【摘要】 目的：探讨羊膜培养液对角膜上皮细胞中血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)表达的影响。方法：刮除并收集新鲜兔角膜上皮细胞，传代培养接种于35mm培养皿及自制的羊膜培养皿上。实验分为4组，Ⅰ组(对照组)：无血清的DMEM培养液，Ⅱ组：去上皮的羊膜培养液，Ⅲ组：未去上皮的羊膜培养液，Ⅳ组：将细胞直接接种于无上皮羊膜。作用48h后用Trizol法提取各组样本的总RNA,进行RTPCR一步法反应检测各组VEGF mRNA表达并与βactin比较。结果：正常角膜上皮细胞中有VEGF基因表达，在Ⅲ，Ⅳ组中表达受到明显抑制(P<0.01，n=5)。结论：羊膜培养液明显抑制VEGF mRNA在角膜上皮细胞中的表达。
【关键词】 羊膜;角膜新生血管;血管内皮生长因子;角膜上皮细胞
Suppression of VEGF expression in rabbit corneal epithelial cells cultured in human amniotic membrane
Jun Li, Xiang Ma
Department of Ophthalmology, The 3rd Peoples Hospital of Dalian, Dalian 116037, Liaoning Province, China;Department of Ophthalmology, the First Affiliated Hospital of Dalian Medical University, Dalian 116011, Liaoning Province, China
Abstract AIM: To detect the suppression effects of culture supernatant of human amniotic membrane on the expression of vascular endothelial growth factor(VEGF) in rabbit corneal epithelial (RCE) cells.METHODS: The normal RCE cells were cultured for 7 days until there were confluences on the cultures. The cells were subcultured to plastic(3 groups,ⅠⅢ, Ⅰ was control) and naked amniotic membrane (Ⅳ group) cultures respectively. Then one of plastic group (Ⅱ group) was cultured by culture supernatant of human amniotic membrane without epithelial layer as its medium; another plastic group (Ⅲ group) was cultured by culture supernatant of human amniotic membrane with epithelial layer as its medium; the other two groups (Ⅰand Ⅳgroup) were still cultured by DMEM (free from FBS components ).After 48 hours, the total RNAs of these cultures were extracted and detected by RTPCR.RESULTS:There were expressions of VEGF mRNA from Ⅰ group and Ⅱ group, however, the expressions were significantly suppressed in Ⅲ group(using culture supernatant of human amniotic membrane with epithelial layer as its medium) and Ⅳ group (RCE cultered directly on naked amniotic membrane) (P<0.01, n=5).CONCLUSION: Amniotic membrane transplantation is partly through depressing the mRNA expression of VEGF in corneal cells to reduce the corneal neovascularization of injuried corneal surface. And it may be an efficient method of curing corneal neovascularization in clinic.
