UVA致人皮肤成纤维细胞急性和慢性光损伤模型的建立
发布时间：2018-12-15 05:47
【摘要】：目的:探讨人皮肤成纤维细胞(human dermal fibroblasts,HDFs)急性和慢性光损伤模型建立的方法。方法:体外培养原代人皮肤成纤维细胞,选取第4~8代的细胞进行实验。用长波紫外线(UVA)单次照射建立HDFs急性光损伤模型,荧光倒置显微镜观察不同剂量UVA照射后第1、2、3天HDFs的形态变化;CCK-8法检测照射后HDFs的增殖活性。慢性光损伤HDFs模型采用8-甲氧沙林(8-MOP)避光孵育细胞24h,随后以含8-MOP的磷酸盐缓冲液(PBS)置换培养基,行9J/cm~2 UVA照射,照射完成后换Dulbecco改良Eagle培养基(DMEM)(含10%胎牛血清)避光传代培养,21d后显微镜下观察HDFs形态;衰老相关-β-半乳糖苷酶(SA-β-Gal)染色法计算衰老细胞率。结果:单次UVA照射导致HDFs增殖率下降,与UVA剂量呈正相关。UVA在剂量为≤10J/cm~2时,细胞存活率保持在85%;而UVA剂量≥15J/cm~2时细胞存活率明显降低;≥20 J/cm~2时存活率为50%左右,至25 J/cm~2时仅为约25%。慢性光损伤HDFs诱导组(即UVA+MOP组)几乎所有细胞均出现体积变大、细胞颗粒增加等细胞老化的特征性改变;SA-β-Gal染色细胞的阳性率95%。结论:UVA单次照射可成功建立HDFs急性光损伤模型,UVA联合8-MOP构建HDFs慢性光损伤模型。
[Abstract]:Objective: to investigate the method of establishing acute and chronic light injury models of human skin fibroblasts (human dermal fibroblasts,HDFs). Methods: primary human skin fibroblasts were cultured in vitro. The acute light damage model of HDFs was established by single ultraviolet (UVA) irradiation. The morphologic changes of HDFs were observed by fluorescence inverted microscope on the 2nd day after different doses of UVA, and the proliferative activity of HDFs was detected by CCK-8 method. The HDFs model of chronic light injury was incubated with 8-methoxaclene (8-MOP) for 24 h, then the culture medium was replaced with phosphate buffer containing 8-MOP (PBS) and irradiated with 9J/cm~2 UVA. After irradiation, the Dulbecco modified Eagle medium, (DMEM) (containing 10% fetal bovine serum, was replaced and cultured without light. After 21 days, the morphology of HDFs was observed under microscope. Senescence associated-尾-galactosidase (SA- 尾-Gal) staining was used to calculate the aging cell rate. Results: the proliferation rate of HDFs was decreased by single UVA irradiation, which was positively correlated with the dose of UVA. When the dose of UVA was 鈮，
本文编号：2380088
[Abstract]:Objective: to investigate the method of establishing acute and chronic light injury models of human skin fibroblasts (human dermal fibroblasts,HDFs). Methods: primary human skin fibroblasts were cultured in vitro. The acute light damage model of HDFs was established by single ultraviolet (UVA) irradiation. The morphologic changes of HDFs were observed by fluorescence inverted microscope on the 2nd day after different doses of UVA, and the proliferative activity of HDFs was detected by CCK-8 method. The HDFs model of chronic light injury was incubated with 8-methoxaclene (8-MOP) for 24 h, then the culture medium was replaced with phosphate buffer containing 8-MOP (PBS) and irradiated with 9J/cm~2 UVA. After irradiation, the Dulbecco modified Eagle medium, (DMEM) (containing 10% fetal bovine serum, was replaced and cultured without light. After 21 days, the morphology of HDFs was observed under microscope. Senescence associated-尾-galactosidase (SA- 尾-Gal) staining was used to calculate the aging cell rate. Results: the proliferation rate of HDFs was decreased by single UVA irradiation, which was positively correlated with the dose of UVA. When the dose of UVA was 鈮，
本文编号：2380088
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