黄芩素对人肝癌细胞株SMMC-7721体外迁移侵袭的影响
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摘要
目的:通过观察黄芩素(Baicalein,BAI)对人肝癌细胞株SMMC-7721体外迁移侵袭能力及埃兹蛋白(Ezrin)、基质金属蛋白酶-9(Matrix Metalloproteinase-9,MMP-9)、血管内皮生长因子(Vascular Endothelial Growth Factor , VEGF)表达的影响,探讨黄芩素对人肝癌细胞株SMMC-7721体外迁移侵袭的作用及其可能机制。方法: 1.不同浓度(5.0μmol/L、10.0μmol/L、20.0μmol/L、40.0μmol/L、80.0μmol/L、160.0μmol/L)黄芩素作用SMMC-7721细胞(24h、48h、72h)用水溶性四氮唑-1(Water Soluble Tetrazolium-1,WST-1)比色法检测细胞的增殖活性,按公式计算细胞的增殖抑制率(Inhibition Ratio,IR),IR=(1-实验组平均OD值/对照组平均OD值)×100%;2.划痕愈合试验(Wound Healing Assay)观察浓度分别为20.0μmol/L、40.0μmol/L黄芩素对SMMC-7721细胞迁移运动能力的影响;3.体外侵袭实验(Invasion Assay)运用侵袭小室(Transwell Chambers)检测浓度分别为20.0μmol/L、40.0μmol/L黄芩素作用于SMMC-7721细胞48h后对细胞侵袭能力的影响;4.逆转录-聚合酶链反应(Reverse Transcription-Polymerase Chain Reaction,RT-PCR)检测黄芩素作用于SMMC-7721细胞48h后对Ezrin、MMP-9、VEGF mRNA表达的影响;5.免疫细胞化学(Immunocytochemistry,ICC)技术检测浓度分别为20.0μmol/L、40.0μmol/L黄芩素作用于SMMC-7721细胞48h对Ezrin、MMP-9、VEGF蛋白表达的影响;6.实验结果用SPSS 16.0进行统计分析,以P<0.05作为有统计学意义。结果:1.与对照组比较,浓度分别为5.0μmol/L、10.0μmol/L、20.0μmol/L、40.0μmol/L、80.0μmol/L、160.0μmol/L黄芩素作用于SMMC-7721细胞24h、48h、72h后均对SMMC-7721细胞的增殖有抑制作用(P<0.05);随着黄芩素作用时间的延长和浓度的增加,黄芩素对SMMC-7721细胞增殖的抑制作用逐渐增强,呈明显的时间和剂量依赖性(P<0.05);2.划痕愈合实验显示浓度分别为20.0μmol/L、40.0μmol/L黄芩素作用于SMMC-7721细胞24h后,细胞划痕愈合宽度占原宽度的比例分别为11.2%±1.8%,8.6%±1.6%;黄芩素作用48h后,划痕愈合宽度占原宽度的比例分别为56.8%±2.5%,28.5%±1.9%;分别与24h、48h对照组(47.1%±2.3%,90.1%±2.1%)比较,差异均有统计学意义(P<0.05)。3.体外侵袭实验显示浓度为40.0μmol/L黄芩素作用于SMMC-7721细胞48h后,穿过人工基底膜胶(Matrigel)的细胞数为54.7±18.5个/视野,与对照组(131.4±32.9个/视野)比较差异有统计学意义(P<0.05)。4.RT-PCR结果显示,浓度分别为20.0μmol/L、40.0μmol/L黄芩素作用于SMMC-7721细胞48h后抑制Ezrin、MMP-9、VEGF mRNA在细胞中的表达,Ezrin、MMP-9、VEGF mRNA的相对灰度值(Relative Gray Value,RGV)与相应对照组比较,差异有统计学意义(P<0.05)。5.免疫细胞化学技术显示浓度分别为20.0μmol/L、40.0μmol/L黄芩素作用SMMC-7721细胞48h后抑制Ezrin、MMP-9、VEGF蛋白的表达,Ezrin、MMP-9、VEGF蛋白表达的积分光密度值(Integral Optical Density,IOD)较相应对照组明显降低(P<0.05)。结论:1.黄芩素可能抑制人肝癌细胞株SMMC-7721增殖呈时间和浓度依赖性;2.黄芩素可能抑制人肝癌细胞株SMMC-7721的迁移侵袭能力; 3.黄芩素可能下调人肝癌细胞株SMMC- 7721 Ezrin、MMP-9、VEGF mRNA和蛋白的表达;4.黄芩素可能通过直接或间接下调Ezrin、MMP-9、VEGF mRNA和蛋白的表达而发挥其抑制人肝癌细胞株SMMC- 7721体外迁移侵袭的作用,其具体机制尚不清楚。
Abstract: Objective: To investigate the possible mechanism that baicalein inhibits the migration and invasion, by detecting the expressions of Ezrin, matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) in human hepatocellular carcinoma cell line SMMC-7721. Methods: 1. SMMC-7721 cells were treated with different baicalein concentration(5.0μmol/L,10.0μmol/L,20.0μmol/L,40.0μmol/L,80.0μmol/L and 160.0μmol/L) for 24h,48h and 72h,water soluble tetrazolium-1 (WST-1) assay were used to measure cell proliferation.The growth inhibition ratio(IR) was calculated by the formulation : IR = (1 - average OD value of experimental group / average OD value of control group)×100% .2. Wound-healing assay was performed to observe the migration of SMMC-7721 cells treated with baicalein of 20.0,40.0μmol/L for 24h,48h.3. Transwell chamber assay was performed to observe the invasion of SMMC-7721 cells treated with baicalein of 20.0μmol/L,40.0μmol/L for 48h in vitro. 