二甲基砷酸对辽宁绒山羊皮肤成纤维细胞的毒性作用以及诱导细胞凋亡的机制

Toxic Effects of Dimethyl Arsenic Acid on Skin Fibroblasts of Liaoning Cashmere Goats and the Mechanism of Inducing Cell Apoptosis

旨在用类金属毒性物质——砷对辽宁绒山羊皮肤成纤维细胞的染毒试验,探索砷对绒山羊皮肤成纤维细胞毒性作用和其诱导细胞凋亡的机制,为后续研究砷对绒山羊皮肤细胞生长的影响提供理论依据。本研究随机选取1只7月龄各项机能正常,体重为25 kg左右的雄性辽宁新品系绒山羊,取其腹部皮肤,进行细胞培养。将二甲基砷酸药物配制成11个不同浓度组(0、0.1、0.2、0.4、0.8、1、5、10、25、50、100 mmol·L^(-1)),对细胞量为3×10^(4)个·mL^(-1)皮肤细胞进行药物染毒24、48、72、96 h后,经MTT法进行3次重复试验,得到绒山羊皮肤成纤维细胞的增殖与抑制情况以及后续试验所选用的时间点和4个浓度组(对照、促进、临界、半抑制浓度(IC50)),进一步借助免疫荧光检测观察细胞骨架形态变化;彗星试验检测细胞DNA损伤情况;流式细胞仪检测细胞凋亡;透射电镜观察细胞质与细胞器的变化;测定细胞内荧光染料Mito-Tracker Green的染色强度分析线粒体跨膜电位(ΔΨm)以及观察分析溶酶体的数量情况。结果,24 h时不同浓度的OD值均能明显地反映出二甲基砷酸对绒山羊皮肤成纤维细胞的增殖和抑制趋势,而48、72、96 h时增殖作用逐渐减弱甚至消失,24 h时细胞增殖与抑制效果最佳,因此选取24 h时的对照组(未做任何处理)、促进浓度组(0.8 mmol·L^(-1))、临界浓度组(1 mmol·L^(-1))、IC50浓度组(38.68 mmol·L^(-1))进行后续的试验。24 h时,剂量范围0.1~1 mmol·L^(-1)的二甲基砷酸促进绒山羊皮肤成纤维细胞增殖,此阶段细胞骨架形态完整,细胞DNA未出现拖尾现象,凋亡率较小,线粒体数目增多,膜结构清晰,嵴致密,形态完整,膜电位升高,溶酶体数量增多;浓度大于1 mmol·L^(-1)的二甲基砷酸抑制绒山羊皮肤成纤维细胞生长,引起明显的细胞毒性(P<0.01),此时细胞骨架形态发生改变,DNA未出现拖尾现象,凋亡率显著上升,线粒体发生肿胀,形态不规则,嵴断裂溶解,膜电位降低,溶酶体数量减少。IC_(50)组细胞骨架形态差异很大,细胞DNA出现彗星尾,最多最明显(P<0.01),细胞凋亡数目明显增多,膜电位明显降低(P<0.01),溶酶体数量显著下降(P<0.01)。本研究探索了二甲基砷酸对辽宁绒山羊皮肤成纤维细胞的毒性作用以及诱导细胞凋亡的机制,表明一定浓度的二甲基砷酸对辽宁绒山羊皮肤成纤维细胞具有毒性作用,并具有进一步诱导细胞凋亡作用。

By metal-like toxic substance-arsenic exposure to the skin fibroblasts of Liaoning cashmere goats,the toxic effects of arsenic on cashmere goat skin fibroblasts and the mechanism of inducing cell apoptosis were explored,which aimed to provide a theoretical basis for further studying the growth effects of arsenic on cashmere goat skin cells.One 7-month-old male Liaoning new line cashmere goat with normal functions and the weight of about 25 kg was randomly selected,its abdominal skin was taken and the cells were cultured.The dimethyl arsenic acid was formulated into 11 different concentration groups(0,0.1,0.2,0.4,0.8,1,5,10,25,50,100 mmol·L^(-1)),and 3×10^(4) cells·mL^(-1) skin cells were exposed to the drug for 24,48,72,and 96 h.After repeating the experiment by MTT method for 3 times,the proliferation and inhibition of cashmere goat skin fibroblasts were detected,the time points and 4 concentration groups(Control,Promotion,Critical,50%inhibitory concentration(IC_(50)))for the subsequent experiment were set up.The changes of cytoskeleton,cytoplasm and organelle were observed by immunofluorescence and TEM(transmission electron microscopy),respectively.The comet assay was used to detect DNA damage.Flow cytometry(FCM)was used to detect the changes of cell apoptosis.The mitochondrial transmembrane potential(ΔΨm)and the number of lysosomes were observed and analyzed by the staining intensity of the fluorescent dye Mito-Tracker Green.The OD values of different concentrations at 24 h could clearly reflect the proliferation and inhibition trend of dimethyl arsenic acid on cashmere goat skin fibroblasts,while the proliferation effect at 48,72,and 96 h was gradually weakened or even disappeared,and the cell proliferation and inhibition effect was the best at 24 h.Therefore,the control group(without any treatment),the promotion group(0.8 mmol·L^(-1)),the critical group(1 mmol·L^(-1)),and the IC50 group(38.68 mmol·L^(-1))for 24 h were selected.Based on all results,when skin fibroblasts were treated with dimethyl arsenic acid(0.1-1 mmol·L^(-1))for 24 h,the cytoskeleton was intact,the cell DNA was not tailed,and the apoptosis rate was low,the number of mitochondria increased,the membrane structure was clear,the cristae was dense,the morphology was complete,the membrane potential and the number of lysosomes increased.When the concentration of dimethyl arsenic acid was greater than 1 mmol·L^(-1),it inhibited the growth of cashmere goat skin fibroblasts and caused obvious cytotoxicity(P<0.01),at the same time,the cytoskeleton morphology changed,the DNA was not tailed,the apoptotic rate significantly increased,the mitochondria swollen,the morphology was irregular shape,the cristae was broken and dissolved,the membrane potential reduced,and the number of lysosomes reduced.The cytoskeleton morphology of IC50group was very different,and cometary tails were most obvious in the DNA of the cells(P<0.01),the number of apoptosis significantly increased,the membrane potential significantly decreased(P<0.01),and the number of lysosomes significantly decreased(P<0.01).In this study,the toxic effect of dimethyl arsenic acid on the skin fibroblasts of Liaoning cashmere goats and the mechanism of inducing cell apoptosis were explored,which indicated that a certain concentration of dimethyl arsenic acid had a toxic effect on the skin fibroblasts of Liaoning cashmere goats and further induced apoptosis.