摘要
目的：建立D-半乳糖（D—gal）致体外培养的小鼠皮肤成纤维细胞（MSFs）损伤模型，为研究增生性瘢痕和皮肤衰老等提供实验依据。方法：原代培养KM乳鼠皮肤成纤维细胞，先用免疫细胞化学技术（SABC）法特异染色波形蛋白作细胞鉴定，然后采用10吕／L的D—gal作用于细胞。培养一定时间制作MSFs损伤模型。苏木精-伊红（HE）染色观察MSFs形态变化；四甲基氮唑兰（MTT）法测定MSFs活性；试剂盒法检测MSFs培养液中超氧化物歧化酶（SOD）的活性、丙二醛（MDA）含量和乳酸脱氢酶（LDH）活性；β-半乳糖苷酶（B—Gal）染色观察模型细胞的酶活性。结果：经SABC法特异鏊定培养细胞为MSFs。D—gal处理细胞YAh后，与正常对照组比较，MsFs形态发生显著变化，MsFs活性明显下降；MSFs的SOD活力明显下降（P〈0．01），而MDA含量和LDH活性显著上升（P〈0．01）；模型细胞培养72h后，β-Gal活性增高。结论：浓度为10g/L的D—gal作用MSFs24h后，对细胞具有明显氧化损伤作用，且在48h内模型细胞稳定，72h后模型细胞出现衰老生物学标志。该模型细胞为今后开展相关研究提供了实验细胞平台。
Objective To set up D - galactose（ D - gal） - injured mouse skin fibroblasts（MSFs） Pathological model, and to provide the evidence for researching hyperplastic scar and skin aging. Methods The MSFs were cultured in vitro. We identified the mouse skin fibroblasts by dyeing Vimentin specifically. We add a series concentration D - gal on the MSFs which we have cultured. Then we observed the Pathological cell models made by 10g/L D - gal. We surveyed the Morphological structure of MSFs by HE staining; The vitality of MSFs was measured by MTT method ; The vitalities of SOD and LDH, the content of MDA was surveyed by kit methods ; We detected the vitality of β - Gal of the model cells by staining. ResuIts The cells were identified as MSFs by SABC method specially. The group used D - gal in 24 hours has significant results compared with the control group, the shapes of the model cells became slender obviously, the vitality of the cells dropped ; The vitality of SOD fell significantly （ P 〈 0.01 ）, the content of MDA and the vitality of LDH ascended markedly（ P 〈 0.01 ）. We also observed the vitality of β - Gal of the model ceils was went up in 72 hours. Conclusions lOg/L D- gal can made Oxidative harm significantly to the MSFs in 24 hours, the model was stable within 48 hours, and the senescent biological markers were appeared in 72 hours. The stable model cells provided a experimental platform for the related research work in future.
基金
上海中医药大学校级项目资助
