摘要
目的探讨0．1％二甲基亚砜（DMSO）对δ-氨基酮戊酸诱导HaCaT细胞光动力学效应的影响。方法将HaCaT细胞分成实验组（0．1％DMSO）和对照组（未加DMSO），加入2mmol／L δ-氨基酮戊酸并于37℃避光孵育3h。采用高效液相色谱荧光法（HPLC—FLD）检测胞内原卟啉Ⅸ（PpⅨ）水平，用激光共聚焦显微镜（LSCM）观察胞内PpⅨ荧光强度，同时检测细胞增殖（噻唑蓝法测定吸光度A值）与存活率；以632．8nm波长激光照射HaCaT细胞后继续培养12h，通过流式细胞仪检测细胞凋亡率与坏死率。结果HPLC—FLD检测实验组和对照组HaCaT细胞质内PpⅨ水平分别为（0．57±0．05）ug／L和（0．44±0．04）ug，L（t=2．79，P〈0．05），LSCM观察实验组胞内荧光强度高于对照组，吸光度A值分别为0．54±0．06、0．51±0．07（t=1．51，P〉0．05），细胞存活率分别是（96．18±2，25）％，（94．64±2．40）％（X^2=1．84，P〉0．05）。照光后，实验组和对照组的细胞凋亡率分别为2．2％，1．5％（r=4．05，P〈0．05），细胞坏死率分别为8．9％和0．1％（r=8．23，P〈0．05）。结论0．1％DMSO可促进δ-氨基酮戊酸所致的HaCaT细胞光动力学效应。
Objective To investigate the effect of 0.1% dimethyl sulfoxide （DMSO） on photodynamic reaction in a human keratinocyte cell line, HaCaT, induced by 5-aminolevulinic acid. Methods HaCaT cells were cultured in vitro with or without the presence of 0.1% DMSO at 37 ℃ and stimulated by δ-aminolevulinic acid （ALA） for 3 hours with light flee. Then, cellular protoporphyrin Ⅸ （Ppix） concen- tration in, fluorescence intensity in, proliferation and survival rates of HaCaT cells were determined by high performance liquid chromatography with fluorescence detection （HPLC-FLD）, laser scanning confocal microscopy （LSCM）, MTT colorimetric method and trypan blue staining, respectively. Also, a portion of HaCaT cells were treated with ALA-based photodynamic therapy （ALA-PDT） and irradiated by 632.8-nm laser, and 12 hours later, cellular apoptosis and necrosis were detected by flow cytometry with annexin V/PI staining. Results Increased concentrations of Ppix were found in DMSO-pretreated HaCaT cells compared with untreated cells （0,57 ± 0.05 ug/L vs 0.44 ± 0.04 ug/L, P 〈 0.05）, while no statistical difference was observed in fluorescence intensity expressed as the absorbance at 570nm （0.54± 0.06 vs 0.51 ± 0.07, t = 1.51, P 〉 0.05 ） or cell survival rate （ （96.18 ± 2.25 ）% vs （94.64 ± 2.40）%, X^2= 1.84, P 〉 0.05 ） between DMSO-pretreated and untreated cells. After ALA-PDT, the apoptosis and necrosis rate were significantly increased in DMSO-treated HaCaT cells compared with untreated HaCaT cells （2.2% vs 1.5%, X^2 = 4.05, P 〈 0.05; 8.9% vs 0.1%, A,2 = 8.23, P 〈 0.05）. Conclusion Low concentration （0.1%） of DMSO could enhance the effect of ALA-PDT on HaCaT cells.
出处
《中华皮肤科杂志》
CAS
CSCD
北大核心
2009年第9期628-631,共4页
Chinese Journal of Dermatology
