Ma Xiang, Bazan Haydee, Li Jun
Department of Ophthalmology, First Affiliated Hospital of Dalian Medical University, Dalian 116011, China.
Zhonghua Yan Ke Za Zhi. 2003 Dec;39(12):753-6.
To investigate the effects of the conditioned culture medium (CCM) of human amniotic membrane (AM) on corneal neovascularization (CNV) induced by basic fibroblast growth factor (bFGF) in mice.
AM (with epithelium side up) was cultured in EGM basic medium for 3 days, and the CCM was collected. The CCM consisted of three groups: control (EGM only), AM with epithelium (AM) and AM without epithelium (De-AM). Corneal neovascularization was induced in mice by using micropocket assay with Hydron polymer pellets containing 100 ng bFGF. Migration and proliferation of human umbilical cord vein endothelial cells (HUVEC) were measured in Boyden chambers and by CyQUANT fluorescence binding assay, respectively. The levels of tissue inhibitors of metalloproteinase 1 and 2 (TIMP-1, TIMP-2) in the CCM were determined by ELISA assay.
CNV induced by bFGF was significantly suppressed by the CCM of amniotic membrane. When CCM was applied as an eyedrop 4 times a day for 7 days, the area of CNV was (2.48 +/- 0.76) mm(2), (0.64 +/- 0.52) mm(2) and (1.96 +/- 0.65) mm(2) in control, AM and De-AM groups, respectively. The migration and proliferation of HUVEC were strongly inhibited by the CCM of AM with epithelium, while the De-AM had no effect on the migration of HUVEC cells. A high level of TIMP-2 was found in AM group, but not in De-AM group. There were no differences in the amount of TIMP-1 in medium among these three groups.
CCM of amniotic membrane significantly suppresses the CNV induced by bFGF. One of the mechanisms of CCM-mediated suppression is that a high level of TIMP-2 protein is secreted or released into the CCM by AM, which can inhibit the migration and growth of vascular endothelial cells.
研究人羊膜条件培养基（CCM）对小鼠碱性成纤维细胞生长因子（bFGF）诱导的角膜新生血管化（CNV）的影响。
将羊膜（上皮面朝上）在EGM基础培养基中培养3天，收集条件培养基。条件培养基分为三组：对照组（仅含EGM）、含上皮的羊膜（AM）组和不含上皮的羊膜（去上皮羊膜，De-AM）组。采用含100 ng bFGF的Hydron聚合物微球微孔试验诱导小鼠角膜新生血管化。分别通过Boyden小室法和CyQUANT荧光结合试验检测人脐静脉内皮细胞（HUVEC）的迁移和增殖情况。采用酶联免疫吸附测定法（ELISA）检测条件培养基中金属蛋白酶组织抑制剂1和2（TIMP-1、TIMP-2）的水平。
羊膜条件培养基显著抑制了bFGF诱导的角膜新生血管化。当每天滴眼4次，持续7天时，对照组、AM组和De-AM组的角膜新生血管化面积分别为（2.48±0.76）mm²、（0.64±0.52）mm²和（1.96±0.65）mm²。含上皮羊膜的条件培养基强烈抑制了HUVEC的迁移和增殖，而去上皮羊膜对HUVEC细胞的迁移没有影响。AM组中发现高水平的TIMP-2，而De-AM组中未发现。这三组培养基中TIMP-1的含量没有差异。
羊膜条件培养基显著抑制bFGF诱导的角膜新生血管化。条件培养基介导抑制作用的机制之一是羊膜分泌或释放高水平的TIMP-2蛋白到条件培养基中，从而抑制血管内皮细胞的迁移和生长。
