Bevacizumab对体外培养猪视网膜色素上皮细胞的毒性作用

目的研究bevacizumab（avastin）对猪视网膜色素上皮（RPE）细胞的毒性作用,进一步探讨该药物的安全剂量。方法 30只猪眼RPE细胞培养于含质量分数20%胎牛血清的DMEM培养基中,透射电镜下观察细胞内含大量色素,免疫荧光检测抗角蛋白抗原阳性。实验分为对照组（0g/L bevacizumab）和实验组（0.125、0.25、0.5、1.0、1.25、2.0、2.5g/L bevacizumab）。MTT比色法测定不同终质量浓度（0、0.125、0.25、0.5、1.0、1.25、2.0、2.5g/L）bevacizumab对RPE细胞增生的影响;流式细胞术检测bevacizumab作用72h后细胞凋亡和细胞周期的变化,倒置相差显微镜和透射电子显微镜下观察细胞形态和超微结构的改变。结果 2.0~2.5g/L bevacizumab作用72h后,与对照组比较RPE细胞的生长均有明显抑制（t=1.300,P=0.000;t=1.300,P=0.000）,且呈质量浓度依赖性;实验组S期细胞较对照组减少,细胞凋亡率与对照组比较差异无统计学意义（χ2=1.870,P=0.240）。光学显微镜下,2.0g/L bevacizumab组细胞形态无明显变化。透射电子显微镜下,2.0g/L bevacizumab组细胞微绒毛减少,细胞质内出现空泡和髓鞘样变性。结论大剂量bevacizumab可抑制猪眼RPE细胞的有丝分裂,抑制细胞增生,明显破坏细胞的超微结构。

Toxic effect of bevacizumab on cultured pig retinal pigment epithelium cells in vitro

Background Intravitreal injection of bevacizumab is used to treat various ocular neovascular diseases,while its potential toxic effect to ocular tissue is not well known.ObjectivePurpose of present study was to investigate the potential toxic effect of bevacizumab on cultured pig retinal pigment epithelium cells.MethodsRetinal pigment epithelium（RPE）cells were isolated from 30 pig eyes and cultured in DMEM containing 20% fetal bovine serum and identified by cytokeratin（AE1/AE3）and Evans blue staining under the fluorescence microscope.The third generation of cells with the density of 3×103/mL were treated by different concentrations of bevacizumab（0,0.125,0.25,0.5,1.0,1.25,2.0,2.5g/L）respectively and was observed under the phase contrast microscope and transmission electron microscope.20μL（5g/L）methylthiazolyl-tetrazolial（MTT）was added into medium in 12,24,48 and 72 hours after cultured respectively to evaluate the cell activity after action of bevacizumab.The changes of cellular cycle and apoptosis were measured by flow cytometry（FCM）analysis in 2.0 and 2.5g/L bevacizumab treated group.ResultsThe RPE cells achieved the fusion in 3 and 5 days with abundant cellular organs and showed the positive response for cytokeratin.In 12,24 and 48 hours after culture,no significant differences were found in the A570 value of RPE cells among different concentrations of bevacizumab（F=1.230,P=0.455;F=1.270,P=0.675;F=1.322,P=0.159）.In 72hours after culture,the A570 value of RPE cells was significantly declined in bevacizumab of 2.0-2.5g/L groups compared with control group（t=1.300,P=0.000;t=1.300,P=0.000）.The percent of the cells in S phase was 7.34% in 2.0g/L bevacizumab group and 2.34% in 2.5g/L bevacizumab group,showing an evident decrease in comparison with control group（18.10%）（P0.05）.There was no statistically significant difference in cellular apoptosis rate among control group,2.0g/L bevacizumab group and 2.5g/L bevacizumab group（P0.05）.The morphology of RPE cells was near normal at 72hours in 2.0g/L bevacizumab group under the light microscope.But the irregularity of cell shape,many prominences and lots of vacuoles changes were seen in 2.0g/L bevacizumab group under the transmission electron microscope.ConclusionHigh-dose of bevacizumab can arrest the S phase karyomitosis of RPE cells,inhibit the cellular proliferation and destroy the cellular structure in vitro,indicating the cytotoxicity of bevacizumab on RPE cell.