中华皮肤科杂志
中華皮膚科雜誌
중화피부과잡지
Chinese Journal of Dermatology
2012年
8期
577-581
,共5页
冯新%曹一鑫%王建力%陈莉
馮新%曹一鑫%王建力%陳莉
풍신%조일흠%왕건력%진리
肝素,低分子量%细胞系,肿瘤%细胞增殖%细胞运动%血管内皮生长因子类
肝素,低分子量%細胞繫,腫瘤%細胞增殖%細胞運動%血管內皮生長因子類
간소,저분자량%세포계,종류%세포증식%세포운동%혈관내피생장인자류
Heparin,low-molecular-weight%Cell line,tumor%Cell proliferation%Cell movement%Vascular endothelial growth factors
目的 研究低分子肝素对体外培养的皮肤鳞状细胞癌A431细胞株生物学行为的影响.方法 通过不同浓度的低分子肝素处理A431细胞,筛选出最佳实验浓度.用最佳浓度低分子肝素处理A431后,分别用细胞活力检测试剂盒(CCK-8)检测细胞活性,用流式细胞仪检测细胞增殖；用划痕、Transwell小室和黏附试验分别检测A431细胞的迁移和黏附；实时荧光定量逆转录-聚合酶链反应、Western印迹和双抗体夹心酶联免疫吸附法检测细胞内血管内皮细胞生长因子(VEGF)表达水平,进行方差分析与t检验.结果 低分子肝素影响癌细胞活力的最佳浓度为200 IU/ml,用其处理A431细胞后,细胞活性降低,细胞周期阻滞,GI期细胞比率显著增加,S期细胞比率明显降低；低分子肝素组A431细胞增殖指数在48 h和72 h分别为23.41±5.51和11.76±5.13,均显著低于相应未处理组(分别为48.62±4.50和46.86±3.51,两组比较,t值分别为6.14和9.78,P值均＜0.05).低分子肝素组A431细胞中VEGF mRNA表达(48 h和72 h分别为10.16±0.07和4.11±0.01)显著少于相应未处理组(分别为18.77±0.11和17.39±0.05,两组比较,t值分别为114.38和451.10,P值均＜0.05),VEGF蛋白表达(分别为0.16±0.01和0.12±0.01)、VEGF分泌量(分别为67.17±3.34 ng/L和28.14±3.14 ng/L)亦显著少于相应未处理组(分别为0.20±0.01和0.21±0.01,122.63±23.17 ng/L和86.76±1.18 ng/L,P值均＜0.05).低分子肝素组A431细胞黏附率在48 h和72 h分别为29.7％±1.92％和17.5％±0.79％,均显著低于相应未处理组(分别为36.9％±0.35％和34.6％±0.96％,P值均＜0.05),并在24h、48 h和72 h后显著抑制了细胞迁移.结论 低分子肝素通过降低A431细胞活性、减少细胞VEGF表达,抑制A431细胞的增殖、迁移和黏附.
目的 研究低分子肝素對體外培養的皮膚鱗狀細胞癌A431細胞株生物學行為的影響.方法 通過不同濃度的低分子肝素處理A431細胞,篩選齣最佳實驗濃度.用最佳濃度低分子肝素處理A431後,分彆用細胞活力檢測試劑盒(CCK-8)檢測細胞活性,用流式細胞儀檢測細胞增殖；用劃痕、Transwell小室和黏附試驗分彆檢測A431細胞的遷移和黏附；實時熒光定量逆轉錄-聚閤酶鏈反應、Western印跡和雙抗體夾心酶聯免疫吸附法檢測細胞內血管內皮細胞生長因子(VEGF)錶達水平,進行方差分析與t檢驗.結果 低分子肝素影響癌細胞活力的最佳濃度為200 IU/ml,用其處理A431細胞後,細胞活性降低,細胞週期阻滯,GI期細胞比率顯著增加,S期細胞比率明顯降低；低分子肝素組A431細胞增殖指數在48 h和72 h分彆為23.41±5.51和11.76±5.13,均顯著低于相應未處理組(分彆為48.62±4.50和46.86±3.51,兩組比較,t值分彆為6.14和9.78,P值均＜0.05).低分子肝素組A431細胞中VEGF mRNA錶達(48 h和72 h分彆為10.16±0.07和4.11±0.01)顯著少于相應未處理組(分彆為18.77±0.11和17.39±0.05,兩組比較,t值分彆為114.38和451.10,P值均＜0.05),VEGF蛋白錶達(分彆為0.16±0.01和0.12±0.01)、VEGF分泌量(分彆為67.17±3.34 ng/L和28.14±3.14 ng/L)亦顯著少于相應未處理組(分彆為0.20±0.01和0.21±0.01,122.63±23.17 ng/L和86.76±1.18 ng/L,P值均＜0.05).低分子肝素組A431細胞黏附率在48 h和72 h分彆為29.7％±1.92％和17.5％±0.79％,均顯著低于相應未處理組(分彆為36.9％±0.35％和34.6％±0.96％,P值均＜0.05),併在24h、48 h和72 h後顯著抑製瞭細胞遷移.結論 低分子肝素通過降低A431細胞活性、減少細胞VEGF錶達,抑製A431細胞的增殖、遷移和黏附.
