摘要背景 二甲基亚砜(DMSO)是常用的药物助溶剂,但DMSO体积分数较高时对细胞有毒性,可能影响对受试药物的正确评价,所以确定不同眼组织细胞对DMSO的敏感性十分重要. 目的 探讨DMSO对体外培养的多种眼组织细胞的最小毒性体积分数,为眼部药物的体外筛选提供依据. 方法 采集青紫蓝兔的视网膜色素上皮(RPE)细胞进行培养,并利用免疫组织化学法进行鉴定.分别对人视网膜色素上皮细胞株(ARPE19)、巩膜成纤维细胞(S75-Fron)、Müller细胞(MIO-M1)、人晶状体上皮细胞(HLEC)、人眼葡萄膜黑色素瘤细胞(OCM-1)、人脐静脉血管内皮细胞(HUVEC)进行体外培养.取160μl DMSO溶液和9.84 ml含体积分数2％胎牛血清(FBS)的RPM I1640或DMEM/F12或DMEM培养液配制体积分数分别为1.6％、1.0％、0.8％、0.4％、0.2％和0.1％ DMSO的RPMI1640、DMEM/F12和DMEM培养液,在培养的各种细胞中分别加入不同体积分数的DMSO培养液作用96 h,采用MTS法检测各组细胞的吸光度(A)值. 结果 培养的兔原代RPE细胞对细胞角蛋白(CK)和HMB45呈黄绿色荧光,细胞质和细胞核内S100阳性反应呈红色荧光.随着DMSO体积分数的增加,各细胞活性(A值)逐渐降低,随着DMSO体积分数的增加,ARPE19、S75-Fron、HLEC、OCM-1、HUVEC和原代RPE细胞活性(A值)均明显下降,差异均有统计学意义(均P＜0.05),当DMSO体积分数≥0.8％时,RPE细胞活性均明显低于空白对照组,差异均有统计学意义(均P＜0.05),但MIO-M1细胞株活性的差异无统计学意义(F=0.830,P=0.547).DMSO对ARPE19、HUVEC、HELA、HLEC、MIO-M1、OCM-1、原代RPE细胞和S75-Fron细胞最低有毒体积分数分别为0.8％、0.1％、0.8％、＞1.6％、＞1.6％、0.2％、0.2％和0.2％,HUVEC对DMSO的毒性最敏感,在0.1％ DMSO情况下即显示出毒性(P=0.02),而MIO-M1细胞在1.6％ DMSO的情况下仍未显示出毒性作用(P=0.39).HUVEC和原代RPE细胞活性随DMSO体积分数的增加而下降,S75-Fron细胞活性在DMSO体积分数为0.1％时开始下降,之后虽然DMSO体积分数增加,但细胞活性保持平稳,当DMSO体积分数增至1.6％时,S75-Fron细胞活性进一步下降,MIO-M1细胞活性在不同体积分数的DMSO组无明显变化. 结论 DMSO在体外实验中的细胞毒性随体积分数的增大而增强,在达到助溶效果的情况下应选用最小体积分数的DMSO,同时设与样品中DMSO相同体积分数的DMSO对照.

abstractsBackground Dimethyl sulfoxide(DMSO) is a commonly used adjuvant to promote testing drug solubility to prepare multi-levels testing drug concentrations.DMSO is cell type-dependent cytotoxic and its toxicity can interfere the testing drug evaluation.Determining its safe concentration on commonly used cell types is important for ocular drug development.Objective This study was to determine the minimal toxic concentration of DMSO for in vitro ocular cell lines in a simulated drug screening setting.Methods Retinal pigment epithelial (RPE) cells were isolated from one pigmented rabbit and primarily cultured.Human RPE cell strain (ARPE19),scleral fibroblasts line (S75-Fron),human Müller cell line (MIO-M1),human lens epithelial cell line (HLEC),human choroidal melanoma cell line (OCM-1),human umbilical endothelial cell (HUVEC) and human HeLa cell line (HELA) were cultured.Different concentrations of DMSO (1.6％,1.0％,0.8％,0.4％,0.2％ and 0.1％) were prepared with 160 μl DMSO solution and 9.84 ml RPMI1640 (or DMEM/F12 or DMEM) containing 2％ fetal bovine serum.Different concentrations of DMSO were added in medium for 96 hours,and the and viability (absorbance) of the cells was detected using MTS to evaluate the cytotoxicity of DMSO.Results Rabbit primary RPE cells showed the yellow-green fluorescence for cytokeratin(CK) and HMB45 red fluorescence for S100.The viability of the cells was gradually declined as the increase of DMSO dose,showing significant differences in ARPE19,S75-Fron,HLEC,OCM-1,HUVEC and primary RPE cells (all at P＜0.05),and when DMSO concentrations were ≥ 0.8％,the cell viabilities were significantly lower.But no significant difference was found in MIO-M1 cells among different doses of DMSO (F=0.830,P=0.547).The minimal toxic concentration of DMSO for ARPE19,HUVEC,HELA,HLEC,MIO-M1,OCM-1,primary RPE cells and S75-Fron was 0.8％,0.1％,0.8％,＞1.6％,＞1.6％,0.2％,0.2％,0.2％,respectively,and HUVEC was more sensitive to the cytotoxicity of DMSO (P=0.02),and MIO-M1 was the least sensitive to DMSO (P =0.39).The viability of HUVEC and primary RPE cells went down with the increase of DMSO dose,and S75-Fron viability started to decline in 0.1％ DMSO and then stabilize with the higher concentrations until 1.6％ DMSO at which the viability showed further decline.Conclusions The tolerability of ocular cells in vitro to DMSO varies depending on the cell types.The minimal toxic concentration ranged from 0.1％ to 1.6％.The result suggests that a concurrent DMSO control should be set up along with the testing compound.