| ForewordIt is a challenge problem to prevent and cure keloid in burn and plastic surgery. Keloids not only make patients disability and loss appearance, but also strongly hurt their heart. With the development of economy, more and more people pursue high quality live. There is no time to delay solving this problem. But the pathogeny of keloid is still not clear and lack of effective remedy. So explore pathogeny, prevention and cure method are a focus in burn and plastic surgery. With the study go deep, more and more medicaments are came in sight of us. China have the independence knowledge property of artemisinin, and domestic scientists are energetically empolderring it. Artemisinin and its ramification has the effect of antimalaria, but its efficacies are far more this. Recently studies show artemisinin have the ablity to restrain keloid fibroblast proliferation, but the mechanismis are not clear. This study want to explore the mechanism of antiproliferation effect to keloid fibroblast after treated by dihydroartemisinin through detecting apoptosis gene and protein in primary culture keloid fibroblast after dihydroartemisinin administered. This may have great meaning to enlarge the spectrum of artemisinin using and will inaugurate a new method to treatment keloid. ObjectiveTo explore bcl-2, p53, survivin and VEGF express in normal skin and keloid tissue. To cultivation human keloid fibroblasts in vitro for further reasearch. To investigate cell proliferation and cell apoptosis of keloid fibroblast induced by dihydroartemisinin. To explore bcl-2, p53, survivin and VEGF change and to investigate mechanism of antiproliferation and cell apoptosis of keloid fibroblast induced by dihydroartemisinin.Methods1. The expression of bcl-2, p53, survivin and VEGF in 14 cases of keloid and 14 cases of normal skin were detected by immunohistochemical assay.2. RPMI-1640 medium was the basic culture medium, being supplemented with fetal bovine serum (10%) using for culturing keloid tissue pieces attaching to the wall of culture flask. Fibroblasts were incubated at 37℃in a humidified atmosphere of 5%CO 2 and 95% air in the incubator. Growth kinetics, Cellular morphologies were observed, and the cultured cells were identified by viment in immunostaining.3. IC50 of dihydroartemisinin to keloid fibroblast was detected by MTT. Cell apoptosis was judged by TUNEL. Activation of caspase-3, caspase-8 and caspase-9 were tested by colorimetry.4. The expression of bcl-2, p53, survivin and VEGF in keloid fibroblast was detected by immunohistochemical assay. The changes of bcl-2, p53, survivin and VEGF gene were detected with RT-PCR and bcl-2, p53, survivin and VEGF protein were detected with Western blot.Results1. Positive rate of bcl-2, p53, survivin and VEGF expression in 14 cases keloid were 78.6%, 71.4%, 57.1% and 71.4% respectively. Among 14 cases of normal skin, positive rate of bcl-2, p53, survivin and VEGF were 14.3%, 21.4%, 14.3% and21.4% respectively. The difference of positive rate of bcl-2, p53, survivin and VEGF expression between two groups were statistical significance(P<0.05). 2. The keloid fibroblasts could grow in 7 to 14 days and proliferate in 48 to 65 days in vitro, and immuno taining of vimentin was positive in cultured fibroblasts.3. IC50 of dihydroartemisinin to keloid fibroblast was 150μmol/L. Obvious cell apoptosis was observed by TUNEL and caspase-3 and caspase-9 were activation in dihydroartemisinin groups.4. The mRNA level of bcl-2 and survivin decreased 39.8% and 58.8% respectively, its protein expression also reduced 32.2% and 40.4% respectively. There were almost no changes in p53 and VEGF mRNA level and protein expression.Conclusion1. The up-regulation of bcl-2, p53, survivin and VEGF in keloid suggested that bcl-2, p53, survivin and VEGF may play an important role in the formation of keloid through its antiapoptosis effect on fibroblast and endothelial cell.2. Human keloid fibroblasts can be cultured in stable condition in vitro, and can be used for further studies.3. Dihydroartemisinin induced cell apoptosis in keloid fibroblast, which may be related to the activation of caspase-9 and caspase-3.4. Dihydroartemisinin induced cell apoptosis in keloid fibroblast, which may be related to the down-regulated of bcl-2 and survivin.
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