血小板源性生长因子-BB对体外培养的牛眼小梁细胞中基质金属蛋白酶-2表达的影响

Effects of platelet-derived growth factor-BB on the expression of matrix metalloproteinase-2 in trabecularmeshwork cells in vitro

背景目前在青光眼的治疗研究中，血小板源性生长因子-BB（PDGF—BB）作为直接作用于小梁网的药物手术刀正处于研究中，期望药物成分能直接作用于小梁网，在不破坏正常房角生理结构的前提下，疏通小梁网房水流出通道，从而达到降眼压的目的。目的探讨PDGF—BB对体外培养的牛眼小梁细胞中基质金属蛋白酶．2（MMP-2）表达的影响及其意义。方法取新鲜牛眼球的小梁组织，以组织块培养法培养小梁细胞，通过形态学观察和神经元特异性烯醇化酶（NSE）染色对培养的细胞进行鉴定。将第3代小梁细胞接种于6孔培养板，在培养基中加入不同质量浓度（0、5．0、12．5、25．0μg／L）的PDGF-BB培养2h，分别采用逆转录PCR（RT—PCR）法和免疫组织化学法观察PDGF—BB对牛眼小梁细胞MMP．2mRNA（相对值）和蛋白在小梁细胞中的表达水平，分析各组培养细胞中阳性信号的吸光度（A）值。结果牛眼小梁组织块培养5～9d时可见细胞游出，第3代小梁细胞可见较多突起，细胞核居中，NSE染色细胞基质中呈绿色荧光。RT—PCR检测结果发现，0、5．0、12．5、25．0Ixg／LPDGF—BB组小梁细胞中MMP-2mRNA／β-actin的A值分别为0．127-＋0．026、0．147-＋0．045、0．178-＋0．053和0．222±0．062，差异有统计学意义（F=56．71，P〈0．05），其中5．0、12．5、25．0μg／LPDGF—BB组小梁细胞中MMP-2mRNA表达量明显高于0μg／LPDGF—BB组，差异均有统计学意义（P〈0．05）。免疫组织化学检测发现，0、5．0、12．5、25．0μg／LPDGF—BB组小梁细胞中MMP-2蛋白表达量（A值）分别为446．12±13．81、1444．65±54．64、2086．18±73．18和3488．65±25．98，差异有统计学意义（F=213．12，P〈0．01），各组间两两比较，差异均有统计学意义（P〈O．05）。结论在体外培养的条件下，PDGF—BB可促进牛眼小梁细胞中MMP-2的表达，其作用呈剂量依赖的方式。

Background At present,a new drug, platelet-derived growth factor-BB （PDGF-BB） , as a drug knife for the treatment of glaucoma is under study to expect it directly working on the trabecular meshwork without disrupting the normal physiological structure of anterior chamber angle, clearing the trabecular meshwork aqueous outflow channel so as to achieve the purpose of lowering intraocular pressure. Objective This study aimed to investigate the effect of PDGF-BB on matrix metalloproteinase-2 （MMP-2） expression in cultured bovine trabecular meshwork cells. Methods Trabeular tissue was obtained from fresh bovine eyes, and trabcular meshwork cells were cultured and passaged using explant method. Cultured cells were identified by morphological evaluation and neuronspeeific enolase （NSE） staining. The third generation of cells were inoculated to 6-well plate, and different concentrations （0,5.0,12.5,25.0μg/L） of PDGF-BB was added into the medium for 2 hours. Expression levels （A value） of MMP-2 mRNA （MMP-2 mRNA/β-actin） and protein in the cells were assayed by reverse transcription polymerase chain reaction （RT-PCR） and immunochemistry, respectively. Results Trabcular meshwork cells appeared 5-9 days after cultured. The third generation of cells presented with many process and showed the green influence in cytoplasm. MMP-2 mRNA/β-actin value （A） was 0. 127 -＋ 0. 026, O. 147 -＋ O. 045, O. 178 ＋ O. 053 and 0. 222_＋0. 062 in the 0,5.0,12.5,25.0 μg/L PDGF-BB group,respectively, showing a significant difference amongthem （F=56.71 ,P〈0.05） ,and the MMP-2 mRNA/13-actin value in the 5.0,12.5,25.0 μg/L PDGF-BB group was elevated in comparison with that of the 0 ixg/L PDGF-BB group （all P〈0.05）. The expression value （A value） of PDGF-BB protein in the cells was 446. 12-＋13.81,1444.65-＋54.64,2086. 18-＋73. 18,3488.65-＋25.98 in the 0,5.0, 12.5,25.0 p,g/L PDGF-BB group,respectively,with a significant difference among the four groups （F= 213. 12 ,P〈 0.01）,and the expression value （A value） of PDGF-BB protein was gradually increased with the ascend of concentration of PDGF-BB（ all P〈0.05 ）. Conclusions PDGF-BB can promote the expression of MMP-2 in bovine trabcular meshwork cells in vitro in concentration-dependent manner.