【摘要】：目的:探討人皮膚成纖維細胞(human dermal fibroblasts,HDFs)急性和慢性光損傷模型建立的方法。方法:體外培養(yǎng)原代人皮膚成纖維細胞,選取第4~8代的細胞進行實驗。用長波紫外線(UVA)單次照射建立HDFs急性光損傷模型,熒光倒置顯微鏡觀察不同劑量UVA照射后第1、2、3天HDFs的形態(tài)變化;CCK-8法檢測照射后HDFs的增殖活性。慢性光損傷HDFs模型采用8-甲氧沙林(8-MOP)避光孵育細胞24h,隨后以含8-MOP的磷酸鹽緩沖液(PBS)置換培養(yǎng)基,行9J/cm~2 UVA照射,照射完成后換Dulbecco改良Eagle培養(yǎng)基(DMEM)(含10%胎牛血清)避光傳代培養(yǎng),21d后顯微鏡下觀察HDFs形態(tài);衰老相關(guān)-β-半乳糖苷酶(SA-β-Gal)染色法計算衰老細胞率。結(jié)果:單次UVA照射導(dǎo)致HDFs增殖率下降,與UVA劑量呈正相關(guān)。UVA在劑量為≤10J/cm~2時,細胞存活率保持在85%;而UVA劑量≥15J/cm~2時細胞存活率明顯降低;≥20 J/cm~2時存活率為50%左右,至25 J/cm~2時僅為約25%。慢性光損傷HDFs誘導(dǎo)組(即UVA+MOP組)幾乎所有細胞均出現(xiàn)體積變大、細胞顆粒增加等細胞老化的特征性改變;SA-β-Gal染色細胞的陽性率95%。結(jié)論:UVA單次照射可成功建立HDFs急性光損傷模型,UVA聯(lián)合8-MOP構(gòu)建HDFs慢性光損傷模型。
[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，

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