【作者】 翟羽；

【导师】 吕占军；

【作者基本信息】 河北医科大学 ， 免疫学， 2003， 博士

【摘要】 研究目的：肿瘤是目前我国疾病死亡的主要原因之一，而原发性肝癌在恶性肿瘤中死亡率又是排在前列，严重威胁人民的健康。目前外科手术切除仍然是肝癌治疗中比较有效的手段，但能行手术切除的仅占全部患者的5.4％～24.3％。对不能手术的中、晚期肝癌患者，单纯使用化疗或放疗，疗效还不理想。二十世纪七十年代，Folkman根据实体瘤生长总伴随并依赖血管生成这一现象，提出了抗血管生成治疗肿瘤的策略。随后的事实证明，实体瘤的恶性增长、浸润及转移确实依赖于新生血管不断提供养料和氧，因此以阻断肿瘤细胞诱发的血管生成来抑制、治疗实体肿瘤的思路被越来越多的人所接受。在对白血病等血液病的治疗中，抗血管生成治疗也显示出一定的效果。体内的血管生成是一个多步骤的现象，参与多种生理和病理过程，如：胚胎发生，月经周期，伤口愈合，肿瘤的发生、发展和转移等。抗血管生成疗法就是要阻止或减弱肿瘤细胞诱发的血管生成现象的发生。与传统的放、化疗相比，抗血管生成治疗一般并不会损伤正常血管，具有副作用较小的优点。其次，针对血管内皮细胞的抗血管生成疗法从原理上讲应该不会或只会引起很低的抗药性反应。此外，抗血管生成疗法从原理上说可适用于所有实体肿瘤的治疗。这些特点使得抗血管生成疗法在肿瘤治疗中的应用，呈现出很有前途的发展趋势。大多数肝癌随肿瘤体积增大，血管密度增加，表现为肝动脉供血的多血供型，我们推测采用抗血管生成疗法治疗肝癌可能会取得较好的抑瘤效果。此外，对一些不能手术的大肝癌，行肝动脉结扎或栓塞治疗时，如果配合抗血管生成治疗也可能为部分患者争取到手术的机会和时间。研究发现，一些已有的药物就具有一定的抗血管生成和抑制肿瘤生长的作用，但它们确切的抑瘤效果目前尚无定论，它们抗肿瘤的分子机制及适用范围还未阐明。基于以上想法，我们观察了几种现有的约物对小鼠H₂₂肝癌细胞株在体内、外生长及对H₂₂移植瘤血管生成的影响，旨在探讨这些药物治疗肝癌的可能性。中文摘要 实验方法和结果: 1、药物筛选实验:观察了地塞米松、反应停、鱼精蛋白、肝素、阿司匹林五种药物对体外培养的小鼠肝癌玩2细胞、正常小鼠肝细胞和人血管内皮细胞的增殖及VEGF mRNA表达的影响。通过药物对体外培养细胞增殖的影响，反应该药物对肿瘤和/或血管内皮细胞是否具有直接杀伤作用;通过对培养细胞VEGF mRNA表达的影响，反应该药物是否对血管生成有直接的抑制作用;并观察了三种不同剂量的药物对体内玩:移植瘤生长的影响。方法:1)、体外增殖实验:玩:细胞、正常小鼠肝细胞和人脐静脉内皮细胞（ECV一304）分别用20%NBS一DN田M培养基和1%NBS一D侧田M培养基配为1 xl护/ml，96孔培养板培养。每孔加入细胞悬液100川，处理孔分别加入不同浓度的药物20贝，对照孔加等体积的生理盐水，用相应培养基补足体积到200贝。培养48h后，MTT法检测各孔550nln的吸光度。加入的地塞米松终浓度分别为9.68脚ol/L，24.21卿ol/L，48.41脚01几，96.82脚ol/L，242.06卿ol/L，484.12阿oljL，968.24娜ol/L;鱼精蛋白终浓度为10 mg/IJ，25 mg/L，50 mg/L，100m留L，250 mg/L，500 mg/L，1 000 mg几;肝素终浓度为5 U/inl，12.5 U/inl，25U/inl，50LI/ml，125U/ml，25OU/ml，500U/ml。反应停和阿司匹林对玩:细胞和ECV一304细胞增殖的影响只做了高血清浓度培养实验，培养48h后，做显微计数。反应停终浓度为0.19 nunol/L，0.48 mmol/L，0.97 mmol/L，1.94m幻nol几，4 .84 rnrnoljL，9.68力。IOoljL，19.36r口nlol/L;阿司匹林终浓度为0 .67 ranooljL，1 .67 mmol/L，3.33 ron以ol/L，6.66力。刀nol/L，16.65 mmoljL，33.30 rn刃。o比，66.60们。幻以。比。2)、培养细胞的VEGF mRNA表达:玩:细胞用20%NBs.DN田M培养基配为1 xl护/ml，24孔培养板培养。每孔加入细胞悬液1 000川，处理孔分别加入不同浓度的药物200川，对照孔加入等体积生理盐水，每孔再补加入800川培养基。加入的地塞米松终浓度分别为9.68卿ol/L，24.21林mol/L，48.41卿0比，96·82卿。比;鱼精蛋白终浓度为10mg/L，25mg/L，50mg/L，100mg/L，250 mg/L;肝素终浓度为5 U/ml，12.SU/ml，25 U/ml，50 U/ml，125 U/ml，250U/ml;反应停终浓度为0.19 mmol/L，0.48rn力。ol/L，0.97 mmol/L，l.94r口rnol/L，4.84 mmol/L;阿司匹林终浓度为0.67 mmol/L，1.672中文摘要mmol/L，3 .33 mmol/L，6.66 mmol/L，16.65 rn们nol/L。ECV一304细胞不「l正常小鼠肝细胞VEGF mRNA表达实验，只设了对照组和加入96 .82林m。讥地塞米松的处理组。培养48h后，换为无血清DMEM培养基培养，24h后收集培养细胞，提取RNA，以GAPDH做内参，做RT一PCR。PCR产物做5%队GE，银染显色。结果用图像分析系统做图像分析，计算各样本的VEGF相对表达量。3)、体内剂量试验:BALB/c小鼠每只皮下接种玩:细胞1 X 10v/0.2 ml，随机分为给药组和对照组。从分组当天开始，给药组小鼠每只分别腹腔注射地塞米松2.5m叭g、sm眺g、10m叭g;反应停50m叭g、200m?更多还原

