Observtion Of The Ultra Structure Of Demodex, Analyzing DNA Polymorphism Of Gene Patterns And Experimental Study On The Effect Of Stemona Tincture Extract On Demodex In Vitroin Southern Area Of Sichuan Province

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.