南方医科大学学报
南方醫科大學學報
남방의과대학학보
JOURNAL OF SOUTHERN MEDICAL UNIVERSITY
2015年
6期
826-831
,共6页
夏丹%邓淦明%滕萍英%谢郁%李耀民%王春梅%陈淑洁%陈敏芳%麦荣嘉%廖海燕%石玲玉%欧丽妍%陈启伟%陈晓光%周晓红
夏丹%鄧淦明%滕萍英%謝鬱%李耀民%王春梅%陳淑潔%陳敏芳%麥榮嘉%廖海燕%石玲玉%歐麗妍%陳啟偉%陳曉光%週曉紅
하단%산감명%등평영%사욱%리요민%왕춘매%진숙길%진민방%맥영가%료해연%석령옥%구려연%진계위%진효광%주효홍
日本血吸虫%血吸虫病%SjP40%虫卵肉芽肿%免疫荧光定位
日本血吸蟲%血吸蟲病%SjP40%蟲卵肉芽腫%免疫熒光定位
일본혈흡충%혈흡충병%SjP40%충란육아종%면역형광정위
Schistosoma japonicum%schistosomiasis%Sjp40%egg granuloma%immunofluorescence localization
目的：观察Sjp40在感染新西兰白兔肝脏沉积虫卵及其肉芽肿病变形成中的表达情况，并进行免疫定位。方法日本血吸虫尾蚴感染新西兰白兔，收集未感染组、29 dpi组和45 dpi组肝脏，Trizol法提取各组肝脏总RNA，以日本血吸虫甘油醛-3-磷酸脱氢酶（GAPDH）为内参，Taqman探针qRT-PCR检测Sjp40 mRNA的表达；提取各组肝脏总蛋白，以硫酸铵盐析法和Protein G免疫亲和柱层析法纯化的抗Sjp40-McAb 9G7和抗弓形虫tSAG1-McAb Y3A8（对照抗体），western blot检测Sjp40蛋白的表达；制备肝脏石蜡切片，HE染色观察肝脏虫卵肉芽肿的形成与发展，进一步以免疫荧光技术进行Sjp40在肝脏沉积虫卵及其肉芽肿组织中的定位。结果日本血吸虫感染兔呈急性肝肉芽肿病变期的45 dpi肝脏沉积虫卵中Sjp40 mRNA水平显著高于29 dpi尚未形成虫卵肉芽肿结节的肝脏沉积的未成熟虫卵（P<0.05），westernb blot证实了Sjp40蛋白在29 dpi和45 dpi兔肝脏中的表达。免疫荧光显示：Sjp40在29 dpi兔肝脏中定位于未成熟虫卵内，而45 dpi可见感染兔肝脏中沉积大量成簇内含毛蚴的成熟虫卵及其周围肉芽肿组织均有明显荧光。结论 Sjp40在日本血吸虫感染兔肝脏沉积虫卵中的转录水平随虫卵发育成熟而显著增加，在感染兔急性肉芽肿病变期（45dpi）呈现由虫卵向其周围肉芽肿组织扩散现象，提示该分子在血吸虫肉芽肿形成与发展过程中具有重要作用。
目的：觀察Sjp40在感染新西蘭白兔肝髒沉積蟲卵及其肉芽腫病變形成中的錶達情況，併進行免疫定位。方法日本血吸蟲尾蚴感染新西蘭白兔，收集未感染組、29 dpi組和45 dpi組肝髒，Trizol法提取各組肝髒總RNA，以日本血吸蟲甘油醛-3-燐痠脫氫酶（GAPDH）為內參，Taqman探針qRT-PCR檢測Sjp40 mRNA的錶達；提取各組肝髒總蛋白，以硫痠銨鹽析法和Protein G免疫親和柱層析法純化的抗Sjp40-McAb 9G7和抗弓形蟲tSAG1-McAb Y3A8（對照抗體），western blot檢測Sjp40蛋白的錶達；製備肝髒石蠟切片，HE染色觀察肝髒蟲卵肉芽腫的形成與髮展，進一步以免疫熒光技術進行Sjp40在肝髒沉積蟲卵及其肉芽腫組織中的定位。結果日本血吸蟲感染兔呈急性肝肉芽腫病變期的45 dpi肝髒沉積蟲卵中Sjp40 mRNA水平顯著高于29 dpi尚未形成蟲卵肉芽腫結節的肝髒沉積的未成熟蟲卵（P<0.05），westernb blot證實瞭Sjp40蛋白在29 dpi和45 dpi兔肝髒中的錶達。免疫熒光顯示：Sjp40在29 dpi兔肝髒中定位于未成熟蟲卵內，而45 dpi可見感染兔肝髒中沉積大量成簇內含毛蚴的成熟蟲卵及其週圍肉芽腫組織均有明顯熒光。結論 Sjp40在日本血吸蟲感染兔肝髒沉積蟲卵中的轉錄水平隨蟲卵髮育成熟而顯著增加，在感染兔急性肉芽腫病變期（45dpi）呈現由蟲卵嚮其週圍肉芽腫組織擴散現象，提示該分子在血吸蟲肉芽腫形成與髮展過程中具有重要作用。
목적：관찰Sjp40재감염신서란백토간장침적충란급기육아종병변형성중적표체정황，병진행면역정위。방법일본혈흡충미유감염신서란백토，수집미감염조、29 dpi조화45 dpi조간장，Trizol법제취각조간장총RNA，이일본혈흡충감유철-3-린산탈경매（GAPDH）위내삼，Taqman탐침qRT-PCR검측Sjp40 mRNA적표체；제취각조간장총단백，이류산안염석법화Protein G면역친화주층석법순화적항Sjp40-McAb 9G7화항궁형충tSAG1-McAb Y3A8（대조항체），western blot검측Sjp40단백적표체；제비간장석사절편，HE염색관찰간장충란육아종적형성여발전，진일보이면역형광기술진행Sjp40재간장침적충란급기육아종조직중적정위。결과일본혈흡충감염토정급성간육아종병변기적45 dpi간장침적충란중Sjp40 mRNA수평현저고우29 dpi상미형성충란육아종결절적간장침적적미성숙충란（P<0.05），westernb blot증실료Sjp40단백재29 dpi화45 dpi토간장중적표체。면역형광현시：Sjp40재29 dpi토간장중정위우미성숙충란내，이45 dpi가견감염토간장중침적대량성족내함모유적성숙충란급기주위육아종조직균유명현형광。결론 Sjp40재일본혈흡충감염토간장침적충란중적전록수평수충란발육성숙이현저증가，재감염토급성육아종병변기（45dpi）정현유충란향기주위육아종조직확산현상，제시해분자재혈흡충육아종형성여발전과정중구유중요작용。
