川南地区蠕形螨的超微结构观察、基因组DNA遗传多态性初步分析及百部酊萃取液对虫体体外作用的实验研究

发布时间：2018-05-21 03:19

本文选题：人体蠕形螨 + 碘化丙啶　；参考：《泸州医学院》2010年硕士论文

【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。
[Abstract]:Objective: to study the DNA polymorphism of human Demodex mites in southern Sichuan by careful and precise morphological observation of the ultrastructure of Demodex. To study the relationship between the genetic genes of Anopheles vector and Demodex human.) to study the killing effect and drug safety of Baibu tincture extract on Demodex in vitro. Methods 5ug/ml iodidepyridine (5ug/ml) was used to stain the insect body for 15 min. The specimens were observed under fluorescence microscope and laser confocal microscope respectively. The dehydration of gradient alcohol and tert-butanol were observed by gradient alcohol and tert-butanol. Five random primers were designed. Random amplified polymorphic DNA(random amplified polymorphic DNA, RAPD) was used to amplify genomic DNA of Anopheles minimus, Anopheles dirus and Demodex hair follicles. The amplification product was analyzed by 1.5% agarose gel electrophoresis. The mite was fixed by transparent adhesive paper. The drug was sprayed between the adhesive paper and the glass to observe the killing effect of Baibu tincture extract on human Demodex in vitro. The skin and cornea of guinea pigs were treated with Baibou tincture extract. The allergic reaction was observed and the safety of the drug was determined. Results (1) under the fluorescence microscope, there was a strong binding force between the insect and pyridine iodide, the fluorescence signal distributed evenly on the surface of the insect somatic cells, and the morphology of the insect was fully displayed, and the ultrastructure of the human Demodex mites was more clearly and meticulously displayed by the scanning electron microscope (SEM). Laser confocal microscope (LSCM) scanned the insect and reconstructed the image in three dimensions. The true, complete and intuitive three-dimensional morphology of Demodex was shown by 5 random primers. The number of amplified bands of the two species of Anopheles and Demodex were different. Two species of Anopheles had a better effect on P2 amplification, while Demodex hair follicles had a better effect on P4 amplification. Three bands were amplified from primer P2 pair of Anopheles minimus at 150600 and 1300 BP, while a bright diffuse band was amplified from Anopheles inferioris at 100,500bp. One band was amplified from Anopheles minimus by primer P3 at 200bp, a bright diffuse band was amplified by Anopheles inferioris at 100,400bp, and two bands were amplified by 200300bp from Demodex hair follicle mites. Primer P4 amplified 4 bands at 150400500750 BP, Anopheles inferioris amplified a bright diffuse band at 100,400bp, and Demodex hair follicles amplified 3 bands in 200400600bp. One band was amplified from primer P5 pair at 200 BP and a bright diffuse band was amplified from Anopheles dirus at 100,400 BP. Anopheles dirus, Anopheles minimus and Demodex hair follicle had two similar zonal patterns (200400bp.n.) 100 pieces of tincture extract began to die at 3h, all died at 6h, and the animal drug sensitivity test was negative. Conclusion fluorescence microscope, scanning electron microscope and laser confocal microscope can all show the morphological characteristics of human Demodex. The laser confocal microscope can show the three-dimensional image of the insect. 2) there are not only close genes in genetics of Anopheles mosquitoes and Demodex, but also large gene differences. The extract of Baibu tincture has direct mite killing effect, which is safe and effective.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R384.4

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