利用脂多糖体外构建细菌性角膜炎基质细胞模型

In vitro modeling of bacterial keratitis in stromal cells using lipopolysaccharide

目的建立有效的细菌性角膜炎体外细胞模型,探讨其对角膜基质细胞的影响。方法采用贴壁法分离培养SD大鼠角膜基质细胞,以1000细胞·100μl^-1·孔^-1接种并使用CCK8测定不同浓度(0.4、2、10、50、250、1250 mg/L)脂多糖(LPS)对细胞1、2、3、4、5 d内的量效作用曲线,对照组使用PBS(n=3)。根据量效曲线选定建模浓度后,分别设置模型组与对照组(n=3)。光镜观察培养5 d角膜基质细胞形态变化。以5000细胞·100μl^-1·孔^-1接种,使用CCK8测定LPS作用6、12 h对角膜基质细胞短期活性影响。划痕实验检测LPS作用6、12 h角膜基质细胞迁移率;ELISA检测角膜基质细胞炎症因子TNF-α、IL-6分泌水平;免疫荧光染色鉴定胶原分泌水平;免疫印迹检测基质金属蛋白酶9(MMP9)表达变化。结果与对照组比较,自LPS作用1 d开始,相同时间点时10、50、250、1250 mg/L角膜基质细胞活性均降低(均P<0.05)。为满足细胞炎症诱导的需要,选用出现细胞增殖抑制效应的低药物浓度10 mg/L LPS建立体外炎症细胞模型。细胞体外培养5 d时,与对照组比较,模型组角膜基质细胞形态由梭形变为多边形。调整细胞种植密度后,短期的体外活性检测表明,与对照组比较,模型组角膜基质细胞在LPS作用6 h和12 h后出现细胞活性抑制现象[6 h:0.036±0.006比0.061±0.006,12 h:0.033±0.004比0.053±0.005,均P<0.05]。6 h和12 h时模型组角膜基质细胞的体外迁移速率明显低于对照组[6 h:(6.77±3.22)Pixel/h比(16.52±1.18)Pixel/h,12 h:(8.41±1.45)Pixel/h比(17.09±0.75)Pixel/h,均P<0.05]。LPS作用48 h后,与对照组比较,模型组角膜基质细胞炎症因子分泌水平升高[TNF-α:(446.71±20.59)pg/ml比(289.27±26.44)pg/ml,IL-6:(146.26±17.34)pg/ml比(65.94±10.11)pg/ml,均P<0.05];MMP9表达量增加[0.884±0.052比0.385±0.036,P<0.05];Ⅰ、Ⅵ型胶原含量降低[Ⅰ型:(0.46±0.15)×10^5 Pixel^2比(1.21±0.53)×10^5 Pixel^2,Ⅵ型:(2.63±2.07)×10^6 Pixel^2比(7.08±1.52)×10^6 Pixel^2,均P<0.05],Ⅲ型胶原含量升高[(3.69±0.73)×10^5 Pixel^2比(1.12±0.40)×10^5 Pixel^2,P<0.05]。结论LPS诱导角膜基质细胞可模拟角膜炎中细胞炎症反应及变化,为研究细菌性角膜炎的机制和治疗方法提供有效的体外细胞模型。

Objective To establish an effective cell model of bacterial keratitis in vitro and to investigate its effect on corneal stromal cells.Methods The corneal stromal cells of SD rats were isolated and cultured by adherence method,seeded at a density of 1000 cells per 100μl per well,and CCK8-measured for the dose-effect of lipopolysaccharide(LPS)treatment on the corneal stromal cells at LPS doses of 0.4,2,10,50,250,1250 mg/L on days 1 through 5.In the control group,phosphate-buffered saline(PBS)was used instead of LPS(n=3).After selecting the modeling concentration according to the dose-effect curve,the model group and the control group were set(n=3).The morphology of corneal stromal cells were observed under light microscope at 5 days of culture.Then,with the corneal stromal cells seeded at a density of 5000 cells per 100μl per well,CCK8 assay was used to determine the effect of 6 h and 12 h LPS treatment on short-term activity of corneal stromal cells.Scratch test was used to detect the corneal stromal cell migration rate at 6 and 12 hours of LPS treatment.ELISA was used to detect the secretion of inflammatory factors TNF-αand IL-6 by corneal stromal cells.Immunofluorescence staining was used to measure the levels of collagen secretion.The expression of matrix metalloproteinase 9(MMP9)was examined with Western blotting.Results Compared with the control group,the activities of corneal stromal cells treated with 10,50,250,and 1250 mg/L LPS were lower at any given time point starting from frist day of the treatment(all P<0.05).To allow for induction of cell inflammation,the minimum LPS concentration(10 mg/L)that elicited cell proliferation-inhibitory effect was used to establish the cell inflammation model in vitro.Compared with the control group,the morphology of corneal stromal cells in the model group changed from fusiform to polygonal at five days of cell cultured in vitro.As shown by in vitro test of short-term activity,after modulating the cell seeding density,the corneal stromal cells in the model group exhibited suppressed cell activity at 6 h and 12 h after LPS treatment,compared with the control group[6 h:0.036±0.006 vs 0.061±0.006,12 h:0.033±0.004 vs 0.053±0.005,both P<0.05].At 6 h and 12 h,the migration rates of corneal stromal cells in the model group was significantly lower than those in the control group[6 h:(6.77±3.22)Pixel/h vs(16.52±1.18)Pixel/h,12 h:(8.41±1.45)Pixel/h vs(17.09±0.75)Pixel/h,both P<0.05].At 48 hours after LPS treatment,compared with the control group,the corneal stromal cells in the model group presented increased secretion of inflammatory factors[TNF-α:(446.71±20.59)pg/ml vs(289.27±26.44)pg/ml,IL-6:(146.26±17.34)pg/ml vs(65.94±10.11)pg/ml,both P<0.05],lower MMP9 expression[0.884±0.052 vs 0.385±0.036,P<0.05],higher contents of typesⅠandⅥcollagens[TypeⅠ:(0.46±0.15)×10^5 Pixel^2 vs(1.21±0.53)×10^5 Pixel^2,TypeⅥ:(2.63±2.07)×10^6 Pixel^2 vs(7.08±1.52)×10^6 Pixel^2,both P<0.05],and increased content of typeⅢcollagen[(3.69±0.73)×10^5 Pixel^2 vs(1.12±0.40)×10^5 Pixel^2,P<0.05].Conclusion LPS-treated corneal stromal cells may mimic inflammatory response and changes in keratocytes with keratitis.This provides a useful cell model in vitro for mechanistic and therapeutic studies on bacterial keratitis.