RT-PCR 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.<br>选取6只3月龄新西兰白兔，经全身麻醉后，通过心脏内注射致死剂量戊巴比妥钠（sodic pentobarbital）实施安乐死。随后进行角巩膜缘环切除，分离结膜组织，摘除晶状体与房水，获取角膜标本。在无菌条件下，从角膜基质层分离后弹力层（Descemet’s membrane），使用含OptiMEM-I培养基、8%胎牛血清（fetal bovine serum, FBS）与1%抗生素的基础稳定培养基（basal stabilizer medium, SM）进行冲洗。从后弹力层剥离角膜内皮细胞（corneal endothelia），取约5 mm²的组织片段，接种于包被Matrigel、I型胶原（Collagen I）或未包被的培养板中，采用增殖培养基（proliferative medium, PM）进行培养；该增殖培养基包含OptiMEM-I、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%硫酸软骨素与抗生素，培养至细胞汇合度达约90%，培养周期约15天。使用胰蛋白酶消化细胞后，转接至基础稳定培养基中继续培养。通过摄影成像记录细胞的形态学变化。分别提取在增殖培养基与基础稳定培养基中培养的角膜内皮细胞的总RNA，采用终点逆转录聚合酶链反应（RT-PCR）分析特异性角膜内皮细胞标志物的表达情况，检测的标志物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）与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%琼脂糖凝胶中进行电泳，通过摄影成像记录电泳条带结果。采用免疫细胞化学染色分析两种培养条件下角膜内皮细胞中GPC4与Na/K-ATPase的表达情况，使用荧光倒置显微镜采集图像。