董孟佳 许尤琪 沈明勤

摘要：目的 观察健脾活血方对肝癌裸鼠皮下移植瘤模型细胞凋亡和血管生成的影响，探讨其可能的作用机制。方法 建立SMMC-7721肝癌荷瘤裸鼠模型。将60只雄性荷瘤裸鼠随机分为空白组、模型组、5-Fu组和健脾活血方高、中、低剂量组，每组10只。健脾活血方各剂量组给予相应浓度健脾活血方药液灌胃，每日1次，5-Fu组给予5-Fu隔日腹腔注射，连续2周。2周后比较各组裸鼠体质量、瘤质量和抑瘤率，免疫组化检测血管内皮生长因子（VEGF）和VEGF受体2（VEGFR-2）的蛋白表达，计算微血管密度（MVD），qRT-PCR检测Caspase-3和生存素（Survivin）的mRNA表达。结果 与模型组比较，各给药组VEGF、VEGFR-2蛋白表达，MVD和Caspase-3、Survivin mRNA表达均降低，其中5-Fu组和健脾活血方高剂量组作用最明显（P<0.05）。结论 健脾活血方可能通过抑制血管生成和诱导细胞凋亡作用抑制肿瘤生长。

关键词：健脾活血方；肝癌；血管生成；细胞凋亡；小鼠

DOI：10.3969/j.issn.1005-5304.2018.02.009

中图分类号：R285.5 文献标识码：A 文章编号：1005-5304（2018）02-0038-04

Study of Anti-tumor Effect of Jianpi Huoxue Prescription on SMMC-7721 Hepatoma Mice DONG Meng-jia1， XU You-qi2， SHEN Ming-qin3

1. Nanjing University of Chinese Medicine， Nanjing 210023， China；

2. NO.2 Hospital of TCM of Jiangsu Province， Nanjing 210023， China；

3. Jiangsu Province Institute of Chinese Medicine， Nanjing 210028， China

Abstract： Objective To observe the effects of Jianpi Huoxue Prescription on the subcutaneous transplantation of tumor cell apoptosis and angiogenesis； To discuss its possible mechanism of action. Methods SMMC-7721 hepatoma mice models were established. 60 tumor bearing male mice were randomly divided into blank group， model group， 5-Fu group， Jianpi Huoxue Prescription high-， medium- and low-dosage groups， with 10 rats in each group. Model group and Jianpi Huoxue Prescription high-， medium- and low-dosage groups were given medicine with relevant concentrations for gavage， once a day. 5-Fu group was given 5-Fu for intraperitoneal injection every other day. The weight， tumor weight and tumor inhibitory rate of each group were compared two weeks later. The protein expressions of VEGF and VEGFR-2 were detected by immunohistochemistry， microvessel density （MVD） were calculated， and the expressions of Survivin and Caspase-3 mRNA were detected by RT-qPCR. Results Compared with model group， protein expressions of VEGF and VEGFR-2， MVD， Caspase-3 and Survivin mRNA decreased in Jianpi Huoxue Prescription high-， medium- and low-dosage groups， and the most obvious differences were in 5-Fu group and Jianpi Huoxue Prescription high-dosage group （P<0.05）. Conclusion Jianpi Huoxue Prescription can inhibit tumor growth by inhibiting angiogenesis and inducing apoptosis.

Keywords： Jianpi Huoxue Prescription； hepatoma； angiogenesis； apoptosis； mice

健脾活血方臨床用于治疗肝癌，具有抑制肿瘤生长、提高生存质量和对化疗减毒增效的作用[1]。本实验观察健脾活血方对肝癌裸鼠皮下移植瘤模型肝癌细胞凋亡和血管生成的影响，探讨其可能的作用机制，为临床应用提供依据。

1 实验材料

1.1 细胞株和动物

SMMC-7721人肝癌细胞株，南京凯基生物科技发展有限公司；SPF级BALB/c（nu/nu）裸鼠60只，雄性，5～7周龄，体质量18～20 g，南京沐途医疗科技有限公司，动物许可证号SYXK（苏）2016-0010，饲养于南京中医药大学SPF级动物实验室。

1.2 药物及制备

健脾活血方（黄芪30 g，太子参30 g，茯苓10 g，白术10 g，山药10 g，茵陈15 g，薏苡仁30 g，莪术15 g，石打穿15 g，半枝莲15 g），南京中医药大学附属第二医院制剂中心制备，饮片浸泡2 h，煮沸后改文火煎煮15 min，纱布过滤取汁，药渣加水再次煎煮30 min，2次药汁混合，再浓缩并定容；……