4.The expressions of Ezrin, MMP-9 and VEGF mRNA in SMMC-7721 cells treated with baicalein of 20.0μmol/L,40.0μmol/L for 48h was analyzed through reverse transcription- polymerase chain reaction (RT-PCR). 5.The expressions of Ezrin, MMP-9 and VEGF protein in SMMC-7721 cells treated with baicalein of 20.0μmol/L,40.0μmol/L for 48h was detected by means of immuno- cytochemistry technique.6.All the experimental results were analyzed by ststistical software SPSS16.0. Results:1. Compared with the control group, the growth inhibition ratio of SMMC-7721 cells was significantly increased after treated by baicalein at different concentrations (5.0μmol/L, 10.0μmol/L, 20.0μmol/L,40.0μmol/L,80.0μmol/L,160.0μmol/L) for 24h, 48h and 72h (P<0.05); the proliferation of SMMC-7721 cells were significantly inhibited, all the inhabition ratio compared with those cause by different times and drug concentrations, and showed a time and dose dependent manner(P<0.05).2. Wound-healing assays displayed that the percent wound closure was 11.2±1.8, 8.6±1.6,56.8±2.5 and 28.5±1.9,respectively,in SMMC-7721 cells treated with baicalein of 20.0,40.0μmol/L for 24h, 48h, it showed a significant decreased compared with control group (47.1±2.3,90.1±2.1, P<0.05).3.A significant decreased was observed in the number of SMMC-7721 cells per field permeating the artificant membrane in the group treated with bacalein of 40.0μmol/L for 48h compared with the control group(54.7±18.5 vs 131.4±32.9,P<0.05)by means of Transwell assay.4.Reverse transcriptase-polymerase chain reaction detected that the expressions of Ezrin, MMP-9 and VEGF mRNA in SMMC-7721 cells of 20.0μmol/L and 40.0μmol/L group were significantly down-regulated,the relative gray values of Ezrin, MMP-9 and VEGF were significantly decreased compared with the control group(P<0.05).6.The expressions of Ezrin, MMP-9 and VEGF protein in SMMC-7721 cells of 20.0μmol/L and 40.0μmol/L group were significantly down-regulated,the integral optical density of Ezrin, MMP-9 and VEGF were significantly decreased compared with the control group (P<0.05)using immunocytochemistry technique.Conclusions:1. Baicalein may inhibit the proliferation of human hepatocellular carcinoma cell line SMMC-7721 in a time and dose dependent manner;2.Baicalein may inhibit the invasion and migration of human hepatocellular carcinoma cell line SMMC-7721; 3.Baicalein may inhibit the mRNA and protein expressions of Ezrin,MMP-9 and VEGF; 4. Baicalein may induce partly a antineoplastic effectiveness via directly or indirectly inhibiting expressions of Ezrin, VEGF and MMP-9,but the mechanism is unknown.
