CAJ | 학술논문

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-2 mRNA(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5～9 d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、25.0μg/L PDGF-BB组小梁细胞中MMP-2 mRNA/β-actin的A值分别为0.127±0.026、0.147±0.045、0.178土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、25.0 μ g/LPDGF-BB组小梁细胞中MMP-2 mRNA表达量明显高于0μg/L PDGF-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、25.0 μg/L PDGF-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＜0.05).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.
배경 목전재청광안적치료연구중,혈소판원성생장인자-BB(PDGF-BB)작위직접작용우소량망적약물수술도정처우연구중,기망약물성분능직접작용우소량망,재불파배정상방각생리결구적전제하,소통소량망방수류출통도,종이체도강안압적목적. 목적 탐토PDGF-BB대체외배양적우안소량세포중기질금속단백매-2(MMP-2)표체적영향급기의의.방법 취신선우안구적소량조직,이조직괴배양법배양소량세포,통과형태학관찰화신경원특이성희순화매(NSE)염색대배양적세포진행감정.장제3대소량세포접충우6공배양판,재배양기중가입불동질량농도(0、5.0、12.5、25.0μg/L)적PDGF-BB배양2h,분별채용역전록PCR(RT-PCR)법화면역조직화학법관찰PDGF-BB대우안소량세포MMP-2 mRNA(상대치)화단백재소량세포중적표체수평,분석각조배양세포중양성신호적흡광도(A)치. 결과 우안소량조직괴배양5～9 d시가견세포유출,제3대소량세포가견교다돌기,세포핵거중,NSE염색세포기질중정록색형광.RT-PCR검측결과발현,0、5.0、12.5、25.0μg/L PDGF-BB조소량세포중MMP-2 mRNA/β-actin적A치분별위0.127±0.026、0.147±0.045、0.178토0.053화0.222±0.062,차이유통계학의의(F=56.71,P＜0.05),기중5.0、12.5、25.0 μ g/LPDGF-BB조소량세포중MMP-2 mRNA표체량명현고우0μg/L PDGF-BB조,차이균유통계학의의(P＜0.05).면역조직화학검측발현,0、5.0、12.5、25.0 μg/L PDGF-BB조소량세포중MMP-2단백표체량(A치)분별위446.12±13.81、1444.65±54.64、2086.18±73.18화3488.65±25.98,차이유통계학의의(F=213.12,P＜0.01),각조간량량비교,차이균유통계학의의(P＜0.05).결론 재체외배양적조건하,PDGF-BB가촉진우안소량세포중MMP-2적표체,기작용정제량의뢰적방식.
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 Trabcular 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 neuronspecific enolase (NSE) staining.The third generation of cells were inoculated to 6-well plate,and different concentrations (0,5.0,12.5,25.0 μg/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 ± 0.026,0.147 ± 0.045,0.178 ± 0.053 and 0.222±0.062 in the O,5.0,12.5,25.0 μg/L PDGF-BB group,respectively,showing a significant difference among them (F =56.71,P＜0.05),and the MMP-2 mRNA/β3-actin value in the 5.0,12.5,25.0 μg/L PDGF-BB group was elevated in comparison with that of the 0 μg/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 μg/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.