抗血管内皮生长因子抗体对肝癌的放射增敏作用

抗血管内皮生长因子抗体对肝癌的放射增敏作用

                   作者：郑青平,陈龙华,石玉生

【关键词】  肝肿瘤;放射疗法;抗VEGF单抗;放射增敏

　　Radiosensibility of human hepatocellular carcinoma xenografts enhanced by antivascular endothelial growth factor monoclonal antibody

　　【Abstract】 AIM: To investigate the inhibiting effects of combining antivascular endothelial growth factor (VEGF) monoclonal antibody (mAb) therapy with radiotherapy on the growth of hepatocellular carcinoma xenografts in vivo. METHODS: Human hepatocellular carcinoma cell line, SMMC7721, was implanted sc in mice. The mice with xenografts were divided into 4 groups, radiation (RT) 20 Gy group, RT 20 Gy plus mAb group, RT 30 Gy plus mAb group and control group. AntiVEGF mAb was injected ip on alternate days for a total of 6 injections at a dosage of 50 μg/mouse. Single radiation doses were given 24 h after the 4th injection of mAb. Tumor diameters were measured and tumor volume was calculated. Mice were sacrificed 2 wk after the 6th injection and the intratumoral microvessel density was determined by counting the stained vessels with immunohistochemistry. RESULTS: Radiation significantly reduced the growth and the microvessel density of tumors. The inhibitory effects were more significant when radiation was combined with antiVEGF mAb, and RT 30 Gy plus mAb was more effective than RT 20 Gy plus mAb. For RT 20 Gy group, RT 20 Gy plus mAb group and RT 30 Gy plus mAb group, the inhibitory rates of tumor weight were 75.3%, 83.9% and 94.7%, respectively. CONCLUSION: AntiVEGF mAb enhances the radiosensibility of human hepatocellular carcinoma xenografts.The strategy is one of the approaches for hepatocellular carcinoma treatment.

　　【Keywords】 hepatocellular carcinoma; radiotherapy; antiVEGF mAb; radiosensibility

　　【摘要】 目的: 观察抗血管内皮生长因子抗体(VEGF mAb)与不同剂量放射联合对肝癌裸鼠移植瘤生长的抑制作用. 方法: 将SMMC7721肝癌细胞种植于裸鼠皮下,成瘤动物分为单纯放射20 Gy组、放射20 Gy+抗体组、放射30 Gy+抗体组和对照组,腹腔给予抗VEGF mAb 50 μg/只，隔日1次,共6次,放射剂量一次性给予.测量肿瘤直径并计算瘤体积,抗体给药结束后2 wk处死动物,免疫组化法测肿瘤微血管密度(MVD). 结果: 放射能抑制肿瘤生长,减少肿瘤MVD,放射与抗体结合对肿瘤生长的抑制作用更显著，且放射30 Gy+抗体比放射20 Gy+抗体效果更好.单纯放射20 Gy组、放射20 Gy+抗体组和放射30 Gy+抗体组的瘤质量抑制率分别为75.3%,83.9%和94.7%,差异有统计学意义. 结论: 抗VEGF mAb对放射治疗肝癌移植瘤有增敏作用,是肝癌综合治疗的有效途径之一.

　　【关键词】 肝肿瘤;放射疗法;抗VEGF单抗;放射增敏

　　0引言

　　放射治疗肿瘤的目的是尽可能提高肿瘤的放射剂量以杀灭瘤细胞,但同时正常组织和器官所受辐射量也相应提高,增加了放射治疗的副反应,因而限制了通过提高放射剂量来控制肿瘤的作用.肝癌是放射欠敏感肿瘤,而正常肝组织对放射线较敏感.单纯放射治疗肝癌的疗效并不理想［1］.有报道［2］抗血管内皮生长因子(vascular endothelial growth factor, VEGF)抗体可以抑制肝癌裸鼠移植瘤生长,我们观察抗VEGF mAb与不同放射剂量联合对肝癌移植瘤生长的抑制作用,以了解两者联合应用的机制与方法.

