Department of Ophthalmology, Eberhard-Karl University, Tuebingen, Germany. Efdal.Yoeruek @ med.uni-tuebingen.de
Ophthalmic Res. 2010;44(1):43-9. doi: 10.1159/000286339. Epub 2010 Feb 19.
This laboratory study was undertaken to investigate the influence of bevacizumab on apoptosis, Na(+)-K(+)-adenosine triphosphatase (Na(+)-K(+)-ATPase) and zonula occludens 1 (ZO-1) expression on cultured human corneal endothelial cells (HCECs).
Annexin V binding combined with propidium iodide (PI) costaining was used to distinguish viable, early and late apoptotic cells. Immunolocalization of ZO-1 and Na(+)-K(+)-ATPase was performed to analyze intercellular cell integrity after exposure to 5.0 mg/ml bevacizumab for 24 h.
No significant induction of apoptosis or necrosis was seen in HCECs after exposure to 5.0 mg/ml bevacizumab (p = 0.689, p = 0.516, respectively). The mean number of annexin-V-FITC- and PI-positive cells did not change significantly. Additionally, no significant changes in expression were detectable, neither for ZO-1 nor for Na(+)-K(+)-ATPase in comparison with the control. For ZO-1, 70.0% of the cells stained intensely, 24.7% stained moderately, and 5.3% stained weakly in the control group. After exposure to 5.0 mg bevacizumab, only minor changes were observable: 68.8% stained intensely, 25.4% moderately and 5.8% weakly (p = 0.524). For Na(+)-K(+)-ATPase, 19.3% of the cells stained intensely, 59.4% moderately, and 21.3% weakly in the control group. After exposure to 5.0 mg bevacizumab, again only minor changes were observable in the expression pattern: 18.2% stained intensely, 60.3% moderately and 21.5% weakly. The changes were not significant compared with the control (p = 0.492).
Bevacizumab, at concentrations used clinically, did not induce apoptosis or necrosis in HCECs in vitro. Additionally, no alteration of ZO-1 or Na(+)/K(+)-ATPase expression was detected after exposure to 5.0 mg/ml bevacizumab for 24 h.
本实验室研究旨在探讨贝伐单抗对培养的人角膜内皮细胞（HCEC）凋亡、Na(+)-K(+)-三磷酸腺苷酶（Na(+)-K(+)-ATPase）和闭合蛋白 1（ZO-1）表达的影响。
用 Annexin V 结合碘化丙啶（PI）染色来区分活细胞、早期凋亡细胞和晚期凋亡细胞。用免疫组化法检测 ZO-1 和 Na(+)-K(+)-ATPase 的表达，以分析暴露于 5.0 mg/ml 贝伐单抗 24 小时后细胞间的细胞完整性。
HCEC 暴露于 5.0 mg/ml 贝伐单抗后，未见明显的凋亡或坏死诱导（p = 0.689，p = 0.516）。 Annexin-V-FITC 和 PI 阳性细胞的平均数量无明显变化。此外，与对照组相比，ZO-1 和 Na(+)-K(+)-ATPase 的表达也没有明显变化。ZO-1 对照组中，70.0%的细胞强烈染色，24.7%中度染色，5.3%弱阳性染色。暴露于 5.0 mg 贝伐单抗后，仅观察到轻微变化：68.8%强烈染色，25.4%中度染色，5.8%弱阳性染色（p = 0.524）。Na(+)-K(+)-ATPase 对照组中，19.3%的细胞强烈染色，59.4%中度染色，21.3%弱阳性染色。暴露于 5.0 mg 贝伐单抗后，其表达模式也只有轻微变化：18.2%强烈染色，60.3%中度染色，21.5%弱阳性染色。与对照组相比，这些变化无显著性差异（p = 0.492）。
在体外，贝伐单抗在临床使用浓度下不会诱导 HCEC 凋亡或坏死。此外，暴露于 5.0 mg/ml 贝伐单抗 24 小时后，ZO-1 或 Na(+)/K(+)-ATPase 的表达也未发生改变。
