房水对培养的角膜基质细胞的影响

Role of aqueous humor on growth of corneal stromal cells

目的观察房水对角膜基质细胞生长的作用。方法将新西兰大白兔的角膜去除后弹力层、内皮层和上皮层后,得到基质层,用组织块培养法体外培养角膜基质细胞。在实验组的培养基中加入10%房水,对照组使用常规培养基培养。通过CCK8实验测得角膜基质细胞的吸光度(A)值以分析细胞增生的情况。分别在培养基中加入2.5%、5%、10%、15%、20%的房水,用明胶酶谱法检测基质金属蛋白酶(MMPs)的活性。结果倒置显微镜观察可见培养的角膜基质细胞呈多角形或树枝状,与对照组相比,实验组的细胞生长状态良好,培养的角膜基质细胞数量明显增加。明胶酶谱法显示实验组的条带比对照组清晰。实验第1～5天分别测角膜基质细胞的A值,实验组的检测条带明显强于对照组,>10%的房水培养组检测的条带明显强于2.5%、5%房水培养组。CCK8检测表明,培养1～5d实验组的角膜基质细胞A值明显高于对照组,差异均有统计学意义(P<0.05)。结论房水作用于角膜基质细胞后,可促进角膜基质细胞的生长,10%房水对体外培养角膜细胞有促细胞增生的作用。

Background Many elements are found in human aqueous humor, including norepinephrine, epinephrine, dopamine, serotonin and N-acetylserotonin etc. , and these substances have the roles of maintaining metabolism of ocular tissue and regulating intraocular pressure（lOP）. Objective This study was to investigate the role of aqueous humor for the growth of corneal stromal cells in vitro. Methods Corneal stromal tissue removed Deseemet＇ s membrane, endothelium and epithelium was obtained from SPF New Zealand white rabbit, and corneal stromal cells were cultured using explant culture method. 10% aqueous humor were added in the experimental groups, and conventional culture method was used as control group. CCK8 assay was used to detect the absorbency value of corneal stromal cells for the evaluation of cell proliferations. In additional,2. 5 % ,5% , 10% ,15% and 20% aqueous humor were added in the medium respectively, and gelatin zymography was used to detect the activity of matrix metalloproteinases. Results Cultured corneal stromal cells showed the polygon-form or elongate-form under the inverted microscope. The corneal stromal ceils grew well and showed an increased number in the culture group with aqueous humor compared with the culture group without aqueous humor. Gelatin zymography analysis showed the stronger electrophoretie bands in the culture group with aqueous humor compared with the culture group without aqueous humor. CCK8 assay revealed that from 1 day through 5 days after experiment,the absorbance values of corneal stromal cells in the culture group with aqueous humor were significantly higher than the culture group without aqueous humor（P 〈 0.05 ）. Conclusion Addition of aqueous humor in culture medium can promote the growth and proliferation of corneal stromal cells in vitro. The low concentration of aqueous humor has a stronger promoting effect.