纳米金抑制裸鼠肝癌血管生成及肝癌生长

Nanogold inhibits angiogenesis and growth of liver cancer： experiment with mice

目的观察纳米金对裸鼠H22肝癌血管生成及肝癌生长的抑制作用。方法运用原子力显微镜（AFM）观察纳米金与血管内皮生长因子165（VEGF165）作用前后大小变化。AFM表征纳米金作用前后人脐静脉血管内皮细胞（HUVEC）表面超微结构变化。无血清培养HUVEC，加入VEGF165和不同浓度纳米金，作用5rain，Western印迹方法测定血管内皮生长因子受体-2（VEGFR-2）上的磷酸化磷脂酶C（PLC-γl）蛋白。6周龄Balb／c裸鼠20只，从裸鼠右腋皮下注入H22细胞，肿瘤形成约8mm大小，随机分2组，实验组从肿瘤周围及瘤内注入纳米金，每天1次，连续8d，对照组用生理盐水处理。处死裸鼠时测量肿瘤体积及重量，免疫组化染色并计算微血管密度（MVD）。结果AFM检测到纳米金与VEGF165作用后，粒径普遍〉30nm。AFM观察纳米金与VEGF165作用前后内皮细胞超微结构变化，这些变化与血管内皮细胞处于增殖或抑制状态相关。VEGF；65浓度不变（10μg/L），随着纳米金溶液浓度的增加，从125，250，到500nmoL／L，纳米金抑制PLC-11磷酸化越来越明显。肝癌组织内微血管密度分别为，实验组14．27±1．08，对照组23．52±1．36，表明纳米金抑制了H22肝癌血管生成（P〈0．01）。实验组平均肿瘤重量为（1．39±0．08）g，平均瘤体积为（1．37±0．34）cm^3；而对照组平均肿瘤重量为（2．47±0．15）g，平均瘤体积为（2．49±0．78）cm^3；抑瘤率为43．72％，表明纳米金抑制了H22肝癌的生长（P〈0．05）。结论纳米金明显抑制裸鼠肝癌血管生成及肝癌生长，可能与纳米金阻断VEGF165的信号传导有关。

Objective To investigate the effects of nanogold in inhibition of angiogenesis and growth of liver cancer cells. Methods Nanogold was co-incubated with VEGF165 and VDGF121 respectively. Atomic force microscopy （AFM） was used to observe the changes of the form of the particles. Human umbilical vascular endothelial cells （HUVEC） were serum-starved for 24 hours, then co-cultured with VEGF165 ＋ nanogold or VEGF121 ＋ nanogold for 24 h. ATM was used to observe the uhrastructure of the cells. Another HUVEC were serum-starved for 24 hours and then cultured with VEGF165 （ 10 trg/L） 100 μl ＋ nanogold 125,250,and 500 nmol/L 100 μl respectively for 5 min. Then Western blotting was used to detect the phosphorylation protein of phospholipase C （ PLC ） -γ1 on VEGFR-2. Hepatocellular cancer ceils of the line H22 were injected subcutaneously into the right armpits of 20 Balb/c nude mice. When the size of transplanted tumor reached about 8 mm, the mice were divided into 2 equal groups ： experimental group undergoing injection of nanogold into the tumor once a day for 8 days, and control group injected with normal saline. On day 14 the mice were sacrificed with the liver tumors taken out to measure the size and weight. The microvascular density （MVD） of tumor was determined by immunohistochemical staining. Results ATM showed that acted with VEGF165, the size of nanogold became over 30 nm. Treated with VEGF165 the HUVEC became larger with obvious pseudopodiums. However, such changes were obviously milder in those HUVEC treated with nanogold ＋ VEGF165. The PLC-γl phosphorylation level VEGF receoptor-2 was decreased along with the increase of the concentration of nanogold. The MVD of liver cancer tissue in the experimental group was 14.27 ± 1.08, significantly lower than that of the control group [ （ 23.52 ± 1.36）, P 〈 0. 01 ]. The mean weight and volume of tumor of the experimental group were （ 1.39 ± 0. 08 ） g and （ 1.37 ± 0. 34） cm^3 respectively, both significantly lower than those of the control group [ （2.47 ±0. 15） g and （2.49 ±0. 78） cm^3 respectively, both P 〈0. 05] with a tumor growth inhibition rate of 43.72%. Conclusion Nanogold significantly inhibits the angiogenesis and growth of liver cancer cells with the possible mechanism that nanogold inhibits the VEGF165-induced signaling.