【作者】 佘俊萍；

【导师】 王光西；

【作者基本信息】 泸州医学院 ， 免疫学， 2010， 硕士

【摘要】 目的：(1)对人体蠕形螨的超微结构进行细致、精准的形态学观察。(2)研究川南地区人体蠕形螨基因组DNA的多态性,探讨媒介按蚊与人蠕形螨遗传基因间的相互关系。(3)研究百部酊萃取液对人蠕形螨的体外杀灭作用及其药物安全性。方法：(1)采用5ug/ml碘化丙啶(propidine iodide,PI)对虫体进行荧光染色,避光染色15min,分别置于荧光显微镜和激光共聚焦显微镜下观察；2.5%戊二醛固定虫体标本,梯度酒精和叔丁醇脱水,金喷镀后扫描电镜观察。(2)设计5条随机引物,应用随机扩增多态性DNA(random amplified polymorphic DNA, RAPD)技术对微小按蚊、大劣按蚊与毛囊蠕形螨基因组DNA进行随机扩增,扩增产物经1.5%琼脂糖凝胶电泳后分析。(3)采用透明胶纸粘贴法固定蠕形螨,在胶纸与玻片间喷加药物,观察百部酊萃取液对人蠕形螨的体外杀灭作用。采用百部酊萃取液对豚鼠皮肤、角膜给药,观察有无过敏反应,测定药物安全性。结果：(1)荧光显微镜下虫体与碘化丙啶有很强的结合力,荧光信号均匀分布于虫体细胞表面,充分展现虫体形态；扫描电镜更加清晰、细致地展示人体蠕形螨的超微结构；激光共聚焦显微镜将虫体分层扫描,并将图象进行三维重建,真实、完全、直观地展示蠕形螨三维立体形态。(2)5条随机引物对两种按蚊和毛囊蠕形螨扩增的条带数不同,其中两种按蚊对P2扩增的效果较好,而毛囊蠕形螨在P4扩增的效果较好。引物P2对微小按蚊在150、600、1300 bp扩增出3条带,大劣按蚊在100～500 bp扩增出一段明亮弥散条带。引物P3对微小按蚊在200 bp扩增出1条带,大劣按蚊在100～400 bp扩增出一段明亮弥散条带,毛囊蠕形螨在200、300bp扩增出2条带。引物P4对微小按蚊在150、400、500、750 bp扩增出4条带,大劣按蚊在100～400 bp扩增出一段明亮弥散条带,毛囊蠕形螨在200、400、600bp扩增出3条带。引物P5对微小按蚊在200 bp扩增出1条带,大劣按蚊在100～400 bp扩增出一段明亮弥散条带。大劣按蚊、微小按蚊和毛囊蠕形螨三者相似带型共2条(200、400 bp)。(3)百部酊萃取液作用3h蠕形螨开始死亡,作用6h全部死亡,动物药敏试验阴性。结论：(1)荧光显微镜,扫描电镜,激光共聚焦显微镜均能很好的展示人体蠕形螨形态特征,而激光共聚焦显微镜更能展示虫体的三维图像。(2)按蚊和蠕形螨两种节肢动物在遗传学上既存在近缘基因,也存在较大基因差异。(3)百部酊萃取液具有直接杀螨作用,安全有效。更多还原

【Abstract】 Objective:(1) To observe the ultra morphological characteristics of Demodex. (2) To discuss the interrelationship of genetic genes between Anopheles and Demodex according to analyzing the DNA polymorphism of gene patterns of Demodex in southern area of Sichuan province. (3) To study the effect and security of stemona tincture extract in killing the Demodex in vitro. Methods:(1) To observe and photograph Demodex by using fluorescence microscope and confocal laser scaning microscope after fluorescent staining with propidine iodide (PI) for 15 minutes; To observe and photograph Demodex by using SEM after fixation with 2.5% glutaral, dehydration of gradient ethanol and tert-butyl alcohol and gold plating. (2)Five random primers were designed randomly, the DNA of Anopheles and Demodex folliculorum of gene patterns were amplified by random amplified polymorphic DNA technique (RAPD), the products were analyzed by 1.5% agarose electrophoresis (AGE).(3) Demodex were fixed by the cellophane tape adhering method. Drug was sprayed between the cellophane tape and slide. It was used to observe the effect of stemona tincture extract on Demodex in vitro.The stemona tincture extract was given to the skin and cornea of guinea pigs. The allergic reaction was observed and the drug security was evaluated. Results:(1) The Specimens combined with propidine iodide well. Fluorescence signals were distributed uniformly on the surface of the specimens;Demodex can be observed clearly with fluorescence microscope. The ultrastructure of Demodex was displayed by using SEM;High definition three-dimensional picture were gotten by using confocal laser scaning microscope.and finished Three-dimensional reconstructing picture of Demodex was true,complete and objective. (2) Between mosquito and Demodex folliculorum, the band numbers amplified by the five primers were different, Anopheles gene were better amplified by P2 and Demodex folliculorum gene by P4. By P2,3 different bands of Anopheles minimus were amplified in 150、600、1300bp and Anopheles dirus 1 bright wide band in 100～500 bp; By P3, Anopheles minimus were amplified 1 band in 200 bp, Anopheles dirus 1 bright wide band in 100～400 bp, and Demodex 2 bands in 200 and 300 bp; By P4, the Anopheles minimus were amplified 4 bands in 150、400、500and 750bp, the Anopheles dirus 1 bright wide band in 100～400bp, and the Demodex folliculorum 3 bands in 200、400 and 600bp. By P5, the Anopheles minimus were amplified 1 band in 200bp, and the Anopheles dirus 1 bright wide band in 100～400bp. There were 2 similar bands (200、400bp) between the 3 kinds arthropods. (3) Demodex began to die at 3 hours after the application of stemona tincture extract in vitro and died out in 6 hours. No allergic reaction in guinea occurred. Conclusion:(1) Demodex can be identified well by using Fluorescence microscope, Confocal Laser scaning microscope and SEM, and the three-dimensional image can be showed best by using confocal Laser scaning microscope. (2)Genetic similarities and difference are existed in both Demodex and Anopheles. (3)The stemona tincture extract was safe, direct, and effective in killing Demodex.更多还原