The Over Expression Of Mast Cell Tryptase In Keloids

Objectiveto investigate the expression and distribution of mast cell tryptase(MCT) in keloids,and to find out the difference of MCT among keloids,hypertrophic scar and normal skins.IntroductionKeloids develop as a result of a proliferation of dermal tissue following skin injury. They differ from hypertrophic scars clinically and histologically.Clinically,keloids are a deep red or purple color with raised indurated tissue that extends beyond the original wound borders.Hypertrophic scars have a less impressive white or pink color, with firm tissue limited to original wound border.Histologically,keloids are composed of disorganized thick hyalinized collagen with a prominent mucoid matrix, whereas hypertrophic scars are characterized by fewer,more organized collagen fibers with a scanty mucoid matrix.Mast cell tryptase(MCT) is a tetrameric neutral serine protease with a molecular weight of 134 kDa and is the most abundant mediator stored in mast cell granules. The genes encoding mast cell tryptase are located on the short arm of chromosome 16. The enzyme is made up of four non-covalently bound subunits,and each subunit has one active enzyme site.MCT has ability to stimulate proliferation of dermal fibroblasts and to stimulate type al pro-collagen synthesis,which may be the great related to the formation of scars.In our research,we detected the expressing differences of MCT among the keloids,hypertrophic scars and skins by means of fluorescence immunohistochemistry and relative quantification real time pcr. Materials20 keloids from 17 patients aged from 15 to 32,20 hypertrophic scars from 18 patients aged from 10 to 45,and 20 healthy skins as control from 20 patients aged from 8 to 30.All the samples were supplied by Shandong Province Hospital Burn and Platic Department.The scars had developped for 6 months to 5 years and were classified clinically and histologically.All the samples undercovered no theropies before and were put into liquid nitrogen quickly after they were incised.MethodsFluorescence Immunohistochemistry After the frozen samples were sliced to 4-μm sections and fixed,50μl 1:100 Anti-Humin Tryptase mAb Biotin was incubated at 4℃for a whole night,the following day,50μl 1:100 Immunopure Goat Anti-Mouse IgG was incubated at 37℃for lh in darkroom.And then the slices were viewed with a Leica DMIREZ IM50 fluorescence microscope powered by a 100-W mercury lamp and equipped with a Hamamatsu ORCA-ER camera.RNA isolation About 50 mg sample was homogenized in 1 mL RNAiso Reagent and RNA was separated according to the manufacturer's instruction.The amount of the isolated RNA were measured spectrophotometrically,and the purity was checked by the OD260/OD280 values.Electrophoresis was done to make sure the isolated RNA had not degradeted.RNA was dissolved in 20μL of RNase-free H₂O.Reverse transcription The reaction system was composed of 2μl 5×PtimeScript Buffer,0.5μl PtimeScript RT Enzyme Mix I,0.5μlOligodTPrimer(50μM),0.5μl Random 6mers(100μM),500ng Total RNA,and proper RNase Free dH₂O to make sure the total amount is 10μl.All the reagents were added on the ice.Then the system reacted on a Biometra T1 Rt-pcr Instrument(Biometra)with a thermocycle of 37℃15min,85℃5sec and cDNA was stored at -20℃until use.Semi-quantative realtime-pcrβ-Tryptase appears to be the main form of tryptase [10]and we had proliferated its gene TPSB2.Dilute the cDNA by 20 times,and A 20-μL reaction mixture including 10μlSYBR Premix Ex Taq(2×),0.4μlROX Reference Dyeâ…¡,5μl primer(0.8μm),4μl diluted cDNA and 0.6μl dH₂O was added to the wells of the MicroAmp Optical 96-Well Reaction Plate(Applied Biosystems,US),shortly centrifuged(3,000 x g) and then reacted in7500 Real-Time PCR System(Applied Biosystems,US).Products were controlled by SYBR Green I dissociation curves and 2.5%agarose gel electrophoresis to ensure only one single targetspecific product was amplified.ACTB was chosen as a reference house-keeping gene.7500 PCR system software was used to estimate scar mRNA expression as well as the expressing differences of each sample relative to controls and based on an efficiency corrected mathematical model and a pair-wise fixed reallocation randomization test.Statistical analysisExperiments were carried out in triplicates.Data analysis was performed using SPSS10.0 software.Unless otherwise indicated,significance was determined at p＜0.01.ResultsFluorescence Immunohistochemistry Strong fluorescent signals were detected in the collagen fiber bundles of the keloids,espicially in the upper dermis.(figure6) Whereas less or weaker signals were detected in hypertrophic scars and skins.RNA isolation The OD260/OD280 values of the isolated RNA ranged from 1.8 to 2.0,means the high purity of the isolated RNA.Electrophoresis showed two bands of 18s and 28s,and 28s had a stronger fluor intensity,which means the RNA had not degradeted.Real time-pcr The proliferative curve run regularly.Only one hump appeared in the dissociation curve and 2.5%agarose gel electrophoresis showed only one single band of 155bp.(figure8) Both the keloid and hypertrophic scars expressed more tryptase genes than the controls(p＜0.01),and the keloids expressed more tryptase than the hypertrophic scars(p＜0.01).Averagely,there were 5.4- and 2.13-fold increase of tryptase gene in keloids and hypertrophic scars compared to the skins,and 2.5-fold increase in keloids compared to the hypertrophic scars. DiscussionKeloids exhibit 20 times the rate of collagen synthesis of normal skin and three times the rate of hypertrophic scars.In our research,strong fluorescent signals of tryptase were detected between the collagen fiber bundles of the keloids,and rt-pcr showed there were 5- and 1.7-fold increase of tryptase gene in keloids and hypertrophic scars compared to the skins,and 3-fold increase in keloids compared to the hypertrophic scars,considering that tryptase can be mitogenic for fibroblasts from various sources and stimulate collagen synthesis,we consider tryptase as a promoting factor of formation of keloids by stimulating collagen synthesis.