圆锥角膜相关成纤维细胞的培养与鉴定

Cultivation and identification of keratoconus associated-fibroblasts

目的分离、培养并鉴定人圆锥角膜相关成纤维细胞(HKCs)。方法采用组织块贴壁法培养HKCs和正常人角膜成纤维细胞(HCFs),倒置相差显微镜和电子显微镜下分别观察细胞形态和超显微结构,细胞计数试剂盒-8(CCK-8)法检测细胞增生,Westernblot法检测成纤维细胞标志性蛋白α-平滑肌肌动蛋白(α-SMA)及α1-1型胶原蛋白(COL1A1)和α1-3型胶原蛋白(COL3A1)蛋白的表达水平。结果与HCFs相比,HKCs生长速度较快,胶原纤维变细减少,线粒体肿大,嵴消失,高尔基体明显扩张,内质网严重肿胀。培养后不同时间点2种角膜成纤维细胞A值比较,差异均有统计学意义(F组间=5023.13,P<0.01;F时间=38518.16,P<0.01),其中同一时间点HKCs细胞A值均明显大于HCFs细胞A值,差异均有统计学意义(均P<0.01)。HKCs和HFCs中α-SMA的相对表达量分别为120.00±5.77和100.00±0.00,COL3A1的相对表达量分别为158.33±4.41和100.00±0.00,COL1A1相对表达量为88.33±1.67和100.00±0.00,HKCs中α-SMA和COL3A1的相对表达量均较HCFs明显升高,HKCs中COL1A1的相对表达量较HCFs明显降低,差异均有统计学意义(t=-3.46,P<0.05;t=-13.23,P<0.01;t=7.00,P<0.05)。结论成功培养并鉴定HKCs,该细胞适用于建立体外圆锥角膜细胞模型。

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(α-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(F group=5 023.13,P<0.01;F time=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 ofα-SMA,COL3A1 and COL1A1 was 120.00±5.77,158.33±4.41 and 88.33±1.67,respectively in HKCs,the relative expression ofα-SMA,COL3A1 and COL1A1 was 100.00±0.00,100.00±0.00 and 100.00±0.00,respectively in HCFs,the relative expressions ofα-SMA and COL3A1 were significantly increased in HKCs than those in HCFs,the relative expression of COL1A1 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.