Contrast microscopy of corneal endothelial cells proliferation uncoated, collagen and Matrigel-coated plates

Six White New Zealand rabbits 3-month-old were sacrificed under general anesthesia and a lethal intracardiac injection of sodic pentobarbital. A corneoscleral rim excision was made and the conjunctiva was dissected. Lens and aqueous humor were removed and the corneas were obtained. Under sterile conditions, Descemet’s membrane were separated from corneal stroma and rinsed with basal stabilizer medium (SM) containing OptiMEM-I 8% fetal bovine serum (FBS) and 1% antibiotics. Corneal endothelia were peeled off from the Descemet’s membrane and a ~5 mm2 section was cultured in proliferative medium (PM) containing OptiMEM-I 8% FBS, 20 ng/mL of nerve growth factor (NGF), 5 ng/mL of epidermal growth factor (EGF), 200 µg/L of calcium chloride, 20 µg/mL of ascorbic acid, 0.08% chondroitin sulfate, and antibiotics over Matrigel, Collagen I or no coated plate until confluence (~90% of the plate showed adherent cells, ~15 days). The plates were tripzinised and cultured in SM. Morphological changes were photodocumented. RNA was isolated from CEC cultured in PM and SM. Final point RT-PCRs were made to analyze the expression of the specific CECs markers: glypican-4 (GPC4), tight junction protein 1 (TJP1), and CD200; housekeeping gene glyceraldehyde phosphate dehydrogenase (GAPDH) was used. Electrophoresis of PCR products was performed on a 2% agarose gel and the bands were photodocumented. Immunocytochemistry was performed to analyze the presence of GPC4 and Na/K-ATPase in CEC cultured in each condition. Images were obtained with a fluorescence inverted microscope.

6只3月龄新西兰白兔经全身麻醉后，采用心脏内注射致死剂量戊巴比妥钠（sodic pentobarbital）实施安乐死。制备角巩膜缘组织块并分离结膜，摘除晶状体与房水后获取角膜组织。在无菌条件下，从角膜基质层分离出后弹力膜（Descemet’s membrane），并用基础稳定培养基（basal stabilizer medium, SM）冲洗，该培养基包含OptiMEM-I培养液、8%胎牛血清（fetal bovine serum, FBS）及1%抗生素。将角膜内皮细胞从后弹力膜上剥离，取约5 mm²的组织块接种于铺有Matrigel、Ⅰ型胶原（Collagen I）或未包被的培养板中，于增殖培养基（proliferative medium, PM）中培养至细胞汇合度达90%左右，整个培养周期约15天；该增殖培养基包含OptiMEM-I培养液、8%胎牛血清、20 ng/mL神经生长因子（nerve growth factor, NGF）、5 ng/mL表皮生长因子（epidermal growth factor, EGF）、200 μg/L氯化钙、20 μg/mL抗坏血酸、0.08%硫酸软骨素及抗生素。使用胰酶消化培养板中的贴壁细胞，随后转接至基础稳定培养基中继续培养。通过摄影记录细胞的形态学变化。从增殖培养基及基础稳定培养基中培养的角膜内皮细胞（corneal endothelial cells, CEC）中提取总RNA，采用终点RT-PCR技术检测角膜内皮细胞特异性标志物的表达情况，检测靶点包括磷脂酰肌醇蛋白聚糖-4（glypican-4, GPC4）、紧密连接蛋白1（tight junction protein 1, TJP1）及CD200；以甘油醛磷酸脱氢酶（glyceraldehyde phosphate dehydrogenase, GAPDH）作为内参基因。将PCR产物在2%琼脂糖凝胶上进行电泳，通过摄影记录电泳条带。采用免疫细胞化学技术，检测不同培养条件下角膜内皮细胞中GPC4及钠钾ATP酶（Na/K-ATPase）的表达情况，通过荧光倒置显微镜采集相关图像。