【摘要】：目的:建立亞馬遜利什曼原蟲體外培養(yǎng)體系以獲得大量的無鞭毛體供免疫學及分子生物學研究;進一步證明前鞭毛體轉化的無鞭毛體的抗原特異牲、基因表達水平及對宿主的感染特性。方法:我們通過改良培養(yǎng)基,將小牛血清從20%提高到50%,在體外將前鞭毛體轉化為無鞭毛體,并在體外純培養(yǎng)。然后對三種不同來源的無鞭毛體(動物皮損來源、J774巨噬細胞感染來源及前鞭毛體轉化)進行鑒定:用特異單克隆抗體的間接免疫熒光染色分析對不同來源無鞭毛體(P-4和P-8)及前鞭毛體(GP-46/M2)進行染色檢測原蟲特異性抗原的表達;設計了編碼P-4、GP-46及β-微管蛋白的特異引物,用于RT-PCR分析;同時比較了三種來源的無鞭毛體的巨噬細胞感染特性。結果:三種來源的無鞭毛體經光鏡檢查顯示相近的形態(tài)學特征;用特異單克隆抗體的間接免疫熒光染色分析法對三種不同來源的無鞭毛體(P-4和P-8)及前鞭毛體(GP-46/M2)進行鑒定。P-4和P-8單克隆抗體對三種無鞭毛體的染色呈陽性,而且濃度及部位相似。而前鞭毛體僅與P-8單克隆抗體(mAb)有較弱的陽性反應。P-4mAb則無陽性反應。相反,GP-46/M2單克隆抗體與前鞭毛體呈強陽性反應,卻不與無鞭毛體出現強陽性反應;用RT-PCR分析三種來源的無鞭毛體均觀察到P-4的特異性帶,且濃度相似,而在前鞭毛體中則幾乎測不到。相反,在前鞭毛體中則GP-46特異性帶的高表達(325bp),三種來源的無鞭毛體卻是弱表達。另外,三種來源無鞭毛體對巨噬細胞的感染率均接近100%。結論:通過溫度改變(提高)將前鞭毛體轉化為無鞭毛體后,將培養(yǎng)基小牛血清的濃度提高到50%,使無鞭毛體在體外純培養(yǎng)并長期傳代。建立了亞馬遜利什曼原蟲體外培養(yǎng)體系。三種來源的無鞭毛體在形態(tài)學上、在抗原特性方面、P-4基因水平上、對巨噬細胞感染物性方面都具有相同的特性。因此,從前鞭毛體轉化而來的鞭毛體可作為良好的無鞭毛體的豐富來源。供進一步在免疫學等方面對亞馬遜利什曼原蟲的體內及體外研究。
[Abstract]:Objective: to establish an in vitro culture system of Leishmania Amazon in order to obtain a large number of flagellate for immunological and molecular biological studies. It was further proved that the antigen specificity, gene expression level and host infection characteristics of non-flagellum transformed by proflagellum were further proved. Methods: by improving the culture medium, calf serum was increased from 20% to 50%, the proflagellum was transformed into flagellum in vitro, and pure culture was carried out in vitro. And then for three different sources of flagellum (animal skin lesions, J774 macrophage infection source and proflagellum transformation): indirect immunofluorescence staining of specific monoclonal antibodies was used to analyze different sources of non-flagellum (P 鈮，

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