祛瘀生肌方通过Caspase信号通路促进糖尿病溃疡上皮化愈合的机制研究

Diabetes ulcer is one of the most serious complications of diabetes with less safe and effective treatment. Formula of Stasis Correction and Granulation Promotion is confirmed effective for the disease.We propose the hypothesis“ The key mechanism of Formula of Stasis Correction and Granulation Promotion promoting the production of diabetic ulcers epithelial healing is the Bcl-2 gene methylation regulation in Caspase signaling pathways”based on the preliminary results that the Formula inhibit the abnormal keratinocyte proliferation through cell apoptosis in Caspase signaling pathways.In vivo experiment,MSP,RT-PCR,immunohistochemical,Westernblot is used to observe diabetic ulcer animal model,KC Bcl-2 mathylation degree and Caspase3 gene expression,reveal the mechanism of Bcl-2 methylation activating caspase pathway apoptosis to regulate KC proliferation.In vitro experiment,siRNA interference and demethylation is used to observe KC morphology and function intervened by medical serum,verify the effects Bcl-2 methylation regulating Caspase pathway on KC apoptosis and proliferation.In all above,through research, to clarify the relationship between Formula of Stasis Correction and Granulation Promotion promoting the diabetic ulcers epithelial healing and the Bcl-2 methylation regulating the Caspase pathway.This study will further enrich our view of “removal of stasis is beneficial for the growth of new tissues ”.

糖尿病溃疡是糖尿病最严重的并发症之一，缺乏安全有效的治疗药物。课题组运用 “祛瘀生肌方”治疗本病临床疗效显著，在明确该方通过Caspase凋亡途径抑制角质形成细胞异常增殖的基础上，提出“祛瘀生肌方促进糖尿病溃疡上皮化愈合的关键环节是Bcl-2基因甲基化调控Caspase信号通路”的假说。在体实验运用MSP、RT-PCR、免疫组化、Westernblot，观察糖尿病溃疡愈合及角质形成细胞Bcl-2甲基化程度、Caspase3基因表达，揭示Bcl-2甲基化致Caspase通路活化进而调控角质形成细胞增殖的机制；离体实验运用siRNA干扰和去甲基化技术，观察经药血清干预的角质形成细胞形态功能，验证Bcl-2甲基化调控Caspase通路对细胞凋亡、增殖的影响。综上阐明祛瘀生肌方促进糖尿病溃疡上皮化愈合与Bcl-2甲基化调控Caspase信号通路的关系，丰富“祛瘀利于生肌”学术观点的科学内涵。

糖尿病溃疡是糖尿病最严重的并发症之一，缺乏安全有效的治疗药物。课题组运用 “祛瘀生肌方”治疗本病临床疗效显著，前期研究已明确该方通过Caspase凋亡途径抑制角质形成细胞异常增殖。通过本项目的研究，在体实验运用MSP、RT-PCR、免疫组化、Westernblot，观察到“祛瘀生肌方”组糖尿病溃疡愈合程度及角质形成细胞Bcl-2甲基化程度、Caspase3基因表达均高于对照组，揭示Bcl-2甲基化致Caspase通路活化进而调控角质形成细胞增殖的机制；离体实验运用siRNA干扰和去甲基化技术，观察到经“祛瘀生肌方”药血清干预的角质形成细胞增值凋亡，验证了Bcl-2甲基化调控Caspase通路对细胞凋亡、增殖的影响。综上，本研究阐明祛瘀生肌方促进糖尿病溃疡上皮化愈合与Bcl-2甲基化调控Caspase信号通路的关系，初步证实了“祛瘀生肌方促进糖尿病溃疡上皮化愈合的关键环节是Bcl-2基因甲基化调控Caspase信号通路”，为“祛瘀利于生肌”的学术观点提供了新的分子生物学证据，丰富了其科学内涵。