Abstract: Objective: To investigate the possible mechanism that baicalein inhibits the migration and invasion, by detecting the expressions of Ezrin, matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) in human hepatocellular carcinoma cell line SMMC-7721. Methods: 1. SMMC-7721 cells were treated with different baicalein concentration(5.0μmol/L,10.0μmol/L,20.0μmol/L,40.0μmol/L,80.0μmol/L and 160.0μmol/L) for 24h,48h and 72h,water soluble tetrazolium-1 (WST-1) assay were used to measure cell proliferation.The growth inhibition ratio(IR) was calculated by the formulation : IR = (1 - average OD value of experimental group / average OD value of control group)×100% .2. Wound-healing assay was performed to observe the migration of SMMC-7721 cells treated with baicalein of 20.0,40.0μmol/L for 24h,48h.3. Transwell chamber assay was performed to observe the invasion of SMMC-7721 cells treated with baicalein of 20.0μmol/L,40.0μmol/L for 48h in vitro. 4.The expressions of Ezrin, MMP-9 and VEGF mRNA in SMMC-7721 cells treated with baicalein of 20.0μmol/L,40.0μmol/L for 48h was analyzed through reverse transcription- polymerase chain reaction (RT-PCR). 5.The expressions of Ezrin, MMP-9 and VEGF protein in SMMC-7721 cells treated with baicalein of 20.0μmol/L,40.0μmol/L for 48h was detected by means of immuno- cytochemistry technique.6.All the experimental results were analyzed by ststistical software SPSS16.0. Results:1. Compared with the control group, the growth inhibition ratio of SMMC-7721 cells was significantly increased after treated by baicalein at different concentrations (5.0μmol/L, 10.0μmol/L, 20.0μmol/L,40.0μmol/L,80.0μmol/L,160.0μmol/L) for 24h, 48h and 72h (P<0.05); the proliferation of SMMC-7721 cells were significantly inhibited, all the inhabition ratio compared with those cause by different times and drug concentrations, and showed a time and dose dependent manner(P<0.05).2. Wound-healing assays displayed that the percent wound closure was 11.2±1.8, 8.6±1.6,56.8±2.5 and 28.5±1.9,respectively,in SMMC-7721 cells treated with baicalein of 20.0,40.0μmol/L for 24h, 48h, it showed a significant decreased compared with control group (47.1±2.3,90.1±2.1, P<0.05).3.A significant decreased was observed in the number of SMMC-7721 cells per field permeating the artificant membrane in the group treated with bacalein of 40.0μmol/L for 48h compared with the control group(54.7±18.5 vs 131.4±32.9,P<0.05)by means of Transwell assay.4.Reverse transcriptase-polymerase chain reaction detected that the expressions of Ezrin, MMP-9 and VEGF mRNA in SMMC-7721 cells of 20.0μmol/L and 40.0μmol/L group were significantly down-regulated,the relative gray values of Ezrin, MMP-9 and VEGF were significantly decreased compared with the control group(P<0.05).6.The expressions of Ezrin, MMP-9 and VEGF protein in SMMC-7721 cells of 20.0μmol/L and 40.0μmol/L group were significantly down-regulated,the integral optical density of Ezrin, MMP-9 and VEGF were significantly decreased compared with the control group (P<0.05)using immunocytochemistry technique.Conclusions:1. Baicalein may inhibit the proliferation of human hepatocellular carcinoma cell line SMMC-7721 in a time and dose dependent manner;2.Baicalein may inhibit the invasion and migration of human hepatocellular carcinoma cell line SMMC-7721; 3.Baicalein may inhibit the mRNA and protein expressions of Ezrin,MMP-9 and VEGF; 4. Baicalein may induce partly a antineoplastic effectiveness via directly or indirectly inhibiting expressions of Ezrin, VEGF and MMP-9,but the mechanism is unknown.
引文
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