　　1材料和方法

　　1.1材料Balb/cnu/nu小鼠20只(南方医科大学实验动物中心),5～6 wk龄,雄性,体质量19.0～22.0 g, SPF层流柜分笼饲养; SMMC7721人肝癌细胞株（武汉大学中国典型培养物保藏中心）; Monoclonal antiVEGF antibody, produced in mouse, clone 26503.11（Sigma公司）;培养箱（37℃,50 mL/L CO2）,倒置显微镜(XDP1,上海光学仪器厂),Varian 23EX医用直线加速器（美国）,RPMI1640细胞培养液（含100 mL/L胎牛血清,青霉素1×105 U/g,链霉素100 mg/L）,胰蛋白酶,免疫组化SABC法相关试剂（武汉博士德公司）.

　　1.2方法

　　1.2.1癌细胞培养SMMC7721人肝癌细胞株于25 mL培养瓶中繁殖传代,细胞贴壁长满瓶壁后移入250 mL培养瓶继续传代繁殖,取指数生长期细胞胰酶消化,2000 r/min离心2 min,收集细胞用生理盐水悬浮,显微镜下以细胞计数板计算细胞密度,生理盐水定容细胞密度为2×1010/L.

　　1.2.2癌细胞种植参考文献［3］方法,裸鼠于饲养环境适应1 d后种植癌细胞,取裸鼠右后肢大腿外侧皮下为种植点,1 mL注射器抽取0.2 mL细胞悬液（含4×106个细胞）注入种植点皮下.

　　1.2.3动物分组及肿瘤生长观察肿瘤种植后1 wk, 可观察到全部动物有明显皮下肿块形成,第10日肿块直径约5 mm.将动物随机分为4组,即单纯放射20 Gy组、放射20 Gy+抗体组、放射30 Gy+抗体组和对照组,每组5只.用游标卡尺测量肿块长径（a）及横径（b）,隔日测量1次,肿瘤体积(V)=a×b2/2.

　　1.2.4抗体给予及放射治疗以0.2 μm针式滤器过滤除菌的PBS液溶解抗VEGF mAb至100 mg/L,于肿瘤种植后10 d分组后腹腔注射抗VEGF mAb, 50 μg/只,隔日1次,共6次.单纯放射组及对照组同样途径给予等体积量PBS.第4次给予抗VEGF mAb后的第2日行放射治疗,放疗机房紫外线消毒,受照动物四肢以细绳固定在小木板上,照射野大小3 cm×3 cm,源皮距100 cm,6 MeV电子线,剂量率4 Gy/min,单纯放射组剂量20 Gy,放射+抗体组剂量分别为20 Gy和30 Gy,全部放射剂量一次性给予.

　　1.2.5瘤体称质量抗体给药结束后继续观察肿瘤生长2 wk,于肿瘤种植后34 d处死动物,仔细分离肿块周边皮肤及非肿瘤组织,瘤体称质量,瘤质量抑制率(%)=（1－实验组平均瘤质量/对照组平均瘤质量）×100.

　　1.2.6免疫组化微血管密度(MVD)测定将肿瘤组织切成3 mm厚片状,40 g/L甲醛固定24 h,脱水石蜡包埋,切片厚5 μm.免疫组化SABC法:一抗为多克隆兔抗鼠CD34抗体,工作浓度1∶100稀释.即用型HighSABC免疫组化试剂盒,具体操作按试剂盒说明进行,DAB染色,苏木素复染,树胶封片.选血管内皮细胞染色密集区于高倍镜(×200)下计数染色细胞,每片计数5个视野取平均值.

　　统计学处理 SPSS 10.0统计分析软件,应用重复测量数据方差分析处理肿瘤体积变化数据,多个独立样本非参数检验处理瘤质量抑制数据和MVD数据,P<0.05为差异有统计学意义.