| BackgroundKeloids are benign dermal fibroproliferative tumors unique to humans. They are defined as abnormal scars growing continuously and invasively beyond the confines of the original wound. The continuous proliferation of fibroblast and excessive accumulation of extracellular matrix (ECM) proteins should be the key features of keloids. Keloids, which can lead to dysfunction, affect appearance of patients, and accompanied by local symptoms, for instance itch and pain.There are many abnormalities of keloid fibroblasts when compared with the normal dermal fibroblasts.①Keloid fibroblasts over express fibronectin and type I procollagen, that leads to overproduction of extracellular matrix. And there is reduced degradation of procollagen poly peptides.②Keloid fibroblasts show reduced growth factor requirements for proliferation. Cell culture studies with keloid-derived fibroblasts demonstrated an altered cytokine expression, such as an increased expression of transforming growth factor (TGF)-β1, TGF-β2, and vascular endothelial growth factor (VEGF). There possibly are elevated levels of platelet derived growth factor (PDGF)-a receptors.③Another abnormalitie of keloid fibroblasts is dysregulation of apoptosis, which is a important factor related to keloid formation may be. Keloid fibroblasts fail to undergo physiologically programmed cell death, and the ratio of apoptosis is lower than normal fibroblast’s.There are several treatment modalities which are useful for the management of keloids, though no single modality is completely effective. The most commonly used modalities are surgical excision, radiotherapy, lasers, intralesional steroids, silicone gel sheet, and5-fluorouracil (5FU). They may be used either singly or, as is done more commonly, in combinations.Radiotherapy, lasers, intralesional steroids, and5-fluorouracil (5FU) can induce the apopotosis and inhibit the proliferation of keloid fibroblasts. But there is a research hold that current therapies are only partially effective because they either induce senescence in too few cells or in enough number of cells, but at the same time inducing death (apoptosis or necrosis) of other cells which are probably the source of a new repair cycle (proliferation). Although the molecular mechanisms of pathogenesis in keloids have not yet been cleared up, controlling of fibroblasts proliferation and apoptosis might be considered as an effective method of keloid therapeutics.Recently, there is a research found that compound Astragalus and Salvia miltiorrhiza extract (CASE) inhibits cell proliferation, invasion and collagen synthesis in keloid fibroblasts by mediating TGF-β/Smad pathway.Salvia miltiorrhiza is one of the main compositions of CASE. Tanshinone (Ⅰ, Ⅱ A, ⅡB) and cryptotanshinone (Ⅰ, Ⅱ) can be isolated in Salvia miltiorrhiza. Tanshinone Ⅱ A, which is characterized by an ortho-quinone C-ring, has many potential therapeutic functions.From recent studies, researchers found that tanshinone ⅡA induce apoptosis and inhibit proliferation of many kinds of tumor cells, such as leukemia THP-1cell, hepatocellular carcinoma cells, HeLa cells, breast cancer cells, colon adenocarcinoma cells, and so on. And there was research found that tanshinone ⅡA can inhibit hypertrophic scar of rabbit ear. Other research found that tanshinone ⅡA effectively inhibit benign stricture of bile duct fibroblasts proliferation and collagen type I secretion, and tanshinone ⅡA can induce the expression of MMP-9mRNA, promote collagen degradation, thereby inhibiting the formation of bile duct scar. Tanshinon ⅡA may have same effect on keloid fibroblast.Keloids, one kind of pathologic scar, are unique to humans. There is no perfect animal model, which make the research for keloids harder. Animal models were described by animals and scientific artifices to cause keloids. They were divided into:①Keloid induction in animals;②Keloids implant in immunodeficient animals;③Building animal models of keloid by using the method of tissue engineering.The animal models built by keloids implant in immunodeficient animals (athymic mice) were firstly reported by Shetlar. Then Estrem and Kischer and their mates conducted some similar researches, and they succeeded too. Following this trend, some researchers from our country use these animal models in their studies, and they verified the feasibilities of the models. More than that, implantation of human keloids into athymic mice sustains true viability and morphology of the keloids, and is a relatively ideal animal model to study keloids.ObjectiveTo investigate effect of Tanshinone ⅡA on cell proliferation, apoptosis and cell cycle of fibroblasts derived from keloids. Build the animal models by implantation of human keloids into athymic