羊膜对人视网膜色素上皮细胞增生和分化的影响  

Effects of amniotic membrane on proliferation and differentiation of human retinal pigment epithelial cell

作　　者：王瑶[1] 宫华青[1] 杨玲玲[1] 王茜[1] 周庆军[1] 王宜强[1] 

机构地区：[1]山东省眼科研究所 山东省眼科学重点实验室 省部共建国家重点实验室培育基地,青岛266071

出　　处：《中华实验眼科杂志》2012年第9期786-790,共5页Chinese Journal Of Experimental Ophthalmology

基　　金：山东省自然科学基金项目（JQ200908）;山东省自然科学基金青年基金项目（Q2008C02）

摘　　要：背景人视网膜色素上皮（RPE）细胞移植治疗视网膜变性性疾病是目前的研究热点之一，许多生物载体可制备RPE细胞单层，但多存在着细胞毒性、稳定性差及免疫反应等问题。目的检测羊膜对人RPE细胞增生和分化的影响，探讨其作为人RPE细胞层载体进行移植的可能性。方法将RPE细胞系ARPE-19细胞株用含质量分数10％胎牛血清的DMEM／F12培养基进行培养和传代，8～12代细胞用于实验。将传代细胞分为2个组，一组细胞接种于去上皮羊膜作为实验组，另一组细胞直接在培养孔内进行培养作为对照组。分别于接种后24、48、72、96h行MTT实验，测定RPE细胞的吸光度（A492）值以评估两组细胞在增生能力方面的不同；培养3周后的细胞层行苏木精-伊红染色，检测细胞在不同培养载体上是否存在形态学方面的区别；分别收集同一孔中生长于羊膜和培养板表面的细胞，采用逆转录聚合酶链反应（RT—PCR）法检测色素上皮衍生因子（PEDF）、钙黏蛋白（N—cadherin）、β-连环蛋白（β—catenin）以及细胞连接相关蛋白的表达情况；同时收集培养3周时的细胞行透射电子显微镜和扫描电子显微镜检查，比较不同载体培养的ARPE-19细胞在超微结构方面的区别。结果与对照组相比，实验组细胞增生的速度明显变缓，两组细胞增生情况比较差异有统计学意义（F=41．760，P=0．000）。苏木精-伊红染色结果显示，同一培养孔内实验组的细胞密度明显低于对照组，说明羊膜对ARPE-19细胞增生的影响不是通过细胞分泌的可溶性因子的作用。RT—PCR结果显示，实验组细胞连接相关蛋白claudin 1、N—cadherin和PEDF mRNA在培养细胞上的表达水平均明显高于对照组，差异均有统计学意义（t=15．828，P=0．000；t=6．839，P=0．002；t=14．667，P=0．000）；Connexin 43的表达低于对照组，差异有统计学意义（t=3�Background Human retinal pigment epithelial （RPE cell transplantation treating retinal degenerative diseases is a researching topic, and the source of human RPE ceils is a key problem. Many biological carriers can be used for the preparation of RPE cell layer. However, some advantages, such as cytotoxicity, lack of stability and immunologic reaction etc. are still existed. To study an ideal biological carrier is very important. Objective This experimental was to determine the effects of amniotic membrane on the proliferation and differentiation of human RPE cells and the possibility as a scaffold for RPE cell transplantation. Methods ARPE- 19 cell line cells were cultured and passaged in DMEM/F12 medium with 10% fetal bovine serum, and 8 - 12 generation of cells were used. The cells were divided into two groups. One group of ceils were incubated on the denuded amniotic membrane, and the other group of ceils were cultured in the medium （control group）. MTT was performed to detect the A,92 value of RPE cells for the evaluation of cell proliferation ability 24,48,72,96 hours after culture. Cell morphology was compared by histopathological examination 3 weeks after culture. The mRNA expression of pigment epithelium-derived factor （PEDF） ,N-cadherin,β-catenin and cell connection related proteins in the ceils of both groups were assayed using reverse transcription polymerase chain reaction （ RT-PCR）. Ultrastructure of the cells was observed under the transmission and scan electronic microscope 3 weeks after culture. Results The number of ARPE-19 ceils cultured on denuded amniotic membrane was decreased significantly in comparison with the normal culture plate（ F=41. 760,P= 0. 000）. Histopatholy also showed that the cell density on amniotic membrane was lower than of normal ceils on plate surface. Moreover,the expression level of claudin 1 mRNA,N-eadherin mRNA and PEDF mRNA were significantly up-regulated in denuded amniotic membrane group in comparison with control group （t= 15. 828,P=0. 000;t=6. 8

关 键 词：羊膜 视网膜色素上皮细胞 增生 分化 

分 类 号：R774.1[医药卫生—眼科]