【Abstract】 Objective: Tumor is a main reason for die of disease, and the mortality of original hepatoma is highest in malignant tumors. At present, surgery removal is still the most useful method of hepatoma treatment, but only 5.4 %~24.3 % of sufferer has opportunity to do the operation. Now, there still has no ideal curative effect on medium or late hepatoma with traditional chemical therapy or radiation therapy. In 1970’s, Folkman brought forward a novel anticancer strategy-anti-angiogenesis based on the phenomena that cancer growth is accompanied with neovascularization. Then it has been proved that tumor growth, development and metastasis did depend on neovascularization. Now more and more peoples have accepted the anti-angiogenesis strategy. Even in treatment to leucocythemia, anti-angiogenesis method has some effect. In vivo, angiogenesis is concerned with many physiological and pathological courses, such as female reproductive system, wound healing, growth and metastasis of tumors. Anti-angiogenesis is just to reject or weaken the neovascularization induced by tumor cells. Compared with traditional chemical and radiation methods, anti-angiogenesis in usually does not injure normal vessel and has less side effects; moreover, the therapy directing to endothelial cells, in theory, has no or little acquired resistance, and in mechanism the therapy fits to all kinds of solid tumors. Because of those reasons, a very good future of anti-angiogenesis strategy is shown in curing cancer. Most of hepatoma depends on angiogenesis, so it is presumed that anti-angiogenic therapy will have better effect in treating hepatoma. Furthermore, if the liver artery ligation or embolus to some suffers having the inoperable larger hapatoma were combined with anti-angiogenic treatment, the opportunity of operation for these patients would be increased. In reports, some drugsthat have already been used in other diseases have the anti-angiogenesis and anti-tumor activity too, but the exact curative effect, molecular mechanism and the scope of application are still unknown. In those opinions, we detected the effects of some drugs on tumor growth and angiogenesis of grafted Hi2 cells in mouse to study the possibility of them to cure hepatoma.Methods and results:1. Drug screening: The effects of five drugs, dexamethasone, thalidomide, protamine, heparin and aspirin, on cell proliferation and VEGF expression in cultured H22 cells, normal hepatic cells, and human umbilical vein endothelial cells (ECV-304) were observed. The proliferation experiment suggested the direct inhibition of drug to the cells; the VEGF expression suggested the ability of drug to inhibit angiogenesis; we also observed the effects of three dosages of drugs on tumor growth in mouse H22 model to judge the efficiency of drug inhibition. Methods: 1) Proliferation in vitro: H22 cells, normal hepatic cells and ECV-304 cells were suspended at a concentration of 1X105/ ml in 20 % NBS-DMEM or in 1 % NBS-DMEM medium. The cell suspension was distributed into 96 micro well plates. Each well included 100 ul of cell suspension, 20 ul of drug at different concentrations and 80 ul of relevant culture medium, then the final volume was 200 ul. In control wells 20 ul normal saline was substituted for drug solution. After 48 h in culture, the absorbency was detected at 550 nm by MTT method. The last concentration of dexamethasone was 9.68 umol/L, 24.21 umol/L, 48.41 umol/L, 96.82 umol/L, 242.06 umol/L, 484.12 umol/L, and 968.24 umol/L; of protamine was 10 mg/L, 25 mg/L, 50 mg/L, 100 mg/L, 250 mg/L, 500 mg/L, and 1 000 mg/L; of heparin was 5 U/ml, 12.5 U/ml, 25 U/ml, 50 U/ml, 125 U/ml, 250 U/ml, and 500 U/ml. Thalidomide and aspirin were added only in 20 % NBS-DMEM when H22 cells and ECV-304 cells were cultured. After 48 h, cell counting was done with microscope. The last concentration of thalidomide was 0.19 mmol/L, 0.48 mmol/L, 0.97 mmol/L, 1.94 mmol/L,104.84 mmol/L, 9.68 mmol/L, and 19.36 mmol/L; of aspirin was 0.67 mmol/L, 1.67 mmol/L, 3.33 mmol/L, 6.66 mmol/L, 16.65 mmol/L更多还原