目的:采用高效毛细管电泳(HPCE)的毛细管区带电泳(CZE)分离与检测兔房水中各蛋白组成,观察房水对培养角膜基质细胞的作用。方法:CZE测定新西兰白兔房水蛋白成分:运行缓冲液硼酸钠-硼酸缓冲溶液(pH=8.7、9.1、9.4);毛细管柱温20℃;检测波长200 nm;压力进样0.5 psi,进样时间10s;运行电压20 kV。第1代培养角膜基质细胞进行DMEM/F12培养,加入体积分数为10%的房水,CCK-8检测24 h后细胞增殖情况,每天倒置显微镜下观察细胞生长情况。结果:应用pH=9.4的硼酸钠-硼酸缓冲溶液可获得分离效果较理想的兔房水毛细管电泳峰图。加入房水24 h后CCK8检测吸光度增高(P〈0.05),角膜基质细胞生长良好。结论:HPCE可快速有效方便检测兔房水蛋白成分,体积分数为10%的兔房水可促进培养的兔角膜基质细胞生长与增殖。
Aim:To detect the protein constitutes of rabbit aqueous humor with capillary zone electrophoresis(CZE) of high performance capillary electrophoresis(HPCE) and the effect of aqueous humor on the growth of keratocytes. Methods: Samples of rabbit aqueous humors were analyzed using CZE.The running buffers were borate-borax(pH=8.7,9.1 and 9.4).Sample application was 10s,applied voltage 20kV,detection at 200nm,sample pressure was 0.5psi and column temperature was maintained at constant 20°C.Rabbit keratocytes were cultured in vitro and 10% rabbit aqueous humors were added in the culture medium DMEM/F12.The cell proliferations after 24 hours were examined by the CCK-8 assay.Phase contrast microscope examination was performed daily.Results: A number of resolved peaks were obtained from aqueous humor of rabbit in running buffers of borate-borax(pH= 9.4).The absorbance values of the CCK8 assay for supplementary aqueous humors after 24 hours were significantly high(P0.05).Rabbit keratocytes grew well.Conclusion:CZE is rapid,effective and convenient for the detection of the protein constitutes of rabbit aqueous humor.10% aqueous humors promote the growth and proliferation of keratocytes.