| Purpose : To clarify the effect of Heibu ointment to the fibroblast histomorphology,the cell proliferation and apoptosis and the collagen synthesis of hypertrophic scar of rabbit ear animal modle,to make the mechanism clear that Heibu ointment remedy and prevent the hypertrophic scar,provide the theoretical and experimental basis for the external use of Heibu ointment. Material and method:Twenty-four white rabbits were randomly divided into normal control group, modle group and Heibu ointment treatment group .No disposition on the normal control group. The modle group and Heibu ointment treatment group were used to establish the modles of hypertrophic scar in ears.After twenty-one days,the modles were successful.Heibu ointment was plastered on the hypetrrophic scarring of Heibu ointment treatment group once every three days, it was continued fifty-six days. The material was respectively taken on the second,the fourth,the sixth and the eighth week in order to meassure the indicators.Visual inspecte the change of the skin and scars in the scar forming and therapeutic process; Observe the change of hypertrophic scars pathological morphology by HE staining technique with optical microscope,calculate the Hypertrophic Index and the fibroblastic density ; Observe the change of collagen in the scar tissue by VG staining technique calculate the collagen areal density; Measure the expression of proliferation cell nuclear antigen (PCNA) protein and apoptosis by immunohistochemistry technique, Observe the fibroblast proliferation with optical microscope, calculate PCNA positive expression rate; Situ test the apoptosis by TUNNEL technique, Observe the fibroblast apoptosis with optical microscope, calculate the apoptosis rate; Detect the expression of Bax,Bcl-2 mRNA by Real Time PCR technique; Detect the expression of type I and?collagen and Matrix Metallo Proteinases-1 mRNA by Real Time PCR technique; Detect the expression of Transforming growth factor-?1 by Western-blot technique. Results:1.Visual inspecte,after the surface of the wound formation,the wound unioned in twelve to fourteen days.In the course of union,the hard lumps can be touched in twenty to twenty-two days,the hard lumps are rosiness and ceaseless overtop the surface of skin,the hyperplasia scop didn't exceed the original wound scop, the thichness of the hyperplasia were two times as the mormal skin.The hyperplasia reached to the peak in the sixth weeks after epithelialization in the model control group,the thichness of the hyperplasia were three times as the mormal skin. In the Heibu ointment treatment group,as the time of treatment goes on,the scars were gradually soft,the size was gradually reduced , the hyperplasia was gradually flat and the color was gradually pale.2. Inspecte with optical microscope, in the model group, it could be seen that the corium tissues were obvious hyperplasia and thick ,it was made up of a great number of fibroblasts,collagen tissues and vessels.The collagen ranged irregularly,the distribution was nodositas and swirls,it didn't have obvious change as time goes on.But in the Heibu ointment treatment group,as the time of treatment goes on, the vessels, the fibroblasts and the collagens were gradually reduced.The collagens were light red,the range of the collagens was sparse and orderly.3. The effect of Heibu Ointment on the Hypertrophic Index in the hypertrophic scarring models in rabbit ears.Compared with the model group, the Hypertrophic Index was reduced in the Heibu ointment treatment group,from the fourth week of the epithelialization,two groups had significant difference(P<0.01).Moreover,as time goes on, the Hypertrophic Index was reduced more obviously.4. The effect of Heibu Ointment on the fibroblastic density in the hypertrophic scarring models in rabbit ears.The fibroblastic density of the model group was higher than the Heibu ointment treatment group, two groups had significant difference(P<0.01). On the fourth week of the epithelialization,the fibroblastic density was the highest, from then on ,it was gradually lower. The Heibu ointment could lower the fibroblastic density,compared with the model group ,they had significant difference(P<0.05)?5. The effect of Heibu Ointment on the collagen areal density in the hypertrophic scarring models in rabbit ears.The collagen areal density of the model group was obviously higher than the Heibu ointment treatment group, two groups had significant difference(P<0.01). On the sixth week of the epithelialization,the collagen areal density was the highest,from then on,it was gradually lower. The Heibu ointment could lower the collagen areal density,compared with the model group ,they had significant difference from the fourth week of the epithelialization(P<0.01).6. The effect of Heibu Ointment on the PCNA in the hypertrophic scarring models in rabbit ears.Measure the PCNA by immunohistochemistry technique. The result showed: The PCNA positive expression was in the epidermal basal cell layer of the normal skin,it was very little in the corium layer.In the model group, The PCNA positive expression was in the basal cell layer,in the granular cell layer and a great deal in the corium layer,there was significant difference between the model group an the mormal control(P<0.01). The PCNA positive expression of was lower than the Heibu ointment treatment group ,and the two group had significant difference(P<0.05).7. The effect of Heibu Ointment on the apoptosis in the hypertrophic scarring models in rabbit ears.Measure the apoptosis by TUNNEL technique. The result showed: The apoptotic cells could be seen in the fibroblasts and vascular endothelium.There were a few positive cells in the normal skin.The apoptosis rate of the model group didn't have obvious difference with the normal group from the second to the fourth week of the the epithelialization. The apoptosis rate of the model group began to reduce from the sixth week of the the epithelialization, as the time goes on of the epithelialization,the reduction was more obvious,but the