回阳生肌膏对人慢性皮肤溃疡创缘成纤维细胞增殖调控的影响

【作者】 张颖；

【导师】 蒋玉凤； 李萍；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2005， 硕士

【摘要】 慢性皮肤溃疡是临床常见病,可继发于很多疾病,治疗十分棘手。到目前为止,其发病机理仍在探索之中。在针对这种修复失控（修复不足）的众多研究中,成纤维细胞（FB）以其在修复中扮演着不可或缺的重要角色而成为关注、研究的焦点。中医药对此类疾病具有确实疗效,认为本病最终往往发展为久不愈合的阴证创疡,治疗以回阳生肌。本实验即以人皮肤成纤维细胞（HFB）为切入点,选用回阳生肌膏,从细胞分子生物学角度深入研究其作用机制。 本文首先对HFB增殖调控与慢性皮肤溃疡及外用生肌方药治疗慢性皮肤溃疡的研究现状进行了简要综述。在了解研究现状的基础上进行本实验。 实验分为两部分:首先,观察回阳生肌膏水及醇提取物对正常皮肤成纤维细胞（nHFB）和慢性皮肤溃疡创缘成纤维细胞（uHFB）增殖的影响,然后初步探讨它们对uHFB增殖调控的机制。实验结果如下: 首先建立慢性皮肤uHFB细胞系及nHFB细胞系。在倒置相差显微镜下观察两种细胞,形态差别较大:uHFB体积大,胞体薄,分支多,形状不规则,nHFB则体积小,胞体丰满晶亮,呈梭形或多边形,分支少,生长旺盛,排列呈漩涡或火焰状。通过对两种细胞生长曲线及群体倍增时间的测定,显示uHFB较nHFB生长缓慢,倍增时间延长22.79h。 采用alarmar blue摄入法检测回阳生肌膏水及醇提取物对uHFB和nHFB增殖的影响。结果显示,水提物在0.075～2.4 mg/L浓度范围内对nHFB具有促增殖作用（P<0.05或P<0.01）,在0.15～1.2 mg/L浓度范围内对uHFB具有促增殖作用（P<0.05或P<0.01）;醇提物在0.027～0.425 mg/L范围内对nHFB具有促增殖作用（P<0.05或P<0.01）,对uHFB则在0.027～0.213 mg/L范围内有明显促增殖作用（P<0.05或P<0.01）。两种提取物作用于nHFB和uHFB后,均能在一定浓度范围内促进两种细胞的增殖。其中水提物1.2mg/L、醇提物0.213mg/L作用最为明显,因此作为后续各实验的实验浓度。 体外细胞伤口实验是体外细胞迁移和增殖能力总和的体现。建立体外uHFB和nHFB细胞伤口模型,以nHFB形成的伤口为对照,观察回阳生肌膏水及醇提取物对体外细胞伤口愈合的影响。结果显示,实验中uHFB细胞伤口的愈合速度较nHFB创口模型显著减慢,各时间点与nHFB组有显著差异,分别P<0.01;回阳生肌膏水及醇提取物均能显著提高两种细胞伤口的愈合速度;但uHFB用药组仍不及nHFB用药组愈合速度快;并且实验发现醇提物比水提物作用更明显。 在确定回阳生肌膏水、醇提取物具有促进HFB增殖的作用后,对其作用机制进行初步探讨。 （1） 采用流式细胞仪检测回阳生肌膏水、醇提取物对uHFB和nHFB细胞周期的影响:结果显示,作用24小时后,醇提物能够增加nHFB的S期、G₂/M期比例（P<0.01,P<0.05）及增殖指数（Proliferation index, PrI）（P<0.01）,增加uHFB的S期比例及Prl指数（P<0.01）;水提物能够明显增加nHFB的S期比例及PrI指数（P<0.01）,对uHFB的S期、更多还原

【Abstract】 Chronic ulcer of skin (CUS), a common clinical disease, can be secondary to many diseases and be treated difficutly. CUS is a kind of impaired wound, and the healing is out of control. Up to now its pathogenesis is still unknown clearly. Fibroblast, playing an important part in wound healing is becoming a research focus. Chinese medicine has a credible curative effect on CUS. According to Chinese medicine theory, at the delayed healing stage of CUS, the differentiaton is mainly belonging to" Yin", opposite to "Yang", and the principle therapy is improving tissue regeneration by Huiyang therapy, In this study, human skin fibroblasts are observed to explore the mechanism of HYSJ by the method cytology and molecular biology in case of offering evidences for the theory of Chinese medicine improving tissue regeneration.The paper has two parts. The first one is composed of two reviews including proliferating regulation of human dermal fibroblast in CUS, and current situation of therapeutics of topical medicine for CUS. Then on the basis of these studies, we suppose Chinese medicine "Huiyang Shengji Gao (HYSJG)" improving fibroblast proliferation by modulating related transcription factors expression to improve cell cycle.Firstly we observed effects of alcohol and aqueous extracts from HYSJG on proliferations of uHFBs and nHFBs. And then we carried out several pilot studies to confirm our supposal by study cell cycle and expressions of proliferation cell nuclear antigen (PCNA), c-Fos and c-Myc.uHFBs were obtained from wound margins from amputated extremity of CUS patients and nHFBs as control from dital end of same patient. Morphologic characteristics and biological features of both cells were compared by using phase-contrast microscope, growth curve and multiply time of colony. uHFBs were larger and polygonal or abnormal in shape, with thinner cytoplasm and more branches. nHFBs were smaller and fusiform or polygonal in shape, with plump and bright cytoplasm and less blanches. The growth curves suggested that uHFBs grew slower than nHFBs and the multiply time of the colony of uHFBs is 22.79 hours longer than nHFBs.The effects of the extracts from HYSJG on proliferations of uHFBs and nHFBs were measured by alarmar blue assay. The results showed that the proliferations of the two cell lines were both elevated after cultured with aqueous extract and alcohol extract from HYSJG for 24 hours. For alcohol extract, obvious proliferation-promoting concentrations on nHFBs and uHFBs were among 0.027~0.425mg/L and 0.027~0.213 mg/L, respectively (P<0.05 or P<0.01). For aqueous extract, they were among 0.075~2.4 mg/L and 0.15-1.2 mg/L, respectively (P<0.05 or P<0.01). The proliferation rates of both cell lines worked by these two extracts were similar (P>0.05, respectively). The most obvious concentrations (alcohol extract of 0.213mg/L, aqueous extract of 1.2mg/L) were used in the next experiments.Cell wound experiment in vitro embodies the motility and proliferation abilities of cultured cells. Two cell wound models were established using uHFBs and nHFBs in vitro.The effects of the two extracts from HYSJG on wound healing were determined by means of time-lapse photo-images. The result showed that the healing rate of uHFBs wound was slower than that of nHFBs. Even addition with extracts of HYSJG, the migration rate of uHFBs was slower than that of nHFBs. These two extracts could both improve the healing rate of the two ceil wounds. Alcohol extract had better effect than aqueous extract.After confirming the pro-proliferating effects of HYSJG on uHFBs and nHFBs, several pilot studies were made for exploring the possible mechanisms involving in proliferation.Flowcytometry were used for cell cycle analysis. After incubation 24 hours, for nHFBs, alcohol extract of HYSJG could enhance S phase, G2/M phase (P<0.01, P<0.05 versus nHFBs control) and cell proliferation index (PrI, S+ G2/M) (P<0.01, versus nHFBs control). As well as for uHFBs, alcohol extract could increase the percentage of S phase and PrI dramaticaly (P<0.01, versus uHFBs control),更多还原