摘要
目的利用原代培养的人外阴正常皮肤成纤维细胞,观察中药对人外阴皮肤成纤维细胞增殖的影响。方法胰蛋白酶消化、分离及培养人外阴皮肤成纤维细胞;倒置显微镜下观察成纤维细胞生长状态;HE染色观察细胞爬片下的形态及着色;免疫组织化学染色观察成纤维细胞中间丝波形蛋白;测定细胞生长曲线;MTT法测定莪术、黄芪、丹参、川穹及当归5种中药作用后细胞增殖能力。结果生长曲线测定结果显示:传6代内以及传6代内复苏人外阴皮肤成纤维细胞增殖能力均很强。MTT检测结果显示:5种药物在特定浓度范围内均能抑制人外阴皮肤成纤维细胞的增殖。分别作用于人外阴皮肤成纤维细胞72 h后,莪术5、10、100 mg/L浓度组,黄芪500、1 000 mg/L浓度组,丹参1 000 mg/L浓度组,川穹500、1 000 mg/L浓度组,当归100、500、1 000 mg/L浓度组均显示抑制增殖作用,与对照组比较差异有统计学意义(P<0.05),其中莪术和川穹的抑制强度呈剂量依赖性。结论成功建立了体外人外阴皮肤成纤维细胞原代培养及鉴定方法;特定浓度范围内的莪术、黄芪、丹参、川穹和当归能够抑制人外阴皮肤成纤维细胞的体外增殖。
Objective To establish a method for culturing of primary human vulva skin fibroblasts, assess the effects of several Chinese herbs in- cluding Rhizoma zedoariaeon, Astragalus membranaceus, Salvia mihiorrhiza, Szechwan lovage rhizome and Angelica sinensis on the in vitro prolifer- ation of primary human vulva skin fibroblasts and preliminarily explore the mechanism for the treatment of lichen sclerosus et atrophicus. Methods Human vulva skin fibroblasts were cultured after trypsin digestion. The morphology of the fibroblasts was observed under inverted and electron micro- scope, also by HE staining. Immunohistochemical staining was performed to observe the vimentin in fibroblasts. The proliferation of fibroblasts was determined by growth curve and MTr assay. Results Primary human vulva skin fibroblasts were obtained and euhured after trypsin digestion. The morphology and biological characteristics of this obtained fibroblasts were verified by inverted and electron microscope observation and HE, immuno- histochernieal staining. The proliferative ability of the fibroblasts was high within 6 passages after cryopreservation. All of the Chinese herbs could in- hint the prelifemtian of the obtained libroblasts at appropriate concentrations. After cultured in medium with different drugs for 72 h, Rhizoma zedo- ariaeon at the concentration 5,10,100 mg/L, Astragalus membranaceus at the concentration 500, 1 000 mg/L, Salvia mihiorrhiza at the concentra- tion 1 000 mg/L, Szeehwan lovage rhizome at the eoneentratian 500, 1 000 mg/L and Angelica sinensis at the concentration 100,500, 1 000 mg/L showed a inhibition effect on the proliferation of fibroblasts, along with significant differences compared with controls. In addition, the effect of Pdli- mma zedoariaeon and Szechwan lovage rhizome present a dose-dependent manner. Conclusion The method for in vitro culture of primary human vulva skin fibroblasts is successfully established. Rhizoma zedoariaeo, Astragalus membranaeeus, Salvia mihiorrhiza, Szechwan lovage rhizome and Angelica sinensis can inhibit the proliferation of the obtained fibroblasts at appropriate concentrations.
出处
《中国医科大学学报》
CAS
CSCD
北大核心
2014年第10期865-869,共5页
Journal of China Medical University
基金
国家自然科学基金(30973190)
关键词
成纤维细胞
细胞增殖
中药
fibroblast
cell proliferation
traditional Chinese medicine
