本文選題：人體蠕形螨 + 碘化丙啶　； 參考：《瀘州醫(yī)學(xué)院》2010年碩士論文

【摘要】：目的：(1)對人體蠕形螨的超微結(jié)構(gòu)進(jìn)行細(xì)致、精準(zhǔn)的形態(tài)學(xué)觀察。(2)研究川南地區(qū)人體蠕形螨基因組DNA的多態(tài)性,探討媒介按蚊與人蠕形螨遺傳基因間的相互關(guān)系。(3)研究百部酊萃取液對人蠕形螨的體外殺滅作用及其藥物安全性。方法：(1)采用5ug/ml碘化丙啶(propidine iodide,PI)對蟲體進(jìn)行熒光染色,避光染色15min,分別置于熒光顯微鏡和激光共聚焦顯微鏡下觀察；2.5%戊二醛固定蟲體標(biāo)本,梯度酒精和叔丁醇脫水,金噴鍍后掃描電鏡觀察。(2)設(shè)計5條隨機引物,應(yīng)用隨機擴增多態(tài)性DNA(random amplified polymorphic DNA, RAPD)技術(shù)對微小按蚊、大劣按蚊與毛囊蠕形螨基因組DNA進(jìn)行隨機擴增,擴增產(chǎn)物經(jīng)1.5%瓊脂糖凝膠電泳后分析。(3)采用透明膠紙粘貼法固定蠕形螨,在膠紙與玻片間噴加藥物,觀察百部酊萃取液對人蠕形螨的體外殺滅作用。采用百部酊萃取液對豚鼠皮膚、角膜給藥,觀察有無過敏反應(yīng),測定藥物安全性。結(jié)果：(1)熒光顯微鏡下蟲體與碘化丙啶有很強的結(jié)合力,熒光信號均勻分布于蟲體細(xì)胞表面,充分展現(xiàn)蟲體形態(tài)；掃描電鏡更加清晰、細(xì)致地展示人體蠕形螨的超微結(jié)構(gòu)；激光共聚焦顯微鏡將蟲體分層掃描,并將圖象進(jìn)行三維重建,真實、完全、直觀地展示蠕形螨三維立體形態(tài)。(2)5條隨機引物對兩種按蚊和毛囊蠕形螨擴增的條帶數(shù)不同,其中兩種按蚊對P2擴增的效果較好,而毛囊蠕形螨在P4擴增的效果較好。引物P2對微小按蚊在150、600、1300 bp擴增出3條帶,大劣按蚊在100～500 bp擴增出一段明亮彌散條帶。引物P3對微小按蚊在200 bp擴增出1條帶,大劣按蚊在100～400 bp擴增出一段明亮彌散條帶,毛囊蠕形螨在200、300bp擴增出2條帶。引物P4對微小按蚊在150、400、500、750 bp擴增出4條帶,大劣按蚊在100～400 bp擴增出一段明亮彌散條帶,毛囊蠕形螨在200、400、600bp擴增出3條帶。引物P5對微小按蚊在200 bp擴增出1條帶,大劣按蚊在100～400 bp擴增出一段明亮彌散條帶。大劣按蚊、微小按蚊和毛囊蠕形螨三者相似帶型共2條(200、400 bp)。(3)百部酊萃取液作用3h蠕形螨開始死亡,作用6h全部死亡,動物藥敏試驗陰性。結(jié)論：(1)熒光顯微鏡,掃描電鏡,激光共聚焦顯微鏡均能很好的展示人體蠕形螨形態(tài)特征,而激光共聚焦顯微鏡更能展示蟲體的三維圖像。(2)按蚊和蠕形螨兩種節(jié)肢動物在遺傳學(xué)上既存在近緣基因,也存在較大基因差異。(3)百部酊萃取液具有直接殺螨作用,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.
【學(xué)位授予單位】：瀘州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R384.4

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