祛瘀生肌法调控创面胶原代谢的机理研究

This study is aimed at approaching to the mechanism of wound healing adjusting and controlling the metabolism of collagen by using the method of removing blood stasis and accelerating wound healing. .Method: We used modern medical techniques and methods such as imumunocytochemistry, molecular biology, morphology and Flow Cytometer(FCM) etc, in animal experiment and in vitro experiment respectively. Our study focused on the keystone "TGF -β1、TGF -β3? MMP-1? I、III collagen", investigated the synthesis and decomposition of collagen and the results of the intervention of the metabolism of collagen by using Chinese Shengji Huayu formula, in multiple links and multiple layers, during the process of would healing. The fibroblasts from granulation tissue in wound were cultured in vitro and compared with the normal skin fibroblast of suckling rat, which were incorporated with serum containing Shengji formula, Huayu formula, small and big doses of Shengji Huayu formula prepared by serum pharmacology respectively. The cell cycle distribution of fibroblasts were measured by Flow Cytometer; TGF -β1、TGF -β3、MMP-1、types I and III collagens of fibroblasts were determined with immunocytochemistry; the mRNA expression of type I and III collagens of fibroblasts were assessed by Northern blot hybridization; in addition the morphology of fibroblasts were observed under the optical and electron microscopes..Results: Animal experiment results :After observing the expression of TGF -β1、TGF -β3、 MMP-1、 type I、III collagen during the process of wound healing in 3 days,7 days,10days and 15days dynamically. We found that the amounts of TGF -β1 of Shengji Huayu group(big doses and small doses) are lower than that of the model group in third day of wound healing （p<0.05）;the amounts of TGF -β3 of Shengji Huayu group and huayu group are lower than that of model group in tenth day of wound healing（p<0.05）.At the same time, the inspection of the expression of mRNA of TGF -β1、TGF -β3、 MMP-1、 type I、III collagen shows consistency results. The observation of the tendency of the different dynamical index during the process of wound healing shows that Shenji Huayu formula may have the effects on regulating the speed of the metabolism and quality of the collagen of the wound .The results also have been proved by the examination of Flow Cytometer and morphology (under the optical and electron microscopes).In vitro experiment results :Shengji formula could promote the proliferation of fibroblasts and enhance the expression of α1(I) and α1(III) and increase the content of collagen I and III, which is significantly different from the control model fibroblasts（p<0.05）.In contrast, Huayu formula could inhibit the above-mentioned three parameters with significantly different from the control model fibroblasts（p<0.05）.Finally, Shengji Huayu formula has bidirectional regulation upon the three parameters. .Conclusion: This study confirms that the Chinese Shengji Huayu formula, which is the prototype of the method of removing stasis and accelerating wound healing, could directly intervene to the quality of the wound healing during the process of wound healing by regulating the metabolism of the collagen. This result fits to the Chinese medical theory "remove stasis benefits to accelerate wound healing, accelerate wound healing leads to skin restoration without scar", and also provides a scientific proof for the further using this methods in the clinical fields. Key words: Shengji Huayu formula , wound healing , metabolism of collagen

. . 本课题根据著名老中医顾伯华教授经验，在预初临床和实验研究基础希プ〈疵娼涸恢饕方?TGF-b→成纤维细胞→胶原酶→I、III型胶原"，观察祛瘀生肌法对大鼠创面修复过程中及体外培养成纤维细胞胶原代谢各环节的影响，阐明该法创面修复呈皮肤修复的机理，为中药创面修复和防治瘢痕形成提供理论指导。..调控.