【作者】 潘运龙；

【导师】 蔡继业；

【作者基本信息】 暨南大学 ， 生物医学工程， 2007， 博士

【摘要】 目的：近年研究表明纳米金能够阻止具有肝素结合位点的VEGF165与其受体VEGFR-2的结合，并抑制VEGF165的信号传导。本实验利用纳米金的这种新特性，研究纳米金能否抑制血管内皮细胞增殖并抑制裸鼠肝癌血管生成。以往研究认为白介素12（IL-12）通过诱导干扰素-γ（IFN-γ）、干扰素-γ诱导蛋白-10（IP-10）和干扰素-γ诱导单核因子（MIG）的生成，进而抑制肿瘤血管生成。然而，VEGF和Ang-2（血管生成素-2）是目前已知血管内皮细胞增殖的主要促进因子，我们推测IL-12是通过抑制肝癌组织中VEGF和Ang-2基因的表达，抑制血管内皮细胞增殖，发挥其抑制肝癌的血管生成和肝癌生长的作用。这种推测需要用实验加以证实。方法：1．用免疫印迹方法检测纳米金是否能特异性的与bFGF肝素结合位点结合；用纳米金与血管内皮细胞作用，检测细胞下游信号PLC—γ1的变化；用缺乏肝素结合位点的VEGF121作为对照，观察纳米金对血管内皮细胞增殖的影响；用AFM表征纳米金与VEGF165、VEGF121作用前后形貌大小变化，以及纳米金与血管内皮细胞作用前后细胞超微结构变化。将H22肝癌接种到Balb／c裸鼠，用或不用纳米金处理作为治疗组和对照组。用免疫组化方法检测肝癌微血管密度。测量肝癌大小、重量。2．构建含小鼠白介素12（mIL-12）双亚基基因的重组腺病毒（AdvmIL-12），检测重组腺病毒对H22肝癌细胞的感染效率。用ELISA法测定H22细胞中mIL-12蛋白的表达量；观察AdvmIL-12对H22细胞生长的影响。将AdvmIL-12转染的肝癌H22细胞作为实验组，以Adv-EGFP／H22为载体对照组和H22为空白对照组。这些细胞被分批接种到裸鼠肾包膜下，右前肢皮下。用RT-PCR法检测肝癌组织中的VEGF和Ang-2基因的表达；免疫组织化学方法测量肿瘤组织中微血管密度。测量肿瘤大小和重量。比较组间差别，P＜0.05为有显著性差异。结果：1．体外实验表明，纳米金能够与具有肝素结合位点的bFGF结合；抑制VEGF165的信号传导；明显抑制VEGF165促进血管内皮细胞增殖作用。而纳米金对缺乏肝素结合位点的VEGF121没有抑制作用。2．裸鼠体内实验发现，用纳米金处理的实验组微血管密度为14.27±1.08，对照组微血管密度为23.52±1.36，实验组微血管密度较对照组明显减少（t=21.694，P＜0.01）。实验组肿瘤体积较小，重量较轻，与对照组比较有显著性差异，P＜0.05。3．在近生理条件下，AFM检测到纳米金与VEGF165、VEGF121作用前后血管内皮细胞超微结构变化。当血管内皮细胞处于增殖状态时，细胞表面出现许多大小约数百纳米的颗粒，细胞膜边缘明显延伸，伪足明显延长。相反地，当血管内皮细胞被纳米金抑制时，细胞表面颗粒大为减少，伪足缩短。4．在AdvmIL-12感染复数为100、感染时间为2小时情况下，重组腺病毒的转染率为96.41％。实验组细胞上清液中检测到IL-12蛋白的表达量为（89.71±22.05）ng．48h^-1．10⁶ cells^-1。AdvmIL-12对H22细胞的生长无抑制作用。5．裸鼠体内实验表明，实验组肝癌组织中IL-12蛋白的表达量明显高于对照组，而实验组VEGF和Ang-2基因的表达弱于对照组。VEGF基因比值分别为：实验组0.8867±0.1924，载体对照组1.1744±0.1189，空白对照组1.1874±0.1752，P＜0.05。Ang-2基因比值分别为：实验组0.7878±0.1471，载体对照组1.0319±0.1574，空白对照组1.0361±0.1536，P＜0.05。实验组肿瘤平均重量、肿瘤体积和肿瘤微血管密度明显少于对照组，差异有显著性。结论：1．体外实验表明，纳米金能够抑制VEGF165的信号传导，明显抑制VEGF165促进血管内皮细胞增殖作用。而纳米金对缺乏肝素结合位点的VEGF121没有抑制作用。2．在裸鼠体内，纳米金通过阻止VEGF165与VEGFR-2结合，抑制H22肝癌血管生成和肝癌生长。3．AFM探测结果表明，纳米金抑制了VEGF165促血管内皮细胞增殖的作用。4．AdvmIL-12高效感染H22细胞，对体外培养H22细胞的生长无抑制作用。5．在缺乏T细胞的裸鼠体内，IL-12通过抑制肝癌组织中VEGF和Ang-2基因的表达，并抑制血管内皮细胞增殖，发挥其抑制H22肝癌血管生成和肝癌生长的作用。本实验结果将有助于肝癌及其他实体肿瘤的抗血管生成治疗。更多还原

