The Influence Of Connective Tissue Growth Factor On The Expression Of â… ,â…¢ Collagen In Regeneration And Scaring Of Wound Healing

Objective There are two primary ways for wound healing after trauma,one is regeneration and the other is scaring healing.Content of the CTGF,collagen type I and collagen type III vary according to different healing ways.However.in the course of skin regeneration and scaring healing,all of the three types belong to sequential expressions,while the direct application of CTGF to the wound healing as well as the expression of collagen type I and collagen type III are rarely reported.This research aims to test sequential expression differences of CTGF,collagen type I and collagen type III in their process of in regeneration and Wound Healing and also, detect the influence of CTGF on the expression of 1,111 Collagen in regeneration and wound healing,so as to apply this result to practical operations.Materials and methods1. A total of fifty Japanese white rabbits(purchased from Veterinary Research Institute in Gansu Province),both male and female ones with an average weight of 2.5-3.0 kg.After been adaptive fed for 7 days in single cage,these samples were then divided randomly into two groups: CTGF group and CTGF antibody group.25 rabbits were distributed in each of the two groups,where there are blank controllers,also the NS group.The ear ventral fur were removed from ears by 5% sodium sulfide.Afterwards,anesthesia the subject with 2% sodium pentobarbital (1.5ml/kg)through ear vein,then establish a 10.0mmx 10.0mm wedge wound in every ear ventral skin with a knife.The wounds directions from the superficial to deep were choose randomly,the deepest layer reached down as far as cartilage layer.2. Each of the left ear wound in the group of CTGF was injected rhCTGF whose concentration is 100ng/0.1ml with 30G needle,and once in every other day until the wound healed.All right ears wounds were injected normal saline as a blank control.Each of the left ear wound in the group of CTGF was injected rhCTGF whose concentration is 100ng/0.1ml with 30G needle,and once in every other day until the wound healed.All right ears wounds were injected normal saline as a blank control.All ears wound were covered sterile materials and fixed volume.3. The situation of wound healing was observed and took photo as record.Five rabbits were selected randomly at 0 d,4 d,8 d,12 d and 30 d after injured.The two groups specimens were cut from the left ear with five specimens respectively,then chose randomly five right specimens from all ten right ears as contrast.Fixed specimens with 10% neutral formalin,dehydrated after 24h,embedded and sliced in paraffin.Use the Hematoxylin-eosin staining(HE) and immunohistochemistry(IHC) to detect the expression of CTGF,collagen type Ⅰ,type Ⅲ.4. Observing the morphological features and expression of CTGF,collagen type Ⅰ and type Ⅲ in the superficial or deep wound healing process.The slice was divided into 10 segments from deep to superficial, lmm width in every part.The distance to the superficial edge of slice is 0-4mm belongs to the superficial wound,and the distance to the superficial edge of slice is 6-10mm belongs to the deep wound.Every segment was chose randomly five horizons under a microscope at 400 times,then measure the average optical density values vision with Image-Pro-plus 6.0 software.According to the photos of wound healing area calculated wound healing area with Image-Pro-plus 6.0 software,and calculated the rate of wound healing.Analyze the date with SPSS 19.0,using analysis of variance when the number of homogeneity of variance among groups were compared; if not,use the Wilcoxon test were used when the heterogeneity of variance,and when P <0.05,it is considered statistically significant.Results1. The superficial wounds had achived regeneration at 8th day after injury,while the deep wounds significantly reduced and scared at 12 day,and on the 30 day after injury the scar was still obvious.The wound healing was (36.4+6.3)% at 4thd,(81.6±5.8)% at 8thd,and (92.2±5.3)% at 12th d when used rhCTGF,all of which were higher (30.3±5.6)%,(69.8±4.7)% and (85.3± 5.2) in the control group(P<0.05).The wound healing was(58.6±6.9)% at 8th d,(76.8±6.9)% at 12th d when used CTGF antibody.lower respictivly the control group(P<0.05),but nearly equal at 4thday(P>0.05).2. At any points,the expression of the deepen ear wounds are all higher than the shallow ones(P<0.05),but the expression pattern of both sides are generally identical.In the forth day it reached higher(P<0.05) and the eight day ascent to its top(P<0.05).In the twelfth day it began slowed down,the deepen side was still above the average tissue(P<0.05),whilst the shallow side returned to normal level.To the 30th d,content of CTGF in wound equaled to the normal tissue(P>0.05).3. In deep wounds,the expression of collagen type I were higher than superficial side at each time(P<0.05).The expression pattern nearly identical during the first 12 d between deep and superficial woundsrincreased gradually at 4rd d,reached the peak at 8th d,and decreased at 12th d in superficial side,but again increased at 30th d in the deep side(P<0.05).The levels of collagen type 1 in the group of CTGF always higher than the control group,while CTGF antibody group lower than the control group(P<0.05).4. In superficial wounds the expression of collagen type III higher than deep wounds at each time(P<0.05).In the superficial wounds,the levels of collagen type III keep increase until 30th day.In the deep wounds,the expression of collagen type III increased gradually at 4rd day,reached the peak at 8th day,then decreased from 12th day,but still higher than the normal tissue at 30th day(P<0.05).The levels of collagen type III in the group of CTGF always higher than the control group,while CTGF antibody group lower than the control group.5. In deep wounds,both the rate between collagen type I and collagen type III were higher than superficial side at each time(P<0.05).The expression pattern nearly identical during the first 12 days between deep and superficial wounds increased gradually at 4rd day,reached the peak at 8th day,and decreased at 12th d in superficial side.But again increased at 30th d in the deep side(P<0.05).The levels of the rate between collagen type I and collagen type Ⅲ in the group of CTGF always higher than the control group,while CTGF antibody group lower than the control group(P<0.05).Conclusions1. In deep wounds which achieved regeneration healing,the levels of CTGF,collagen type I,the rate between collagen type I and collagen type Ⅲ are higher than the superficial wounds which achieved scar healing,while the expression of collagen type III were exactly at the opposite point.2. CTGF can promote wound healing and increases type Ⅰ,Ⅲ collagen expression and Ⅰ/Ⅲ collagen proportion in the skin wounds of rabbit ear.