Objective To examine the expression profile and immunofluorescence localization of the major egg antigen p40 of Schistosoma japonicum (Sjp40) during granuloma formation in the liver of infected New Zealand white rabbits. Methods New Zealand white rabbits were infected with S. japonicum cercariae, and the livers were harvested at 29 and 45 days post-infection (dpi). The total RNA of the liver tissues was extracted for expression profiling of Sjp40 by quantitative reverse transcription-PCR (qRT-PCR) with GAPDH of S. japonicum as the endogenous reference gene. The expression of Sjp40 in the liver were detected by Western blotting using anti-Sjp40 monoclonal antibody (mAb) 9G7 or anti-Toxoplasma gondii tSAG1 mAb Y3A8 (control) as the primary antibody. Paraffin sections of the liver were prepared for observing egg granuloma formation using HE staining and for indirect immunofluorescence assay of Sjp40 location in the trapped eggs and egg granulomas. Results The level of Sjp40 mRNA in the eggs trapped in rabbit livers was significantly higher at 45 dpi than that at 29 dpi (P<0.05), and Western blotting confirmed the presence of Sjp40 protein in the rabbit livers at both 29 and 45 dpi. Immunofluorescence assay demonstrated localized expression of Sjp40 in the immature eggs in the rabbit liver at 29 dpi, but at 45 dpi fluorescence was detected in clusters of mature eggs containing miracidium and in the surrounding egg granulomas. Conclusion The transcriptional levels of Sjp40 significantly increased with the maturation of eggs trapped in the rabbit livers. Sjp40 protein spread from the eggs to the surrounding egg granuloma at 45 dpi when acute liver granulomatous lesions occur, suggesting that Sjp40 plays a key role in egg granulomas formation in the livers of infected New Zealand white rabbits.
