Cultivation and identification of keratoconus associated-fibroblasts
10.3760/cma.j.issn.2095-0160.2019.02.002
- VernacularTitle:圆锥角膜相关成纤维细胞的培养与鉴定
- Author:
Yani WANG
1
;
Xianning LIU
;
Xiaodong WANG
;
Wei GAO
;
Yong YIN
;
Xiuping ZHU
Author Information
1. 西安市第一医院眼科 陕西省眼科研究所 陕西省眼科学重点实验室 陕西省眼科疾病临床医学研究中心 西北大学附属第一医院 710002
- Keywords:
Keratoconus;
Fibroblasts;
Cell culture;
Identification
- From:
Chinese Journal of Experimental Ophthalmology
2019;37(2):83-87
- CountryChina
- Language:Chinese
-
Abstract:
Objective To culture and identify corneal fibroblasts from human keratoconus patients (HKCs).Methods HKCs and corneal fibroblasts from human healthy controls (HCFs) were cultured by tissue block adherence method.Cellular morphology and ultrastructure were observed by inverted phase contrast microscope and electron microscopy respectively.Cell viability was detected by cell counting kit-8 (CCK-8) assay.α-Smooth muscle actin (αt-SMA),collagen type 1 alpha 1 (COL1A1) and collagen type 3 alpha 1 (COL3A1) protein expression levels were detected by Western blot.This study protocol was approved by Ethic Committee of Xi'an No.1 Hospital (No.1504).Results Compared with HCFs,HKCs showed several distinguishing properties.First of all,its growth speed was faster,with collagen fibers decreased and attenuated.At the same time,mitochondrion swelled and mitochondrial cristae disappeared.Additionally,Golgi apparatus presented significant expansion and endoplasmic reticulum displayed severe swelling.There were statistically significant differences in A values between the two kinds of corneal fibroblasts at different time points after culture (Fgroup =5 023.13,P＜0.01;Ftime =38 518.16,P＜0.01),the A value of HKCs was significantly higher than that of HCFs at the same time point,and the difference was statistically significant (all at P＜0.01).The relative expression of α-SMA,COL3A1 and COL1 A1 was 120.00±5.77,158.33 ±4.41 and 88.33± 1.67,respectively in HKCs,the relative expression of α-SMA,COL3A1 and COL1 A1 was 100.00±0.00,100.00±0.00 and 100.00±0.00,respectively in HCFs,the relative expressions of α-SMA and COL3A1 were significantly increased in HKCs than those in HCFs,the relative expression of COL1 A1 was significantly decreased in HKCs than that in HCFs,with significant differences between them (t =-3.46,P ＜ 0.05;t =-13.23,P ＜ 0.01;t =7.00,P＜0.05).Conclusions HKCs are cultured and identied,which is suitable for establishing in vitro cell model of keratoconus.