목적 연구저분자간소대체외배양적피부린상세포암A431세포주생물학행위적영향.방법 통과불동농도적저분자간소처리A431세포,사선출최가실험농도.용최가농도저분자간소처리A431후,분별용세포활력검측시제합(CCK-8)검측세포활성,용류식세포의검측세포증식；용화흔、Transwell소실화점부시험분별검측A431세포적천이화점부；실시형광정량역전록-취합매련반응、Western인적화쌍항체협심매련면역흡부법검측세포내혈관내피세포생장인자(VEGF)표체수평,진행방차분석여t검험.결과 저분자간소영향암세포활력적최가농도위200 IU/ml,용기처리A431세포후,세포활성강저,세포주기조체,GI기세포비솔현저증가,S기세포비솔명현강저；저분자간소조A431세포증식지수재48 h화72 h분별위23.41±5.51화11.76±5.13,균현저저우상응미처리조(분별위48.62±4.50화46.86±3.51,량조비교,t치분별위6.14화9.78,P치균＜0.05).저분자간소조A431세포중VEGF mRNA표체(48 h화72 h분별위10.16±0.07화4.11±0.01)현저소우상응미처리조(분별위18.77±0.11화17.39±0.05,량조비교,t치분별위114.38화451.10,P치균＜0.05),VEGF단백표체(분별위0.16±0.01화0.12±0.01)、VEGF분비량(분별위67.17±3.34 ng/L화28.14±3.14 ng/L)역현저소우상응미처리조(분별위0.20±0.01화0.21±0.01,122.63±23.17 ng/L화86.76±1.18 ng/L,P치균＜0.05).저분자간소조A431세포점부솔재48 h화72 h분별위29.7％±1.92％화17.5％±0.79％,균현저저우상응미처리조(분별위36.9％±0.35％화34.6％±0.96％,P치균＜0.05),병재24h、48 h화72 h후현저억제료세포천이.결론 저분자간소통과강저A431세포활성、감소세포VEGF표체,억제A431세포적증식、천이화점부.
Objective To evaluate the effect of low-molecular weight heparin (LMWH) on biological behavior of a human cutaneous squamous cell carcinoma cell line A431.Methods To optimize the concentration of LMWH,A431 cells were treated with different concentrations (12.5,25,50,100 and 200 IU/ml) of LMWH for 24,48 and 72 hours followed by CCK-8 assay for the detection of cell viability.Then,A431 cells were cultured with or without the presence of LMWH at 200 IU/ml for 24,48 and 72 hours.Subsequently,flow cytometry was performed to assess cell cycle,real time quantitative PCR (RT-qPCR) and Western blot to quantify the expression of vascular endothelial growth factor (VEGF) mRNA and protein respectively,double-antibody sandwich enzyme linked immunosorbent assay (ELISA) to determine the expression level of VEGF protein in the supernatant of A431 cells,wound-healing assay,Transwell assay,and adhesion assay to observe the migration and adhesivity of A431 cells.Analysis of variance and t test were carried out for statistical analysis.Results The optimal concentration of LMWH was determined as 200 IU/ml according to the CCK-8 assay,and used in the following experiment.The LMWH of 200 IU/ml resulted in a decrease in cell viability,cell cycle arrest,an increase in cell percentage in G1 phase,and a reduction in cell percentage in S phase.The proliferation index was 23.41 ± 5.51 and 11.76 ± 5.13 respectively in A431 cells at 48 and 72 hours after treatment with LMWH of 200 IU/ml,significantly lower than that in untreated A431 cells (48.62 ± 4.50,t =6.14,P ＜ 0.05; 46.86 ± 3.51,t =9.78,P ＜ 0.05).A significant decrease was observed in LMWH-treated A431 cells at 48 and 72 hours compared with the untreated A431 cells in the expression level of VEGF mRNA (10.16 ±0.07 vs.18.77 ± 0.11,4.11 ± 0.01 vs.17.39 ±0.05,t=114.38,451.10,both P＜ 0.05),VEGF protein (0.16 ± 0.01 vs.0.20 ± 0.01,0.12 ± 0.01 vs.0.21 ± 0.01,t =4.90,11.02,both P ＜ 0.05),and in the supernatant level of VEGF protein ((67.17 ± 3.34) ng/L vs. ( 122.63 ± 23.17) ng/L, (28.14 ± 3.14) ng/L vs.(86.76 ± 1.18) ng/L,t =4.10,30.27,both P＜ 0.05).The percentage of adherent cells was 29.7％ ± 1.92％ and 17.5％± 0.79％ in LMWH-treated A431 cells at 48 and 72 hours,respectively,significantly lower than that in untreated A431 cells (36.9％ ± 0.35％,34.6％ ± 0.96％,respectively,both P＜ 0.05).The migration of A431 cells was also obviously inhibited by the treatment with LMWH for 24,48 and 72 hours.Conclusion LMWH may suppress the proliferation,migration and adhesion of A431 cells via downregulating cellular viability and VEGF expression.
