| Object: keloid is an inevitable result of tissue repair in wound healing.Therefore,inhibiting keloid formation has always been a clinical difficulty.However,the donkey skin contains a large amount of collagen fiber,which produces a large amount of collagen when the wound is healed,but its wound will not form hyperplastic keloid because of the excessive deposition of collagen.At present,sodium sulfate and 5-FU were added to donkey skin fibroblasts cultured in vitro to screen the appropriate action concentration and time.After that,the model of wound healing was established on the skin of Xinjiang donkey,and sodium sulfate was applied and injected with 5-FU,to explore the effect of these two drugs on granulation wound healing,and the regulatory mechanism affecting collagen synthesis expression was preliminarily analyzed from the molecular point of view.Methods:(1)Donkey skin fibroblasts were cultured in vitro by tissue block method and identified by HE、Masson、 immunochemical staining,and then cultured in vitro with high concentration group(2000 μg/m L、1500 μg/m L),medium concentration group(1000 μg/m L、500 μg/m L、250 μg/m L)and low concentration group(100 μg/m L、50 μg/m L、10 μg/m L)sodium sulfate and 5-FU culture medium.The RT-q PCR、ELISA method was used to detect the expression and protein concentration of collagen 5-FU in donkey skin fibroblasts.(2)Six Xinjiang female donkeys were selected to establish a donkey skin wound model.Six round wounds with a diameter of 1.5 cm were taken from each donkey along both sides of the back spine.The distance between the wounds was more than 30 cm,three of the ipsilateral wounds were in the treatment group and three on the other side were in the control group.the measured at 0 d、7 d、17 d、100 d after trauma,and the wound healing rate was calculated.HE staining was used to observe the histological changes of the wound.Masson staining was used to observe the proliferation of collagen fibers in the wound.RT-q PCR、ELISA method was used to detect the difference of expression of TGF-β some key genes in signaling pathways of smad7、smad3、COL3A1、COL1A1、COL1A2 and TGF-β1 genes in the donkey wound skin.Results:(1)After 5 d of donkey skin fibroblast culture,it began to crawl out of the edge of the tissue block,and after 25 d it was covered with the whole petri dish.and the proliferation rate of fibroblasts was significantly higher in the medium concentration group than in the high and low concentration group(P<0.05).Accordingly,the apoptosis rate in the medium concentration group was significantly lower than in the other two groups(P<0.05).compared with 24 h,sodium sulfate acting on fibroblasts 48 h can significantly increase the expression of collagen synthesis related genes(P<0.05),250 μg/m L concentration is the best.(2)The inhibition rate of fibroblast proliferation in the high and medium concentration group was significantly higher than that in the other group(P<0.05),and the apoptosis rate in the low concentration group was significantly lower than that in the other two groups(P<0.05).Compared with 12 h,5-FU acting on fibroblasts 24 h can significantly inhibit the expression of collagen synthesis related genes(P<0.05),100 μg/m L concentration is the best.(3)Compared with the control group,the sodium sulfate group accelerated the healing speed of donkey skin wounds and shortened the wound healing time at the granulation stage.The wound healing rate of the sodium sulfate group was significantly higher than that of the control group on 7 d and 17 d(P<0.05),there were keloids on the surface of sodium sulfate group at 100 d;after HE and Masson staining of wound tissues,the collagen structure of sodium sulfate group is dense,the fiber bundle is thick;the sodium sulfate group The relative expression levels of smad3,COL3A1,COL1A1,COL1A2 and TGF-β1 genes were significantly increased(P<0.05),and the smad7 gene was significantly decreased(P<0.05);the protein concentrations of COL1A1,COL1A2 and TGF-β1 in the sodium sulfate group were also significantly higher In the control group(P<0.05).(4)Compared with the control group,the wounds in the 5-FU group during the granulation stage contracted more slowly,and the healing rate was smaller.At 100 d,the surface of the 5-FU group had no keloids;after HE and Masson staining,the 5-FU group had collagen density decreased,the gaps became wider,and the fiber bundles were arranged in an orderly manner;the relative expression levels of smad3,COL3A1,COL1A1,COL1A2,and TGF-β1 in the 5-FU group decreased significantly(P<0.05),the relative expression of smad7 increased significantly(P<0.05);the protein concentrations of COL1A1,COL1A2,and TGF-β1 in the 5-FU group were also significantly lower than those in the control group(P<0.05).Conclusion:(1)The 48 h,donkey skin fibroblasts of cultured in 250 μg/m L sodium sulfate medium could significantly increase the cell proliferation rate and promote collagen synthesis.(2)5-FU Culturing 100 μg/m L and 24 h,can effectively inhibit the proliferation of fibroblasts and collagen synthesis.(3)The application of sodium sulfate on donkey skin wounds can speed up wound healing and shorten the time of wound healing in granulation stage,but after healing,keloids are formed,and up-regulated the relative m RNA expression and protein concentration of genes related to the TGF-β/smad signaling pathway(4)Injection of 5-FU into donkey skin wounds can prolong the healing time of wounds in the granulation stage,but does not form keloids after healing,and down-regulate the relative m RNA expression and protein concentration of genes related to the TGF-β/smad signaling pathway.This experiment provides reference basis for the study of wound healing mechanism and keloid. |
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