Study Of Histochemistry And Enzymohistochemistry Of Three Species Of Demodex And Enzymology And Molecularbiology Of Demodex Caprae

Objective In humans, Demodex folliculorum and Demodex brevis have been implied to play a role in several facial conditions: acne, folliculitis, rosacea-like demodicidosis, baldness and blepharitis. Demodex caprae may make goat skins perforated and goat skins will decline in price. Although there are many studies of Demodex epidemiology and medicine in treating demodicidosis, there are few studies of Demodex histochemistry, pathogenic mechanism and molecular biology.After micromanipulative pretreatment of mite specimen, a suspension staining was carried out. Endosomatic chemical constituents and enzymes of three species of Demodex may be observed using technical skills of histochemistry and enzymohistochemistry. The activity of endosomatic enzymes from Demodex caprae may be detected using technical skills of enzymology. We will screen out the pathogenic factors from the endosomatic structures, chemical constituents and enzymes. A method improved by ourselves will be used for extracting the genomic DNA of Demodex caprae. Above pioneering works will the bases for the future molecular biology and enzymology studies on Demodex.Methods We obtained human mites from human forehead using a scraping, and got Demodex caprae from the content of an incised sebaceous cyst. Under the dissecting microscope, with a special needle made by ourselves, cleaned mites were picked out from washings. After the Demodex had been stored at -20℃for 48 hours, glycogen was stained with Giemsa, lipid was stained with oil red O and DNA was stained with methyl green. The activity of acid phosphatase and alkaline phosphatase were stained by Gomori method. The activity of carboxylesterase was stained by Gomori method with 1-Naphthylacetate. The activity of lipase was stained by Tween method.Clean and fresh Demodex caprae were triturated in a cold special mini-mortar made by ourselves under the dissecting microscope, centrifuged at 4℃. The supernatants contained enzyme activity, refrigerated and used within 10 days. The activity of carboxylesterase of Demodex caprae was detected by spectrophotometer at 600nm, using 1-Naphthylacetate as substrate and Fast Blue B salt as chromophore coupling agent. The activity of protease of Demodex caprae was estimated by gelatin-plate method. We extracted genomic DNA of Demodex caprae with an improved method that was used to extract the genomic DNA of human mite. The DNA would be tested by Agar Gel Electrophoresis and Ultra-violet spectrophotometer.Results Endosomatic structure, glycogen, lipid, DNA, acid phosphatase, alkaline phosphatase, esterase and lipase of three species of Demodex dyed well.The absorbance at 600nm of Demodex caprae extract group was significantly higher than that of negative control group (P<0.01). Extract of Demodex caprae digested a part of gelatin-plat, which did not dye by Coomassie brilliant blue R250. Genomes of Demodex caprae were extracted successfully. The extracted products had an high molecular weight DNA band, which could be observed through Agar Gel Electrophoresis. Measured the DNA by Ultra-violet spectrophotometer, the value of OD₂₆₀/OD₂₈₀ was more than 1.6.Conclusion The self-made suspension staining makes it possible that the Demodex body is stained. It was proved that Mites contain glycogen, lipid, DNA, acid phosphatase, alkaline phosphatase, carboxylesterase and lipase. The activity of carboxylesterase and protease from Demodex caprae is estimated. The result explains why mites feed on ceratin and lipid. Above chemical materials may be the pathogenic factors of demodex. The success of extracting the genomic DNA of Demodex caprae would lay the groundwork for the future molecularbiological studies on Demodex.