| 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),... |
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