Objective To investigate the effect and mechanism of Complex Xueshuantong on the angiogenesis of rhesus choroid-retina endothelial (RF/6A) cells. Methods This experimental research included 4 parts. ①RF/6A cells in logarithmic growth phase were cultured and divided into blank control group, positive control group (0.20 mg/ml protamine) and Complex Xueshuantong groups at different concentrations (0.39 to 50.00 mg/ml). Vascular endothelial growth factor (VEGF) at terminal concentration of 10 ng/ml was added into all groups. The optic absorptions (A values) of each group were measured 24 hours later. ②RF/6A cells in logarithmic growth phase were cultured in Transwell insert and divided into blank control group, Complex Xueshuantong groups at different concentrations of 3.13, 6.25, 12.50 mg/ml. The number of cells that migrated to under the Transwell membrane was counted after cultured for 24 hours. ③RF/6A cells were divided into blank control group, Complex Xueshuantong groups at different concentrations of 0.39, 0.78, 1.56 mg/ml. The number of full-formed tubes was counted after cultured for 12 hours. ④RF/6A cells were divided into blank control group, positive control group of protamine at terminal concentration of 0.20 mg/ml, and Complex Xueshuantong groups at different concentrations of 1.56, 3.13, 6.25 mg/ml. Western Blot and real-time quantitative RT-PCR were used to detect the expression of VEGF and matrix metalloproteinase 2 (MMP-2) protein and mRNA of RF/6A cells after cultured for 24 hours. Data were statistically analyzed by ANOVA method. Results ①The difference of the A values within groups was statistically significant (F=158.669, P＜0.01). Compared with that of blank control group,the A values of Complex Xueshuantong groups at different concentrations from 0.78 to 25.00 mg/ml were significantly reduced (all P＜0.01), the proliferation of RF/6A cells induced by VEGF was inhibited by Complex Xueshuantong at certain concentrations. ②When the concentrations of Complex Xueshuantong were 0, 3.13, 6.25, and 12.50 mg/ml, the number of RF/6A cells that migrated to under the Transwell membrane was 123.3±13.8, 114.3±15.5, 54.0±6.1, and 40.3±10.1, respectively.The difference within groups was statistically significant (F=36.918, P＜0.01). Compared with that of blank control group, the cells of Complex Xueshuantong groups at different concentrations of 6.25 and 12.50 mg/ml were significantly reduced (all P＜0.01), the migration of RF/6A cells was inhibited by Complex Xueshuantong at certain concentrations. ③When the concentrations of Complex Xueshuantong were 0, 0.39, 0.78, and 1.56 mg/ml, the number of tubes formed in Matrigel was 20.3±2.5, 12.7±2.1, 9.7±1.2, and 0.7±0.6, respectively. The difference within groups was statistically significant (F=64.324, P＜0.01). Compared with that of blank control group, the number of tubes in 0.39, 0.78,and 1.56 mg/ml groups were significantly reduced (P＜0.01), the tube formation of RF/6A cells was inhibited