阿托品对大鼠巩膜成纤维细胞增殖和凋亡的影响及其机制

Effects and mechanism of atropine on proliferation and apoptosis of rat scleral fibroblasts

目的 探讨不同浓度阿托品对大鼠巩膜成纤维细胞增殖和凋亡的影响及其机制。方法 取出生24 h的健康雄性SD大鼠眼巩膜组织(均为右眼)进行原代培养。取细胞进行分组:对照组(正常巩膜成纤维细胞),0.1 g·L^(-1)、1.0 g·L^(-1)及5.0 g·L^(-1)阿托品处理组(正常巩膜成纤维细胞分别加入三种不同浓度阿托品溶液),每组5只大鼠原代培养细胞。加药后24 h, CCK-8法检测各组巩膜成纤维细胞活力,流式细胞仪检测各组巩膜成纤维细胞凋亡率,Western blot法检测各组巩膜成纤维细胞光蛋白聚糖、基质金属蛋白酶(MMP)-2、MMP-14和MMP抑制剂(TIMP)-2蛋白表达情况。结果 CCK-8实验结果显示:与对照组相比,0.1 g·L^(-1)、1.0 g·L^(-1)及5.0 g·L^(-1)阿托品处理组细胞活力均增高,其中以1.0 g·L^(-1)阿托品处理组增高最显著,差异均有统计学意义(均为P<0.05)。流式细胞仪检测结果表明:阿托品处理组各组的巩膜成纤维细胞凋亡率与对照组相比,差异均无统计学意义(均为P>0.05)。与对照组相比,阿托品处理组巩膜成纤维细胞中光蛋白聚糖、TIMP-2蛋白相对表达量均升高,其中以5.0 g·L^(-1)阿托品处理组上升最明显(均为P<0.05);与对照组相比,阿托品处理组巩膜成纤维细胞中MMP-2、MMP-14蛋白相对表达量均降低,其中以5.0 g·L^(-1)阿托品处理组下降最明显(均为P<0.05)。结论 阿托品处理能增强体外培养大鼠巩膜成纤维细胞活力,这种效应的产生可能与光蛋白聚糖、MMPs、TIMPs等细胞因子有关。

Objective To explore the effect of different concentrations of atropine on the proliferation and apoptosis of rat scleral fibroblasts and its mechanism.Methods Scleral tissues(all removed from right eyes)of 24 h old healthy male Sprague-Dawley rats were taken for primary culture.The cells were assigned to four groups:control group(normal scleral fibroblasts),0.1 g·L^(-1),1.0 g·L^(-1) and 5.0 g·L^(-1) atropine treatment groups(normal scleral fibroblasts were added with three concentrations of atropine solutions),and each group included primary culture cells from 5 rats.At 24 h after dosing,cell counting kit-8(CCK-8)and flow cytometry were used to detect the viability and apoptosis of scleral fibroblasts in each group.In addition,the protein expressions of Lumican,matrix metalloproteinase(MMP)-2,MMP-14 and tissue inhibitor of metalloproteinase(TIMP)-2 in each group were tested by Western blot.Results CCK-8 results showed that the cell viability of the 0.1 g·L^(-1),1.0 g·L^(-1) and 5.0 g·L^(-1) atropine treatment groups increased compared with that of the control group,and the increase was most obvious in the 1.0 g·L^(-1) atropine treatment group,with statistical significances(all P<0.05).Flow cytometry results revealed that there was no significant difference in scleral fibroblast apoptosis rate between the atropine treatment groups and the control group(all P>0.05).Compared with the control group,the relative expression levels of Lumican and TIMP-2 protein in the atropine treatment groups were increased,and the increase was most remarkable in the 5.0 g·L^(-1) atropine treatment group(all P<0.05).Compared with the control group,the relative expression levels of MMP-2 and MMP-14 protein in the atropine treatment groups reduced,and the reduction was most evident in the 5.0 g·L^(-1) atropine treatment group(all P<0.05).Conclusion Atropine interference can enhance the viability of rat scleral fibroblasts cultured in vitro,and its mechanism may be related to cytokines such as Lumican/MMPs/TIMPs.