中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2011年
12期
1097-1101
,共5页
硫代磷酸甘露醇戊糖%乙酰肝素酶%脐静脉血管内皮细胞
硫代燐痠甘露醇戊糖%乙酰肝素酶%臍靜脈血管內皮細胞
류대린산감로순무당%을선간소매%제정맥혈관내피세포
Phosphomannopentaose sulfate%Heparanase%Umbilical vein vascular endothelial cell
背景 已有动物研究表明,乙酰肝素酶(HPA)在脉络膜新生血管(CNV)中呈高表达,提示其与新生血管的形成密切相关.确定HPA抑制剂是否可影响血管的生成和发展对研究CNV的治疗有重要意义.目的 探讨HPA抑制剂硫代磷酸甘露醇戊糖(PI-88)对HPA的表达及体外培养的人脐静脉内皮细胞(UVEC)增生的作用.方法 人UVEC原代细胞株采用内皮细胞培养基(ECM)进行培养和传代,第3～5代细胞用于实验.用ECM配制PI-88溶液,使其终质量浓度为400.00、200.00、100.00、50.00、25.00、12.50、6.25 mg/L,分别加入内皮细胞培养基培养24、48、72 h后,用四甲基偶氮唑盐(MTT)比色法检测细胞的吸光度(A570)值；并采用免疫组织化学法观察不同质量浓度PI-88作用48 h后细胞中HPA的表达.结果 正常培养的第3代人UVEC呈梭形或多角形.相同质量浓度PI-88作用不同时间培养的人UVEC A570值差异有统计学意义(F=9.656,P=0.002),不同质量浓度PI-88作用相同时间培养的人UVEC A570值差异无统计学意义(F=2.721,P=0.053).PI-88作用24 h,各质量浓度PI-88组与0 mg/L PI-88组UVEC A570值的差异均无统计学意义(P＞0.05),当PI-88的质量浓度＞50.00 mg/L时,培养72 h的人UVEC A570值均明显低于0 mg/L PI-88组,差异均有统计学意义(P＜0.05).PI-88作用延长至48 h和72 h时,在＜50.00 mg/L PI-88组人UVEC A570值均明显高于作用24 h时,差异均有统计学意义(P＜0.05)；但各时间点≥100.00 mg/L PI-88组人UVEC A570值差异均无统计学意义(P＞0.05).HPA在生长旺盛的UVEC细胞质内呈强阳性表达,少数细胞核也有表达；不同质量浓度PI-88溶液干预48 h,25.00 mg/L PI-88组人UVEC中HPA在细胞质内表达明显减弱.结论 PI-88能够抑制人UVEC的增生,尤其是当PI-88的质量浓度＞100.00 mg/L,其对人UVEC增生的抑制作用呈剂量和时间依赖性,其作用机制主要是下调HPA在人UVEC中的表达.
揹景 已有動物研究錶明,乙酰肝素酶(HPA)在脈絡膜新生血管(CNV)中呈高錶達,提示其與新生血管的形成密切相關.確定HPA抑製劑是否可影響血管的生成和髮展對研究CNV的治療有重要意義.目的 探討HPA抑製劑硫代燐痠甘露醇戊糖(PI-88)對HPA的錶達及體外培養的人臍靜脈內皮細胞(UVEC)增生的作用.方法 人UVEC原代細胞株採用內皮細胞培養基(ECM)進行培養和傳代,第3～5代細胞用于實驗.用ECM配製PI-88溶液,使其終質量濃度為400.00、200.00、100.00、50.00、25.00、12.50、6.25 mg/L,分彆加入內皮細胞培養基培養24、48、72 h後,用四甲基偶氮唑鹽(MTT)比色法檢測細胞的吸光度(A570)值；併採用免疫組織化學法觀察不同質量濃度PI-88作用48 h後細胞中HPA的錶達.結果 正常培養的第3代人UVEC呈梭形或多角形.相同質量濃度PI-88作用不同時間培養的人UVEC A570值差異有統計學意義(F=9.656,P=0.002),不同質量濃度PI-88作用相同時間培養的人UVEC A570值差異無統計學意義(F=2.721,P=0.053).PI-88作用24 h,各質量濃度PI-88組與0 mg/L PI-88組UVEC A570值的差異均無統計學意義(P＞0.05),噹PI-88的質量濃度＞50.00 mg/L時,培養72 h的人UVEC A570值均明顯低于0 mg/L PI-88組,差異均有統計學意義(P＜0.05).PI-88作用延長至48 h和72 h時,在＜50.00 mg/L PI-88組人UVEC A570值均明顯高于作用24 h時,差異均有統計學意義(P＜0.05)；但各時間點≥100.00 mg/L PI-88組人UVEC A570值差異均無統計學意義(P＞0.05).HPA在生長旺盛的UVEC細胞質內呈彊暘性錶達,少數細胞覈也有錶達；不同質量濃度PI-88溶液榦預48 h,25.00 mg/L PI-88組人UVEC中HPA在細胞質內錶達明顯減弱.結論 PI-88能夠抑製人UVEC的增生,尤其是噹PI-88的質量濃度＞100.00 mg/L,其對人UVEC增生的抑製作用呈劑量和時間依賴性,其作用機製主要是下調HPA在人UVEC中的錶達.
