。
[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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R383

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