聚乙二醇水凝胶膜为载体的人角膜上皮细胞原代培养研究

doi:10.13281/j.cnki.issn.1004-4469.2016.06.010

眼科

聚乙二醇水凝胶膜为载体的人角膜上皮细胞原代培养研究

郭译远石妍金鑫贤惠敏栾桂霞张红

50001 哈尔滨医科大学附属第一临床医院眼科（郭译远、石妍、金鑫、张红）；150001 哈尔滨医科大学附属第二临床医院内分泌科（贤惠敏）；266300 山东省青岛市胶州市人民医院心内科（栾桂霞）

收稿日期:2016-10-05 出版日期:2016-11-25 发布日期:2016-11-29
通讯作者: 张红，Email：dr.hzhang2007@hotmail.com
基金资助:
黑龙江省杰出青年科学基金（JC2016019）；黑龙江省属科研院所自拟课题项目（201608）

Primary culture of human corneal limbal epithelial cells on poly(ethylene glycol)-based hydrogel films

GUO Yi-yuan¹, SHI Yan¹,JIN Xin¹, XIAN Hui-min², LUAN Gui-xia³， ZHANG Hong¹

1. Department of Ophthalmology, the First Affiliated Hospital of Harbin Medical University, Harbin 150001, China; 2. Department of Endocrinology, the Second Affiliated Hospital of Harbin Medical University, Harbin 150001, China; 3. Department of Cardiology, Jiaozhou People's Hospital, Qingdao 266300, China

Received:2016-10-05 Online:2016-11-25 Published:2016-11-29
Contact: ZHANG Hong, Email: dr.hzhang2007@hotmail.com

摘要/Abstract

摘要：

目的观察人角膜上皮细胞（corneal limbal epithelial cells，CLEC）的生物学特性，探讨CLEC在普通培养皿和聚乙二醇水凝胶膜（poly(ethylene glycol)-based hydrogel films，PHF）上的增殖率差异。设计实验研究。研究对象 人角膜上皮细胞。方法采用组织块培养法体外扩增CLEC，待细胞生长至指数生长期，分别种植于普通培养皿和PHF上，倒置显微镜下观察培养细胞的生长特点，利用免疫荧光对其鉴定，利用CCK-8比色法观察两组细胞相对增殖率（relative growth rate， RGR）的差异。主要指标 光镜下CLEC的生长，AE1染色，CCK-8比色法的吸光度（optical density，OD）值。结果光镜下可见CLEC在普通培养皿和PHF上均能移行扩增、融合成片，在PHF上的生长速度与普通培养皿组相近。两组细胞AE1荧光染色均呈阳性，PHF组阳性率（73.26%±8.84%）与普通培养皿组（70.84%±3.51%）基本相同。PHF组在12 h（0.89±0.06）、24 h（1.13±0.10）、48 h（1.24±0.03）的OD值与普通培养皿组（0.89±0.03、1.08±0.04、1.28±0.09）差异无统计学意义（P=0.79，0.36，0.76）。PHF组细胞在12 h（99.12%±4.81%）、24 h（103.74%±5.55%）、48 h （97.83%±13.37%）的RGR均大于75%，各时间点间差异无统计学意义（P=0.37，0.90）。结论 PHF可以作为体外扩增角膜上皮细胞的良好载体。（眼科， 2016, 25： 405-408）

关键词: 聚乙二醇水凝胶膜, 角膜上皮细胞, 角膜缘干细胞, 免疫荧光, CCK-8

Abstract:

Objective To observe the biological characteristics of corneal limbal epithelial cells (CLEC) and compare the cell proliferation rate on common culture dish and poly(ethylene glycol)-based hydrogel films(PHF). Design Experimental study. Participants Human corneal limbal epithelial cells. Methods CLEC were cultured by explant culture method in vitro and placed on common culture dish or PHF until growth to exponential phase. The growth characteristics of the cultured cells were observed under inverted microscope and using immunostaining method. The cell proliferation rate between the two groups was analyzed using CCK-8 assay. Main Outcome Measures The CLEC growth under light microscope, AE1 staining, the optical density(OD) of CCK-8 assay. Results The two groups of CLEC can migrate，amplify and become confluent. The proliferation speed of PHF group was the same as common culture dish group under optical microscope. AE1 positive rate of PHF group(73.26±8.84%) was the same as common culture dish group(70.84±3.51%). There was no statistically significant difference of the OD between PHF group (0.89±0.06, 1.13±0.10, 1.24±0.03, respectively)and common culture dish group(0.89±0.03, 1.08±0.04, 1.28±0.09, respectively) at 12h、24h、48h (P=0.79, 0.36, 0.76, respectively). The relative growth rate of PHF group at 12h、24h、48h (99.12±4.81%, 103.74±5.55%, 97.83±13.37%, respectively) were all higher than 75% (P=0.37, 0.90, respectively). Conclusion PHF is a good carrier for amplification of corneal limbal epithelial cells. (Ophthalmol CHN, 2016, 25: 405-408)

Key words:

poly(ethylene glycol)-based hydrogel films, crneal limbal epithelial cells, limbal stem cells, immunostaining method, CCK-8

郭译远石妍金鑫贤惠敏栾桂霞张红. 聚乙二醇水凝胶膜为载体的人角膜上皮细胞原代培养研究[J]. 眼科, doi: 10.13281/j.cnki.issn.1004-4469.2016.06.010.

GUO Yi-yuan1, SHI Yan1,JIN Xin1, XIAN Hui-min2, LUAN Gui-xia3， ZHANG Hong1. Primary culture of human corneal limbal epithelial cells on poly(ethylene glycol)-based hydrogel films[J]. Ophthalmology in China, doi: 10.13281/j.cnki.issn.1004-4469.2016.06.010.

聚乙二醇水凝胶膜为载体的人角膜上皮细胞原代培养研究

Primary culture of human corneal limbal epithelial cells on poly(ethylene glycol)-based hydrogel films

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