The Impact From PAI-1-RNAi To Keloid Tissue And Transforming Growth Factors Of SD Rats

Objective: Keloid is a disease caused by excessive proliferation offibroblasts during wound healing which is caused by skin damage andexcessive deposition of extracellular matrix (ECM) at wound area. Previousresearch considered that high expression of PAI-1may participate the formingof keloid directly. In the mean time, transforming growth factor plays animportant role in the occurrence of keloid. This article applies smalldisturbance RNA technology to explore impact from PAI-1-RNAi to keloiodtissue and transforming growth factors of SD rats, which provides a new ideaof pathogenesis and therapy of keloid.Methods:1Establishment and grouping of SD rats back keloid model.(1) Choose amale SD rat.10%aldehyde anaesthetized. Remove some fur at back anddisinfect. Make a1.5cm radius round wound at both sides of spine. Wholeskins defect. Observe healing condition of wound every day.(2)After7days,use micro injector injects wound every other day. Inject high concentrationgroup0.05ml (concentration of laboratory reagent is20nmol/ml), lowconcentration group0.05ml (concentration of laboratory reagent is10nmol/ml)each time. Keloid group and normal group inject equivalent dose of salineuntil wound healed.2Use morphological change and average healing time to measurethickness of keloid. Use HE stain, Masson stain and Van-Gieson stain tocompare histological change of skin. Measuring TGF-β expression levelthrough immunohistochemical staining(SP method).Results:1Average healing time result: Average healing time of highconcentration group, low concentration group, keloid group, normal group is (21.15±1.46)d、(24.92±1.55)d、(27.38±1.19)d、(18.61±1.32)d. Statisticanalysis shows that healing time of normal group is the shortest（P＜0.01）.Healing time of high concentration group is obviously shorter than those oflow concentration group and keloid group（P＜0.01）. Healing time of lowconcentration group is obviously shorter than those of keloid group（P＜0.01）.Healing time of keloid group is the longest（P＜0.01）.（Table1）2Keloid measuring result: Results of high concentration group, lowconcentration group, keloid group and normal group are (1.70±0.07)mm、(1.98±0.07)mm、(2.66±0.06)mm、(1.25±0.05)mm. Statistic analysis showsthat keloid group is obviously larger than high concentration group and lowconcentration group（P＜0.01）.And the low concentration group is obviouslylarger than high concentration group（P＜0.01）. Normal group is the smallest.（P＜0.01）.（Table2）3HE stainDermis layer of keloid group became thick obviously, which is3-4timesthicker than surrounding normal dermis layer. Disorderly thick collagenousfiber can be observed. Collagenous fiber bundle formed large quantity oftuberculum-like structures. Large quantity of inflammatory cells infiltratedand telangiectasis among them.(Fig.2.4)Dermis layer of high concentration group became thick slightly. A few oforderly thick collagenous fiber can be observed. Tuberculum-like structuresare seldom observed. Inflammatory cells infiltrated and telangiectasis amongthem.(Fig.2.2)Dermis layer of low concentration group became thick. Disorderly thickcollagenous fiber can be observed. A few of collagenous fiber bundle formedtuberculum-like structures are observed. Inflammatory cells infiltrated andtelangiectasis among them.(Fig.2.3)Dermis layer of normal group became thick slightly. Collagenous fiberhas clear boarder, densely arranged collagen and smooth structure. Elasticfibers decreased. Inflammatory cells infiltration and telangiectasis are notobserved.(Fig.2.1) 4Masson stainMasson collagenous fiber stain result: collagenous fiber appeared blue.In keloid group, large quantity of disorderly thick deep blue collagenousfiber can be observed in dermis layer. Large quantity of spindle fibroblastsobserved.(Fig.3.4)In high concentration group, color of blue collagenous fiber is relativelylighter. Collagenous fiber arranged sparsely and orderly. Fibroblasts celldecreased.(Fig.3.2)In low concentration group, color of blue collagenous fiber is relativelydarker. Collagenous fiber arranged densely and disorderly. Fibroblasts cell canbe observed.(Fig.3.3)In normal concentration group, collagenous fiber arranged densely andorderly. A few of fibroblasts cell can be observed.(Fig.3.1)5Van-Gieson StainIn keloid group, a few of brown elastic fibers and thick collagenous fibermixed and arranged disorderly.(Fig.4.4)In high concentration group, a few of elastic fibers can be observed,which arranged orderly. The color of it is darker.(Fig.4.2)In low concentration group, a few of light brown elastic fibers can beobserved, which arranged sparsely and mixed with disordered collagenousfiber.(Fig.4.3)In normal group, brown elastic fibers arranged densely. The color of it isdarker.(Fig.4.1)6Immunohistochemistry result: TGF-β1has positive expression innormal skin, keloid epidermis and dermis. It is yellow or brown yellowparticle which is mainly located at basal layer cell of epidermis and fibroblastsof dermis(Fig.5). Judge result by two level scoring method. The comparisonof its positive expression level in four groups（Table3）.Positive rate ofTGF-β1in keloid group is82.61%, in high concentration group is50.00%, inlow concentration group is66.69%, in normal group is28.00%. The positiverate of four groups have significant difference(P=0.001<0．05).Positive expression of TGF-β in normal group is obviously lower than other groups.Positive rate of TGF-β1in keloid group is the highest. Along with increasingof reagent concentration, positive rate of TGF-β1decreased gradually.Conclusion:This research build rat back keloid model successfully. PAI-1-RNAiintervenes keloid and improves its thickness and healing time. Pathomorphismalso gets improved. In the mean time, positive rate which TGF-β1expressedin keloid group is obviously higher than normal group. Along with increasingof reagent concentration, positive rate of TGF-β1expressed in lowconcentration group and high concentration group decreased gradually, whichproves TGF-β1is an important cell factor participated in keloid forming.PAI-1may positively adjust the expression of TGF-β1.