摘要：
目的 观察木犀草素对黑素瘤B16细胞系生长、转移及血管生成拟态形成能力的影响.方法 体外培养黑素瘤B16细胞系,按培养基中木犀草素浓度分为低、中、高剂量组(2.5、5、10 μmol/L)以及对照组(0.1％二甲基亚砜处理),分别通过划痕实验、Transwell侵袭实验、管道形成实验观察各组黑素瘤细胞迁移、侵袭及管道形成能力.建立C57小鼠B16黑素瘤皮下移植瘤模型,随机分为对照组(超纯水灌胃)及木犀草素低(10 mg/kg)、中(20 mg/kg)、高(40 mg/kg)剂量组,每组3只,木犀草素组按上述剂量每天给予灌胃,给药至荷瘤第28天处死小鼠,解剖取小鼠肺及肿瘤组织,观察各组移植黑素瘤生长、转移及血管生成拟态情况;通过免疫荧光、免疫组化实验观察木犀草素对移植黑素瘤血管内皮钙黏蛋白(VE-Cad)、血管内皮生长因子受体1(VEGFR1)、VEGFR2、基质金属蛋白酶2(MMP2)、MMP9表达的影响.多组间均数比较采用单因素方差分析或秩和检验.结果 体外实验结果显示,对照组、低剂量组、中剂量组、高剂量组B16细胞在48 h时相对划痕宽度分别为0.47±0.04、0.64±0.04、0.73±0.03、0.84±0.04,差异有统计学意义(F=34.51,P＜0.001),木犀草素低、中、高剂量组细胞迁移能力均低于对照组(均P＜ 0.05).4组细胞在培养24 h时穿过小室膜的细胞数量分别为281.00±8.79、169.00±15.35、92.00±14.79、57.00±13.72,差异有统计学意义(F=275.30,P＜ 0.001),木犀草素低、中、高剂量组侵袭能力均低于对照组(P＜0.01);管道形成数量分别为20.00±2.77、11.00±1.28、7.00±1.86、2.00±1.32,差异有统计学意义(F=48.61,P＜0.001),低、中、高剂量组均低于对照组(P＜ 0.01).体内实验结果显示,对照组、低剂量组、中剂量组、高剂量组肿瘤终体积分别为(5.10±1.72)、(4.02±2.13)、(2.98±0.92)、(1.49±1.13) cm3,差异有统计学意义(F=28.76,P＜0.001);低、中、高剂量木犀草素组均小于对照组(t值分别为3.86、7.11、13.06,均P＜0.01).CD31-PAS双染结果显示,对照组血管生成拟态形成数量高于木犀草素给药组(P＜0.01).在体内与体外实验中,木犀草素高剂量组细胞及小鼠肿瘤组织内血管生成拟态相关标志物的表达水平均低于对照组(均P＜ 0.05).结论 木犀草素可以有效抑制黑素瘤生长、转移及血管生成拟态的形成.%Objective To evaluate the effect of luteolin on the growth,migration and vasculogenic mimicry formation of a melanoma cell line B16.Methods In vitro cultured B16 melanoma cells were divided into 4 groups:low-,middle-and high-dose luteolin groups treated with 2.5,5,10 μmol/L luteolin respectively,and control group treated with 0.1％ dimethyl sulfoxide (DMSO).Scratch assay,Transwell invasion assay and vascular channel formation assay were performed to assess the migration,invasion of and vascular channel formation by melanoma cells.A model of subcutaneous transplanted B 16 melanoma was established in 12 C57 mice,which were randomly and equally divided into 4 groups:control group gavaged with ultrapure water,low-,middle-and high-dose luteolin groups gavaged with 10,20,40 mg/kg luteolin respectively every day.The above treatment for the tumor-bearing mice lasted till day 28,and then these mice were sacrificed.Meanwhile,the lung and tumor tissues of the mice were excised,and the growth,metastasis and vasculogenic mimicry of transplanted melanoma were observed.Immunofluorescence and immunohistochemical studies were performed to evaluate the effects of luteolin on the expression of vascular endothelial cadherin (VE-cadherin),vascular endothelial growth factor receptor 1 (VEGFR1),VEGFR2,matrix metalloproteinase-2 (MMP-2) and MMP-9 in the transplanted melanoma.Means were compared among several groups by using one-way analysis of variation or rank sum test.Results In vitro study showed that the relative scratch width at 48 hours significantly differed among the control group,low-,middle-and high-dose luteolin groups (0.47 ± 0.04,0.64 ± 0.04,0.73 ± 0.03,0.84 ± 0.04 respectively;F =34.51,P ＜ 0.001),and the migration ability of B16 cells was significantly lower in the low-,middle-and high-dose luteolin groups than in the control group (all P ＜ 0.05).At 24 hours,there were significant differences in the number of cells crossing the Transwell membrane among the control group,low-,middle-and high-dose luteolin groups (281.00 ± 8.79,169.00 ± 15.35,92.00 ± 14.79 and 57.00 ± 13.72 respectively;F =275.30,P ＜ 0.001),and the invasive ability was significantly lower in the low-,middle-and high-dose luteolin groups than in the control group (P ＜ 0.01).Meanwhile,the number of formed vascular channels also differed among the above 4 groups (20.00 ± 2.77,11.00 ± 1.28,7.00 ± 1.86 and 2.00 ± 1.32 respectively;F =48.61,P ＜ 0.001),and the number of vascular channels was significantly lower in the low-,middle-and high-dose luteolin groups than in the control group (all P ＜ 0.01).In vivo study showed that the tumor size significantly differed among the control group,low-,middle-and high-dose luteolin groups (5.10 ± 1.72,4.02 ± 2.13,2.98 ± 0.92,1.49 ± 1.13 cm3 respectively;F =28.76,P ＜ 0.001),and was significantly lower in the low-,middle-and high-dose luteolin groups than in the control group (t =3.86,7.11 and 13.06 respectively,all P ＜ 0.01).CD31-PAS double staining showed that the number of vasculogenic mimicry was significantly higher in the control group than in the low-,middle-and high-dose luteolin groups (all P ＜ 0.01).In vivo and in vitro studies both showed that the expression of vasculogenic mimicry-related markers in the cells or mouse tumor tissues was significantly lower in the high-dose luteolin group than in the control group (P ＜ 0.05).Conclusion Luteolin can effectively inhibit the growth,metastasis and vasculogenic mimicry formation of melanoma.
