核心蛋白多糖对兔晶状体上皮细胞增生的抑制作用

Inhibitive effect of Decorin on proliferation of rabbit lens epithelial cells

目的探讨核心蛋白多糖(Decorin)对兔晶状体上皮细胞(lens epithelial cells,LEC)增生的抑制作用及其剂量-效应与时间-效应的关系,为药物防治后发性白内障提供新的选择。方法 MTT比色法分别测定不同浓度的Decorin(0.1mg·L-1、1.0mg·L-1、10.0mg·L-1)作用于LEC不同时间后的细胞生长抑制率;采用流式细胞技术测定细胞周期,用ELISA法检测各组培养液上清中TGF-β的水平,RT-PCR测定TGF-βmRNA的表达,免疫细胞化学观察α-平滑肌肌动蛋白(α-SMA)。结果 ELISA示每100万个细胞中对照组TGF-β含量为(427.24±41.34)ng,0.1mg·L-1、1.0mg·L-1和10.0mg·L-1Decorin组TGF-β的含量分别为(236.01±28.63)ng、(117.86±18.14)ng、(111.72±26.72)ng,与对照组比较差异均有显著统计学意义(均为P<0.01)。MTT比色法实验结果显示,作用24h对照组抑制率为(3.62±1.34)%,0.1mg·L-1、1.0mg·L-1和10.0mg·L-1Decorin组抑制率分别为(4.14±2.08)%、(12.93±2.47)%、(23.47±1.78)%,LEC生长抑制率增加,差异均有统计学意义(均为P<0.05);随着Decorin作用时间延长,生长抑制率增加,差异均有统计学意义(均为P<0.05)。随药物浓度及作用时间延长,LEC出现明显的G0/G1期阻滞,G0/G1期细胞所占比例明显升高(P<0.05)。RT-PCR示Decorin组TGF-βmRNA表达明显减少,与MTT法检测结果呈现大致相同的趋势,免疫细胞化学观察,对照组α-SMA表达较多,细胞肥大,细胞质呈棕色,Decorin组α-SMA较少表达。Decorin表现出明显的抑制作用。结论 Decorin对LEC有抑制增生和诱导凋亡作用,且呈明显的剂量-效应与时间-效应关系,可望成为后发性白内障的防治药物。

Objective To study the inhibitive effect of Decorin on proliferation of rabbit lens epithelial cells（LEC） and its dose-dependent,time-dependent manner,and offer new options for preventing and treating of posterior capsular opacification. Methods The growth of the LEC after treating with different concentrations（0.1 mg·L-1,1.0 mg·L-1,10.0 mg·L-1） of Decorin were examined by MTT method at different time points,and the growth cycle was checked with flow cytometry,ELISA was used to measure the levels of TGF-β in culture medium supernatant,RT-PCR was applied to identity the expression of TGF-β mRNA,and immunocytochemical methods was used to observe the α-smooth muscle actin（α-SMA）. Results ELISA showed the level of TGF-β in per million cells in control group,0.1 mg·L-1,1.0 mg·L-1 and 10.0 mg·mL-1 Decorin group were（427.24±41.34）ng,（236.01±28.63）ng,（117.86±18.14）ng,（111.72±26.72）ng,the level of TGF-β of the Decorin groups were significantly lower than that of the control group（all P〈0.05）.MTT assay results showed the inhibitive ratio of control groups,0.1 mg·L-1,1.0 mg·L-1 and 10.0 mg·L-1 Decorin group at 24 hours were （3.62±1.34）%,（4.14±2.08）%,（12.93±2.47）%,（23.47±1.78）%,there were statistical differences（all P〈0.05）,and the inhibitive ratios were increased with the time prolonged of Decorin treating,there were statistical differences（all P〈0.05）.With the drug concentration increased and treating time prolonged,LEC apparent G0/G1 phase blocked,the percentage of cells in G0/G1 phase was significantly increased（P〈0.05）.RT-PCR showed the expression of TGF-β mRNA in Decorin group was significantly lower than that of the control group at the same time,which was similar to that detected by MTT.Immunocytochemical method showed the expression of α-SMA in control group was higher than that in Decorin groups,cells were hypertrophy and cytoplasm were brown.The Decorin showed significant inhibition. Conclusions Decorin can effectively inhibit the growth of LEC and induce their apoptosis in dose-dependent and time-dependent manner,which is expected to become the drug for prevention and treatment of posterior capsular opacification.