人羊膜上皮细胞培养液抑制角膜炎症的实验研究  

Experimental study of human amniotic epithelial cell culture solution on inhibiting corneal inflammation

作　　者：李彬斌[1,2] 周清 姚敏[1,2] 张晓玲[1,2] 秦晓艳[1,2] 陈剑[1,2] 

机构地区：[1]暨南大学附属第一医院眼科 [2]暨南大学医学院眼科研究室,广州510630

出　　处：《器官移植》2013年第1期12-18,共7页Organ Transplantation

基　　金：国家自然科学基金青年科学基金项目(81100637);广东省科技计划项目(2009B030801231)

摘　　要：目的探讨人羊膜上皮细胞(human amniotic epithelial cell,HAEC)培养液对角膜炎症反应的抑制作用及机制。方法体外培养及鉴定HAEC和兔角膜上皮细胞,制备HAEC培养液。根据培养兔角膜上皮细胞的培养液成分不同分为无血清改良杜氏培养基(Dulbecco's modified Eagle's medium,DMEM)组(DMEM组)、无血清DMEM与HAEC培养液1∶1混合组(混合组)、HAEC培养液组(HAEC组)。传代的角膜上皮细胞贴壁24h后,用磷酸盐缓冲液润洗后按分组更换为上述3种培养液培养48h后收集细胞。应用酶联免疫吸附试验(ELISA)检测HAEC培养液中白介素-1受体拮抗剂(interleukin-1 receptor antagonist,IL-1Ra)的含量。采用实时定量聚合酶链反应(PCR)检测3组培养液中兔角膜上皮细胞中的白介素(IL)-1β信使核糖核酸(messenger RNA,mRNA)的表达,采用八肽胆囊收缩素(cholecystokinin-octapeptide,CCK-8)法检测3组细胞的增殖情况。结果 HAEC和兔角膜上皮细胞的形态学观察符合上皮细胞特征。HAEC培养液中IL-1Ra的含量为(153.56±0.36)ng/L,无血清DMEM对照中未检出IL-1Ra。3组培养液中,兔角膜上皮细胞的IL-1β mRNA表达由低至高分别为HAEC组、混合组和DMEM组,3组的IL-1β mRNA表达差异均有统计学意义(均为P<0.05)。兔角膜上皮细胞分组培养24、48、72h后,HAEC组的兔角膜上皮细胞吸光值明显高于混合组和DMEM组,DMEM组的兔角膜上皮细胞吸光值在各时间点均低于混合组(均为P<0.05)。结论 HAEC可分泌IL-1Ra抑制角膜上皮细胞IL-1β的表达,减少炎症反应及移植排斥作用,并促进角膜上皮细胞增殖。Objective To investigate the inhibition role and mechanism of human amniotic epithelial cell （HAEC） culture solution on inhibiting corneal inflammation. Methods HAEC and rabbit corneal epithelial cell （RCEC） were cultured and identified in vitro. And HAEC culture solution was prepared. According to various composition of solution for RCEC culture, they were divided into serum-free Dulbecco＇s modified Eagle＇s medium （DMEM） group, serum-free DMEM equal mixed with HAEC solution （mixed） group and HAEC culture solution （HAEC） group. After adhereing to the wall for 24 hours and being washed by phosphate buffered saline （PBS） , RCEC was cultured in one of the 3 solutions for 48 hours, and then the cells were collected. The content of interleukin-1 receptor antagonist （IL-1Ra） in HAEC culture solution was detected by enzyme-linked immune absorbent assay （ELISA）. The expression of messenger ribonueleic acid （mRNA） of interleukin （IL） -1β in RCEC of 3 groups was detected by real-time quantitative polymerase chain reaction （RT-PCR）. The proliferation of RCEC in three groups was detected by cholecystokininoctapeptide （CCK-8）. Results Morphology of I-IAEC and RCEC was consistent with the character of epithelial cell. The content of IL-1Ra in HAEC culture solution was （153.56 ±0. 36） ng/L, while no IL-1Ra was detected in serum-free DMEM as the control. The expression of IL-1 [3 mRNA was the lowest in HAEC group and the highest in DMEM group. There was significant difference in three groups （ all in P 〈 0.05 ）. After culturing for 24, 48, 72 hours, the absorbance value of RCEC in HAEC group was significantly higher than that in mixed group and DMEM group. The absorbance value of RCEC in DMEM group was lower than that in mixed group at every time point （all in P 〈 0. 05）. Conclusions HAEC can secrete IL-1Ra to inhibit the expression of IL-1β, reduce the inflammation and rejection, and promote the proliferation of corneal epithelial cell.

关 键 词：羊膜移植 羊膜上皮细胞 角膜上皮细胞 白介素-1 白介素-1受体拮抗剂 细胞增殖 炎症 免疫排斥 

分 类 号：R617[医药卫生—外科学]