KEYWORDS: amniotic membrane; corneal neovascularization; vascular endothelial growth factor; corneal epithelial cell
0 引言
正常的角膜无血管，角膜新生血管(corneal neovascularization, CNV)常由损伤及各种炎症刺激诱导产生。严重的角膜新生血管化会导致视力下降甚至视力丧失;随着新生血管的长入，角膜的免疫赦免地位丧失。虽然目前有多种方法用于治疗角膜新生血管，但均未获得满意疗效。临床上羊膜角膜移植及羊膜移植眼表重建，均显示其有抑制角膜新生血管的作用[1]，虽然已知羊膜的细胞外基质及可溶性细胞因子可能参与这一作用机制[2,3]，然而具体作用机制仍不明。最近研究表明，羊膜培养液可明显地抑制角膜新生血管化[4]，为进一步理解这一作用机制，我们应用可能释放多种细胞因子的羊膜培养液，对角膜上皮细胞中血管内皮细胞生长因子(VEGF)表达的影响进行研究。
1 材料和方法
1.1 材料
羊膜取自于健康剖腹产孕妇的胎盘，无菌条件下钝性剥离羊膜，用含有抗生素的PBS液(1×105U/L青链霉素)清洗羊膜3次，将羊膜上皮面向上贴附于无菌的醋酸纤维素膜上，放入15cm的玻璃培养皿中,加入DMEM培养基+甘油(V/V，1∶1)混合液，80℃低温冰箱冷冻保存。使用时，将羊膜置于室温解冻,PBS液水化30min，用2.5g/L胰蛋白酶+0.2g/L EDTA混合液在37℃消化30min，用细胞刮刀轻轻刮除羊膜的上皮层后，用PBS液漂洗3次，剪成4cm×4cm大小备用。将35mm培养皿的底钻掉，消毒后备用。将剪好的去掉上皮的羊膜的基底面向上，包于35mm培养皿上,用手术缝线固定。将羊膜为底的培养皿置于60mm培养皿中，加入少量DMEM培养基(含有150mL/L胎牛血清)，于CO2细胞培养恒温箱中过夜。取经无菌处理的新鲜羊膜，用2.5g/L胰蛋白酶+0.2g/L EDTA混合液在37℃消化30min。以细胞刮刀将羊膜的上皮层细胞成分刮掉，将去上皮成分的4cm×4cm大小羊膜3块浸泡于制备好的无血清DMEM细胞培养基30mL中。作用48h后取其上清液待用。取经无菌处理的新鲜羊膜，保留其上皮层，将4cm×4cm大小羊膜3块浸泡于制备好的无血清DMEM细胞培养基30mL中。作用48h后取其上清液待用。
1.2 方法
兔处死后，立即将眼球取出，共30眼。用加有双抗的生理盐水漂洗3次，用剪刀取下透明的角膜部分，浸于生理盐水中，漂洗1次。用手术刀片去掉角膜的内皮层，其余组织上皮面向上移入7.5cm玻璃平皿中，加入2.5g/L胰蛋白酶+0.2g/L EDTA的混合液，37℃下消化25min后，用刀片将角膜的上皮层轻轻刮下，离心收集上皮细胞接种到25mL培养瓶中，以含150mL/L FBS的DMEM培养基培养，2～3d换液1次。培养7d致形成融合性生长，用2.5g/L胰蛋白酶+0.2g/L EDTA的混合液消化后，分别传代接种于35mm培养皿和单纯去上皮羊膜为底物的培养皿组。用含150mL/L胎牛血清的DMEM培养液培养传代细胞形成70%融合性生长后，将各组中的培养液换为无血清的DMEM培养液继续培养12h,以去除其它活性成分的影响。实验分为4组，即Ⅰ组(对照组)：无血清的DMEM培养液，Ⅱ组：去上皮的羊膜培养液，Ⅲ组：未去上皮的羊膜培养液，Ⅳ组：将细胞直接接种于无上皮羊膜。每组设5个平行孔。12h后，接种于35mm培养皿的Ⅱ组中加入去上皮的羊膜培养上清液作为培养液,向Ⅲ组中加入未去上皮的羊膜培养上清液作为培养液，其余2组仍用无血清的DMEM培养基培养，培养48h后，去除各样本中的多余培养液，并向每个35mm培养皿样品中加入Trizol提取液1mL，最终RNA沉淀物加入DEPC水20μL充分溶解，80℃冷冻保存。PCR引物由TaKaRa宝生物工程(大连)有限公司设计并合成,用灭菌水配成20μmol/L的溶液,4℃保存。引物序列：VEGF 130bp，Sense TATTCAAGCCTTCCTGCGTG，Antisense TGAGGTTTGATCCGCAT;βactin 320bp Sense GCTGTCCCTGTACGCCTCTG，Antisense TGCCGATGGTGATGACCTGG，将RTPCR反应产物各取5μL，加样于20g/L浓度的琼脂糖凝胶中进行电泳，80V电压下电泳35min。照相记录结果经扫描通过QuantiScan分析软件处理，以内参照βactin为基准分析计算各条带的相对含量(图1)。
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