by Complex Xueshuantong at certain concentrations and with the concentration being increased, the inhibition was enhanced. ④In blank control group, positive control group, and Complex Xueshuantong groups at the concentrations of 1.56, 3.13, and 6.25 mg/ml, there were significant differences in the relative content of VEGF and MMP-2 protein expression level (F=343.346,1670.505, P＜0.01). Compared with that of blank control group, the other groups were significantly reduced (all P＜0.01), the VEGF and MMP-2 protein expression level of RF/6A cells was inhibited by Complex Xueshuantong at certain concentrations and with the concentration being increased, the inhibition was enhanced. There were significant differences in the relative content of VEGF and MMP-2 mRNA expression level (F=228.130, 208.579, P＜0.01). Compared with that of blank control group, the relative content of VEGF and MMP-2 mRNA expression level of all other groups were significantly reduced (all P＜0.01), the VEGF and MMP-2 mRNA expression level of RF/6A cells was also inhibited by Complex Xueshuantong at certain concentrations and with the concentration being increased, the inhibition was enhanced. Conclusion Complex Xueshuantong could inhibit the angiogenesis of RF/6A cells through the path of suppression of the proliferation, migration, and tube formation of RF/6A cells. The mechanism was likely to be related to the inhibition of the expression of proteins and mRNA of VEGF and MMP-2 in RF/6A cells.%目的 探讨复方血栓通干预猴脉络膜-视网膜内皮细胞(RF/6A)参与血管生成的作用及机理。方法 实验研究。本研究分为四部分：①取对数生长期RF/6A细胞进行培养，设立空白对照组、阳性对照组以及不同浓度的复方血栓通观察组，每组均加入终浓度为10 ng/ml的血管内皮生长因子(VEGF)，阳性对照组加入终浓度0.20 mg/ml的硫酸鱼精蛋白，复方血栓通观察组设立0.39～50.00 mg/ml的多个不同浓度组。培养24h后采用酶联免疫检测仪测定各组吸光度A值，观察不同浓度的复方血栓通对VEGF诱导的RF/6A细胞增殖的影响。②设立空白对照组和终浓度为3.13、6.25、12.50 mg/ml的复方血栓通组，培养24h后检测不同浓度复方血栓通对RF/6A细胞移行的影响。③设立空白对照组和终浓度为0.39、0.78、1.56 mg/ml的复方血栓通组，培养12 h后检测不同浓度的复方血栓通对RF/6A细胞管腔形成的影响；④设立空白对照组、终浓度为0.20 mg/ml的硫酸鱼精蛋白阳性对照组和终浓度为1.56、3.13、6.25 mg/ml的复方血栓通组，培养24h后，运用Western Blot和实时定量逆转录聚合酶链反应(RT-PCR)的方法，分别检测不同浓度的复方血栓通对RF/6A细胞表达VEGF、基质金属蛋白酶2(MMP-2)蛋白和mRNA的影响。对多组计量资料进行单因素方差分析。结果 ①各组间A值差异有统计学意义(F=158.669，P＜0.01)，同空白对照组比较，0.78～25.00 mg/ml浓度的复方血栓通对VEGF诱导的RF/6A细胞增殖具有明显抑制作用（P均＜0.01）；②复方血栓通浓度为0、3.13、6.25和12.50 mg/ml时，RF/6A细胞移行数分别为123.3±13.8、114.3±15.5、54.0±6.1和40.3±10.1，组间差异有统计学意义(F=36.918，P＜0.01)；与空白对照组比较，6.25和12.50 mg/ml浓度的复方血栓通对RF/6A细胞移行具有明显的抑制作用（P均＜0.01）；③复方血栓通浓度为0、0.39、0.78和1.56 mg/ml时RF/6A细胞内皮管腔形成数分别为20.3±2.5、12.7±2.1、9.7±1.2和0.7±0.6，组间差异有统计学意义(F=64.324，P＜0.01)；与空白对照组比较，0.39、0.78和1.56 mg/ml浓度的复方血栓通对RF/6A细胞内皮管腔形成均具有明显的抑制作用（P均＜0.01），且抑制作用随浓度增加而增强；④空白对照组、阳性对照组以及1.56、3.13和6.25 mg/ml浓度的复方血栓通组VEGF和MMP-2蛋白相对含量组间差异均有统计学意义(F=343.346、1670.505，P均＜0.01)；与空白对照组比较，其余各组均具有显著抑制RF/6A细胞VEGF和MMP-2蛋白表达的作用（P均＜0.01），复方血栓通的抑制作用随浓度增加而增强；各组VEGF和MMP-2 mRNA相对含量的差异也有统计学意义(F=228.130，208.579，P均＜0.01)，与空白对照组比较，其余各组均具有显著抑制RF/6A细胞VEGF mRNA表达的作用（P均＜0.01），复方血栓通的抑制作用随浓度增加而增强。结论 复方血栓通可能通过抑制RF/6A细胞增殖、移行以及管腔形成来抑制其参与新生血管生成，其机理可能与抑制RF/6A细胞VEGF、MMP-2的表达有关。
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