mice, and investigate effect of Tanshinone ⅡA on keloids, especially the histological appearance and cell apoptosis.Method1. Keloid tissues were taken from seventeen patients who underwent surgical excision at Shanghai Jiaotong University of Shanghai Ninth People’s Hospital. Preoperative clinical diagnosis is the same as postoperative pathologic diagnosis, both are keloids. All keloid samples obtained in this study had not been previously treated and were not recurred cases.2. Fibroblasts derived from keloid tissues were maintained in DMEM. Only cells from passages one to two were analyzed in this study.3. Fibroblasts cultured with different concentration of Tanshinone ⅡA (Oug/ml,50ug/ml,100ug/ml,200ug/ml) in vitro. To examine cell proliferation, cell apoptosis, and cell cycle at the different time.4. To produce standard curve and examine cell prolieration by CCK-8.5. To analyze cell early apoptosis by flow cytometry and Annexin V-FITC/PI doubled staining. 6. To analyze cell cycle by flow cytometry and PI staining.7. The animal model was built by implanting human keloid into athymic mice. Each of the keloids is8mm×5mm×5mm8. Except3athymic mice which were killed and exmanined on16th day after implantation, other12athymic mice were randomly divided into two groups. One group is experimental group, which is intraperitoneally treated with5mg tanshinone Ⅱ A. The other is control group, which is intraperitoneally treated with normal saline.9. The keloid model was evaluated according to morphologic changes and histology.10. Keloids were examined by HE staining and Masson staining.11. Cell apoptosis was examined by Tunel12. Spss13.0was used in statistical analysis. All data are expressed as mean±standard deviation (SD). Analysis of variance of factorial design was used in comparisons of cell proliferation, cell early apoptosis, and cell cycle G0/G1phase.All experiments were performed in triplicate and were repeated at least three times. Two independent sample t test was used to analyze a statistical significance of keloids volume. A P value<0.05was considered significant.Result1. Standard curve was produced. R2=0.96, regression equation is Y=-359.51+15211.73X (Y is the number of fibroblasts, X is OD value). And this regression equation has statistics significance. So the OD value can reflect the actual number of fibroblast cells. Cell proliferation of experiment group is inhibited with varying degrees. Group of200ug/ml is obviously inhibited after72hours,96hours and120hours. Showed by statistics analysis, the effects from concentration and treating time both both are statistically significant, as well as interaction between them.2. Analysis from flow cytometry and Annexin V-FITC/PI doubled staining, early apoptosis rate of experiment group increased significantly. The rate of200ug/ml group is most obviously increased after120hours, and mean is41.62%, the increased early apoptosis percentage is1030.98%. Showed by statistics analysis, the effects from concentration and treating time both both are statistically significant, as well as interaction between them.3. Analysis from flow cytometry and PI staining, G0/G1phase rate of experiment group is increased with varying degrees. The rate of200ug/ml group is most obvious after120hours, and mean is94.16%, the increased G0/G1phase percentage is25.33%. Showed by statistics analysis, the effects from concentration and treating time both both are statistically significant, as well as interaction between them.4. There is no rejection in all of animal models. Examining all volume of keloids, mean of the experiment group is364.33±50.17mm3, mean of the control group is392.33±32.26mm3. Showed by statistics analysis, the difference is no statistically significant.5. Examining all keloids by HE staining and Masson staining, there is no rejection, and the keloids sustains true viability and morphology of the lesions. Experiment group comparing control group, there is no obvious difference found in HE staining and Masson staining.6. Examining by Tunel, there is almost no apoptosis cell in the control group, except some ones in epidermis layer. In contrast, there are many apoptosis cells in the experiment group.ConclusionTanshinon ⅡA can control fibroblast derived from keloid by suppressing cell proliferation, inducing cell apoptosis and arresting cell cycle. Implantation of human keloids into athymic mice sustains true viability and morphology of the keloids, and is a relatively ideal animal model to study keloids. Keloid animal models intraperitoneally treated with tanshinone ⅡA, there is no obvious difference in gross appearance and hyphology, although tanshinone ⅡA can induce many cell apoptosis in the keloids. |
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