difference between two groups didn't have statistical significance(P>0.05). The apoptotic cells of the Heibu ointment treatment group were more increased than the model group.It could be seen a lot of apoptotic fibroblasts,the apoptosis reached to the peak on the sixth week of the epithelialization,it was still very high on the eighth week of the epithelialization. The apoptosis rate of the Heibu ointment treatment group and the model group had significant difference(P<0.01).8. The effect of Heibu Ointment on the expression of the Bax mRNA in the hypertrophic scarring models in rabbit ears.Detect the expression of the Bax mRNA by Real Time PCR technique.The result showed: The expression of the Bax mRNA of the model group was lower than the normal control group ( P<0.05 ) , moreover,as the time goes on of the epithelialization,the reduction was more obvious,the difference between two groups was more significant from the sixth week of the epithelialization(P<0.01). The expression of the Bax mRNA of the Heibu ointment treatment group was higher than the model group, two groups had significant difference(P<0.01).Moreover,as the time goes on of the epithelialization,the rise was more obvious.9. The effect of Heibu Ointment on the expression of the Bcl-2 mRNA in the hypertrophic scarring models in rabbit ears.Detect the expression of the Bcl-2 mRNA by Real Time PCR technique.The result showed: The expression of the Bcl-2 mRNA of the model group was higher than the normal control group, two groups had significant difference(P<0.01), moreover,as the time goes on of the epithelialization,the rise was more obvious. The expression of the Bcl-2 mRNA of the Heibu ointment treatment group was lower than the model group, two groups had significant difference from the fourth week of the epithelialization ( P<0.01 ) .Moreover,as the time goes on of the epithelialization,the reduction was more obvious. 10. The effect of Heibu Ointment on the expression of the type I collagen mRNA in the hypertrophic scarring models in rabbit ears. Detect the expression of the type I collagen mRNA by Real Time PCR technique. The result showed: The expression of the type I collagen mRNA of the model group was higher than the normal control group, two groups had significant difference(P<0.01), moreover,as the time goes on of the epithelialization,the rise was more obvious. The expression of the type I collagen mRNA of the Heibu ointment treatment group was lower than the model group, two groups had significant difference from the fourth week of the epithelialization(P<0.01).Moreover,as the time goes on of the epithelialization,the reduction was more obvious.11. The effect of Heibu Ointment on the expression of the type?collagen mRNA in the hypertrophic scarring models in rabbit ears.Detect the expression of the type?collagen mRNA by Real Time PCR technique.The result showed: The expression of the type?collagen mRNA of the model group was lower than the normal control group, moreover,as the time goes on of the epithelialization,the reduction was more obvious. Two groups had significant difference from the sixth week of the epithelialization(P<0.01). The expression of the type?collagen mRNA of the Heibu ointment treatment group was higher than the model group, two groups had significant difference(P<0.01).Moreover,as the time goes on of the epithelialization,the rise was more obvious.12. The effect of Heibu Ointment on the expression of the Matrix Metallo Proteinases-1 mRNA in the hypertrophic scarring models in rabbit ears.Detect the expression of Matrix Metallo Proteinases-1 mRNA by Real Time PCR technique.The result showed: The expression of the Matrix Metallo Proteinases-1 of the model group was lower than the normal control group, two groups had significant difference ( P<0.01 ) .Moreover,as the time goes on of the epithelialization,the reduction was more obvious. The expression of the Matrix Metallo Proteinases-1 mRNA of the Heibu ointment treatment group was higher than the model group, two groups had significant difference(P<0.01).Moreover,as the time goes on of the epithelialization,the rise was more obvious. 13. The effect of Heibu Ointment on the expression of the Transforming growth factor-?1 protein in the hypertrophic scarring models in rabbit ears.Detect the expression of the Transforming growth factor-?1 protein by Western-blot technique.The result showed: The expression of the Transforming growth factor-?1 of the model group was higher than the normal control group, two groups had significant difference(P<0.01).Moreover,as the time goes on of the epithelialization,the rise was more obvious. The expression of the Transforming growth factor-?1 of the Heibu ointment treatment group was lower than the model group, two groups had significant difference from the fourth week of the epithelialization(P<0.05), the difference was more significant from the sixth week of the epithelialization(P<0.01).Moreover,as the time goes on of the epithelialization,the reduction was more obvious.Conclusion:1.Heibu ointment could obviously inhibit the fibroblast proliferation and reduce the content of the collagen synthesis,it could reduce the Hypertrophic Index, the fibroblastic density and the collagen areal density significantly,consequently it could obviously inhibit the proliferation of hypertrophic scar tissue.2. Heibu ointment could obviously reduce the PCNA positive expression rate , increase the apoptosis rate , reduce the expression of Bax mRNA and increase the expression of Bcl-2 mRNA in the hypertrophic scar of rabbit ear. Heibu ointment could obviously restrain the fibroblast proliferation and, induce apoptosis in the hypertrophic scar.3. Heibu ointment could obviously reduce the expression of type I and?collagen mRNA, increase the expression of MMP-1 mRNA and reduce the expression of TGF-?1 in the hypertrophic scar of rabbit ear. consequently it could obviously reduce the content of the collagen and increase the content of the collagenase. |
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