Spitzer M S, Wallenfels-Thilo B, Sierra A, Yoeruek E, Peters S, Henke-Fahle S, Bartz-Schmidt K U, Szurman P
University Eye Clinic, Department I, Eberhard-Karls University, Tuebingen, Schleichstrasse 12, Tuebingen, Germany.
Br J Ophthalmol. 2006 Oct;90(10):1316-21. doi: 10.1136/bjo.2006.095190. Epub 2006 May 24.
To evaluate the antiproliferative and cytotoxic properties of bevacizumab, a monoclonal antibody against vascular endothelial growth factor (VEGF), on human retinal pigment epithelium (ARPE19) cells, rat retinal ganglion cells (RGC5), and pig choroidal endothelial cells (CEC).
Monolayer cultures of ARPE19, RGC5, and CEC were used. Bevacizumab (0.008-2.5 mg/ml), diluted in culture medium, was added to cells that were growing on cell culture dishes. Cellular proliferative activity was monitored by 5'-bromo-2'-deoxyuridine (BrdU) incorporation into cellular DNA and the morphology assessed microscopically. For cytotoxicity assays ARPE19, RGC5, and CEC cells were grown to confluence and then cultured in a serum depleted medium to ensure a static milieu. The MTT test was performed after 1 day. The "Live/Dead" viability/cytotoxicity assay was performed and analysed by fluorescence microscopy after 6, 12, 18, 24, 30, 36, and 48 hours of incubation. Expression of VEGF, VEGF receptors (VEGFR1 and VEGFR2) and von Willebrand factor was analysed by immunohistochemistry.
No cytotoxicity of bevacizumab on RGC5, CEC, and ARPE19 cells could be observed after 1 day. However, after 2 days at a bevacizumab concentration of 2.5 mg/ml a moderate decrease in ARPE19 cell numbers and cell viability was observed. Bevacizumab caused a dose dependent suppression of DNA synthesis in CEC as a result of a moderate antiproliferative activity (maximum reduction 36.8%). No relevant antiproliferative effect of bevacizumab on RGC5 and ARPE19 cells could be observed when used at a concentration of 0.8 mg/ml or lower. CEC and ARPE 19 cells stained positively for VEGF, VEGFR1, and VEGFR2. More than 95% of the CEC were positive for von Willebrand factor.
These experimental findings support the safety of intravitreal bevacizumab when used at the currently applied concentration of about 0.25 mg/ml. Bevacizumab exerts a moderate growth inhibition on CEC when used in concentrations of at least 0.025 mg/ml. However, at higher doses (2.5 mg/ml) bevacizumab may be harmful to the retinal pigment epithelium.
评估抗血管内皮生长因子（VEGF）单克隆抗体贝伐单抗对人视网膜色素上皮（ARPE19）细胞、大鼠视网膜神经节细胞（RGC5）和猪脉络膜内皮细胞（CEC）的抗增殖和细胞毒性特性。
使用ARPE19、RGC5和CEC的单层培养物。将在培养基中稀释的贝伐单抗（0.008 - 2.5mg/ml）添加到细胞培养皿中生长的细胞中。通过将5'-溴-2'-脱氧尿苷（BrdU）掺入细胞DNA来监测细胞增殖活性，并通过显微镜评估形态。对于细胞毒性测定，将ARPE19、RGC5和CEC细胞培养至汇合，然后在无血清培养基中培养以确保静态环境。1天后进行MTT试验。在孵育6、12、18、24、30、36和48小时后，进行“活/死”活力/细胞毒性测定并通过荧光显微镜分析。通过免疫组织化学分析VEGF、VEGF受体（VEGFR1和VEGFR2）和血管性血友病因子的表达。
1天后未观察到贝伐单抗对RGC5、CEC和ARPE19细胞的细胞毒性。然而，在贝伐单抗浓度为2.5mg/ml的情况下培养2天后，观察到ARPE19细胞数量和细胞活力适度下降。由于适度的抗增殖活性（最大降低36.8%），贝伐单抗导致CEC中DNA合成的剂量依赖性抑制。当以0.8mg/ml或更低的浓度使用时，未观察到贝伐单抗对RGC5和ARPE19细胞有相关的抗增殖作用。CEC和ARPE 19细胞对VEGF、VEGFR1和VEGFR2染色呈阳性。超过95%的CEC对血管性血友病因子呈阳性。
这些实验结果支持玻璃体内注射贝伐单抗在当前约0.25mg/ml的应用浓度下的安全性。当以至少0.025mg/ml的浓度使用时，贝伐单抗对CEC有适度的生长抑制作用。然而，在更高剂量（2.5mg/ml）时，贝伐单抗可能对视网膜色素上皮有害。