배경 이유동물연구표명,을선간소매(HPA)재맥락막신생혈관(CNV)중정고표체,제시기여신생혈관적형성밀절상관.학정HPA억제제시부가영향혈관적생성화발전대연구CNV적치료유중요의의.목적 탐토HPA억제제류대린산감로순무당(PI-88)대HPA적표체급체외배양적인제정맥내피세포(UVEC)증생적작용.방법 인UVEC원대세포주채용내피세포배양기(ECM)진행배양화전대,제3～5대세포용우실험.용ECM배제PI-88용액,사기종질량농도위400.00、200.00、100.00、50.00、25.00、12.50、6.25 mg/L,분별가입내피세포배양기배양24、48、72 h후,용사갑기우담서염(MTT)비색법검측세포적흡광도(A570)치；병채용면역조직화학법관찰불동질량농도PI-88작용48 h후세포중HPA적표체.결과 정상배양적제3대인UVEC정사형혹다각형.상동질량농도PI-88작용불동시간배양적인UVEC A570치차이유통계학의의(F=9.656,P=0.002),불동질량농도PI-88작용상동시간배양적인UVEC A570치차이무통계학의의(F=2.721,P=0.053).PI-88작용24 h,각질량농도PI-88조여0 mg/L PI-88조UVEC A570치적차이균무통계학의의(P＞0.05),당PI-88적질량농도＞50.00 mg/L시,배양72 h적인UVEC A570치균명현저우0 mg/L PI-88조,차이균유통계학의의(P＜0.05).PI-88작용연장지48 h화72 h시,재＜50.00 mg/L PI-88조인UVEC A570치균명현고우작용24 h시,차이균유통계학의의(P＜0.05)；단각시간점≥100.00 mg/L PI-88조인UVEC A570치차이균무통계학의의(P＞0.05).HPA재생장왕성적UVEC세포질내정강양성표체,소수세포핵야유표체；불동질량농도PI-88용액간예48 h,25.00 mg/L PI-88조인UVEC중HPA재세포질내표체명현감약.결론 PI-88능구억제인UVEC적증생,우기시당PI-88적질량농도＞100.00 mg/L,기대인UVEC증생적억제작용정제량화시간의뢰성,기작용궤제주요시하조HPA재인UVEC중적표체.
Background Experimental study showed that heparanase (HPA)is overexpressed in choroidal neovascularization,suggesting that it may play a role in the pathogenesis of angiogenesis.To certify HPA inhibitor suppress the formation and development of new blood vessel has an important significance for the treatment of choroidal neovascularization.Objective The aim of this study was to investigate the effect of HPA inhibitor on the proliferation of human umbilical vein vascular endothelial cell (UVEC) and the expression of HPA.Methods Hunan UVEC was primarily cultured and passaged and the third generation cells were used in the experiment.Phosphomannopentaose sulfate (PI-88) solution,a HPA inhibitor,was prepared with endothelial cell medium and the end concentrations were 400.00,200.00,100.00,50.00,25.00,12.50,6.25 mg/L respectively.The cells were treated with PI-88 solutions for 24,48 and 72 hours.The growth and proliferation of human UVEC were analyzed using MTT colorimetric assay at absorbance 570 nm.The expression of HPA in the cells was detected by immunochemistry in 48 hours after addition of PI-88.Results Cultured human UVEC showed the fusiform and polygon in shape.The A570 values of human CVEC were significantly different among various concentrations of PI-88 groups (F=2.721,P=0.053 ) and different action time (F=9.656,P =0.002).When PI-88 was administered for 24 hours,the A570 values of human UVEC were insignificantly altered in comparison with the one without PI-88 culture group (P＞0.05 ).However,in 72 hours after experiment,the A570 values were significantly declined as the PI-88 concentration was ＞50.00 mg/L ( P＜0.05 ).When PI-88 was administered for 48 and 72 hours,the A570 values of human UVEC were significantly higher than those of 24 hours in ＜50.00 mg/L groups (P＜0.01 ),but no statistical differences were seen in ＞100.00 mg/L groups among various time points (P＞0.05 ).HPA was intently expressed in the cytoplasm of human UVEC.However,at 48 hours after addition of ＞25.00 mg/L PI-88,the HPA expression was obviously weaker.Conclusions PI-88 can suppress the growth and proliferation of human UVEC at the dose-and time-dependent manner by downregulating the expression of HPA in the cells.
