保存人羊膜对人脐静脉内皮细胞生长状态的影响

Effects of preserved amniotic membrane on cultivating human vascular endothelium in vitro and in vivo

目的:观察保存人羊膜对体外培养及移植状态下人血管内皮细胞生长状况的影响,研究羊膜移植治疗角膜新生血管性疾病的机制。方法:取健康产妇胎盘羊膜,以酶消化加机械分离法去上皮,-80℃甘油保存。使用时复水,平铺于培养板底,干燥备用。1g/L胰蛋白酶灌流消化法收获人脐静脉内皮细胞,以1～1.7×108/L分别接种于培养板及铺有羊膜的培养板孔内培养。倒置显微镜及电镜进行形态学观察。待羊膜上细胞生长成完整单层后,将载有血管内皮细胞的羊膜移植到去内皮细胞的猫眼角膜内皮侧,3mo后取角膜植片行组织病理学观察。结果:羊膜及培养板上培养的细胞均在24h完全贴壁,2～3d形成单层,细胞为铺路石样,Ⅷ因子染色阳性。植入猫眼内的人脐静脉内皮细胞在长达3mo的观察期内仍存活,有些甚至增生成多层排列。结论:体外培养及移植状态下,保存人羊膜都不能抑制血管内皮细胞增殖,其抑制角膜新生血管形成的机制可能不是通过内皮细胞途径。

AIM： To investigate the effect of preserved amniotic membrane on human vascular endothelium so as to understand the anti-angiogenic mechanism of amniotic membrane transplantation （AMT）. METHODS： The amniotic membrane was gained from healthy placenta and de-epithelization by chemical and mechanical method, then was preserved in pure glycerol at -80℃. When they had to be used for cell culture, they were re-washed by PBS and tiled on the plate. Human umbilicus vascular endothelium （HUVEC） were gained from healthy umbilical veins by Ig/L tripsin which was filled in umbilicus vascular at 37℃ for 10min, and then the endothelium were harvested and were seeded on the plates or amniotic membrane, respectively, whose morphs were observed by light and electric microscope. After the endothelium cultured on the amniotic membrane grew to confluent layer, they were transplanted into the cat＇s anterior chamber to replace its native corneal endothelium. Three month later, the corneas were removed and were studied by HE staining to investigate their histology. RESULTS： The cells cultured on two kinds of substrates were all completely adherent to their substrates in about 24h after seeded. About 2-3d later, they could grow to a complete monolayer and looked like cobble-stone, meanwhile whose factor Ⅷ immunooltochemistry stain was positive. The result of HE staining showed that the transplanted endothelium were all survived in 5 cases of experimental cats after 3mo duration, among them there were 2 cases had been found that the transplanted endothelium had proliferated to multi-layers. CONCLUSION： Preserved amniotic membrane could not inhibit the growth of HUVEC either in vivo or in vitro, the mechanism under which AMT could inhibit cornea neovascularization may be based on other points except the inhibition of vascular endothelium.