【Abstract】 Purpose: Recent studies have showed that nanogold could inhibit the binding between VEGF165 （which has heparin binding domain） and its receptor VEGFR-2, conferring the inhibition of signal transduction induced by VEGF165. Here, we investigate whether nanogold could inhibit the proliferation of vascular endothelial cells and the angiogenesis of liver cancer. Previous reports have suggested that interleukin-12 （IL-12） could induce the expression of interferon-γ（INF-γ） and then allow INF-γto induce the expression of IP-10 （inducible protein-10） and MIG （monokine induced by interferon-gama）, and inhibit the angiogenesis of tumor. Whereas the VEGF and Ang-2 are the main factors known by far, promoting the proliferation of vascular endothelial cells. We hypothesize that IL-12 present antiangiogenesis and anticancer effect by inhibiting the expression of Ang and VEGF gene in liver cancer tissue and inhibiting the proliferation of vascular endothelial cells, which needs to be confirmed by experiments.Methods: Immunoblotting was adopted to determine whether nanogold could specifically combine to heparin binding domain of bFGF. After nanogold acted on vascular endothelial cells, the changes of PLC—γ1 were examined. The effects of nanogold on the growth of vascular endothelial cells were assessed, while VEGF121 which has no heparin binding domain was set up as contral. Atomic force microscopy （AFM） was used to examine the sizes of nanogold acting with VEGF165 or VEGF121 and the changes of ultrastructure on cell surface treated with nanogold. The H22 liver cancer cells were transplanted into Balb/c nude mice, and mice were divided into two groups, experimental group treated with nanogold and control group treated with non nanogold. The microvascular density （MVD） was determined by imunohistochemistry method, and sizes and weights of liver cancer were examined after treatment with nanogold or not. 2. AdvmIL-12 was constructed and transfected into H22 cell. The transfection efficiency of AdvmIL-12 on H22 cell was detected. The expression of IL-12 protein in H22 cells was examined with ELISA and the effect of IL-12 on H22 cell growth was assessed. AdvmIL-12 was transfected into H22 cells in the experimental group, while the H22 cells were transfected with adenovirus in vector control group and the H22 cells were not transfected with anything as blank group. Then they were inoculated into the subrenal capsule or subcutaneously into right oxter of nude mouse, respectively. The expression of gene of vascular endothelial growth factor （VEGF） and angiopoietin-2 （Ang-2） in liver cancer tissue was detected by reverse transcription-polymerase chain reaction （RT-PCR）. MVD was determined by imunohistochemistry method, and sizes and weights of liver cancer were measured. The results of P＜0.05 were considered to denote statistical significance.Results:1. Nanogold could bind to bEGF with heparin binding domain and inhibit the signal transduction of VEGF 165 and proliferation of vascular endothelial cells in vitro experiment. In contrast, no significant inhibition of nanogold on VEGF121 was observed.2. In vivo experiment suggested that MVD of liver cancer tissue treated by nanogold was 14.27±1.08, while control group had a MVD about 23.52±1.36. Therefore, the MVD in experimental group decreased significantly compared to that in control group （P＜0.01）. In addition, compared to the control group, the group treated by nanogold had a significantly less tumor weights and volumes （P＜0.05）.3. In nearby physiological conditions, AFM could probe the changes of ultrastructure on vascular endothelial cells surface before and aider the treatment with nanogold and VEGF165、VEGF121. When vascular endothelial cells were being in proliferation, there were many granules about hundreds nm in size on cell surface, the edges of cell membrane extended markedly, and cellular pseudopods prolonged obviously. In contrast, when cellular proliferations were inhibited by nanogold, the number of granules on cell surface decreased clearly, and cellular pseudopods shortened obviously.4. When multiplicity of infection （MOI） was 100 and time of infection （TOI） was 2 hours, the transfection efficiency of recombinant adenovirus was about 96.41%. The expressive quantity of IL-12 in experimental cell supematant was about （89.71±22.05） ng.48h^-1.10⁶cells^-1. Data suggested that no significant inhibition effect of IL-12 against H22 cell growth was observed.5. In vivo experiment, data suggested that, compared to control groups, expression level of IL-12 in AdvmIL-12 group increased significantly. Compared to control group, the expression of VEGF and Ang-2 gene in experimental group decreased significantly. The ratioes of VEGF gene were 0.8867±0.1924 in experimental group, 1.1744±0.1189 in vector group, and 1.1874±0.1752 in blank group, respectively, and P＜0.05. The ratioes of Ang-2 gene were 0.7878±0.1471 in experimental group, 1.0319±0.1574 in vector group, and 1.0361±0.1536 in blank group, respectively, and P＜0.05. And the MVD, weights and volumes of tumor were significantly smaller than those in control groups, and P＜0.05.Conclusions:1. Nanogold could inhibit the signal transduction of VEGF165 and confer the significant inhibition effect against vascular endothelial cell proliferation induced by VEGF 165 in vitro experiment. In contrast, no significant inhibition effect of nanogold against VEGF121 was observed.2. Nanogold could inhibit the angiogenesis and the growth of H22 liver cancer by inhibiting the binding between VEGF165 and VEGFR-2 in vivo.3. AFM showed that nanogold inhibited the effects of VEGF165 on the proliferation of vascular endothelial cells.4. AdvmIL-12 could efficiently transfect into H22 cells and no significant inhibition effect oflL-12 against H22 cell growth was observed in vitro.5. In the nude mouse that absents T cells, IL-12 inhibited the angiogenesis and the growth of H22 liver cancer by inhibiting the expression of VEGF and Ang-2 gene in liver cancer tissue and the proliferation of vascular endothelial cells.These results in this experiment will be helpful for the antiangenesic treatment of liver cancer and other solid tumors.更多还原