维a酸注射液对裸鼠人肝癌细胞smmc-7721移植瘤的抑制作用

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1、中图分类号：R965；R979.1硕士学位论文维A酸注射液对裸鼠人肝癌细胞SMMC-7721移植瘤的抑制作用院（系）、所药学院研究生姓名张磊学科、专业药理学导师姓名刘明华教授二Ο一二年五月目录1维A酸注射液对裸鼠人肝癌细胞SMMC-7721移植瘤的抑制作用11.1中文摘要„„„„„„„„„„„„„„„„„„„„„„„„11.2英文摘要„„„„„„„„„„„„„„„„„„„„„„„„31.3前言„„„„„„„„„„„„„„„„„„„„„„„„„„71.4材料与方法„„„„„„„„„„„„„„„„„„„„„„„101.5结

2、果„„„„„„„„„„„„„„„„„„„„„„„„„„201.6讨论„„„„„„„„„„„„„„„„„„„„„„„„„„271.7结论„„„„„„„„„„„„„„„„„„„„„„„„„„321.8参考文献„„„„„„„„„„„„„„„„„„„„„„„„331.9英汉缩略词对照表„„„„„„„„„„„„„„„„„„„„362致谢„„„„„„„„„„„„„„„„„„„„„„„„„„373综述„„„„„„„„„„„„„„„„„„„„„„„„„„384参考文献„„„„„„„„„„„„„„„„„„„„„„„„46维A酸注射液对裸

3、鼠人肝癌细胞SMMC-7721移植瘤的抑制作用摘要目的：维A酸为目前用于临床肿瘤化学治疗及预防较为成熟的维甲酸类药物之一，可用于肿瘤的化学预防和治疗。本研究在前期研究的基础上，观察维A酸注射液对人肝癌细胞SMMC-7721增殖及裸鼠移植瘤生长的影响，检测肿瘤组织细胞凋亡和caspase-3的表达，旨在阐明维A酸注射液抗肝癌的作用及其可能机制，为其临床应用于肝癌的治疗提供科学的实验依据。方法：（1）取对数生长期的SMMC-7721细胞，用不同浓度的维A酸注射液（终浓度分别为100μg/mL、50μg/mL、25μg/mL

4、、12.5μg/mL、6.25μg/mL、3.125μg/mL）干预48小时，阴性对照为等体积培养基，阳性对照为抗癌药顺铂（cisplatin,DDP，终浓度分别为10μg/mL、1μg/mL、0.1μg/mL），采用CCK-8试剂盒（CellCountingKit-8）检测对人肝癌细胞SMMC-7721增殖的影响；（2）建立人肝癌细胞SMMC-7721裸鼠移植瘤模型，在接种后第15天，去除移植瘤未生长或移植瘤生长过缓、过速的裸小鼠。选择体内已形成瘤块且瘤径约≥50mm3的裸鼠，按实验方案随机分为5组给药。分别为阴性对

5、照组(生理盐水）、阳性对照组(DDP，1mg/kg)和维A酸注射液低、中、高三个剂量组（7.5mg/kg、15mg/kg、30mg/kg）。采用腹腔注射给药，每周连续给药5天后停药2天，共给药4周。每周测量瘤径和体重3次，计算肿瘤体积（tumorvolume,TV）、相对肿瘤体积（relativetumorvolume,RTV）和相对肿瘤增殖率[T/C(%)]，以检测对人肝癌细胞SMMC-7721裸鼠移植瘤肿瘤生长的影响；（3）荷瘤裸鼠连续给药4周后停药l天，再予脱颈处死，取出皮下移植瘤，用10%中性多聚甲醛固定，常规

6、脱水，石蜡包埋，4m1厚连续切片，脱蜡水化，以TUNEL法检测肿瘤组织细胞凋亡，免疫组化检测肿瘤组织caspase-3的表达。结果：（1）维A酸注射液随着浓度的增加，对人肝癌细胞株SMMC-7721增殖的抑制作用增强，呈现出较好的量效关系，IC50为(15.140.46)μg/mL；（2）维A酸注射液治疗组与阳性对照组（DDP）均能不同程度抑制人肝癌细胞株SMMC-7721裸鼠移植瘤的生长，腹腔注射给药后，TV、RTV和T/C%明显下降，T/C（%）均小于60%，且与阴性对照组(生理盐水）比较，P<0.05。与DD

7、P可以减轻祼鼠体重不同，维A酸注射液治疗后裸鼠体重均没有明显减轻，揭示其毒性较小；（3）TUNEL法检测结果显示，阳性细胞胞核呈棕褐色着色，部分细胞表现为核固缩或碎裂，染色质分割成块状或凋亡小体。维A酸注射液治疗组凋亡细胞阳性率明显高于阴性对照组，差异有统计学意义(P<0.01)，阳性对照顺铂对细胞凋亡无明显影响，凋亡细胞阳性率低，与阴性对照组无明显差异(P>0.05)；免疫组化结果显示，与阳性对照顺铂不同，维A酸注射液治疗组肿瘤细胞均有caspase-3表达，胞浆呈现不同程度的棕黄色颗料，caspase-3表达阳性率

8、明显高于阳性对照顺铂和阴性对照组，差异有统计学意义(P<0.01)。结论：维A酸注射液能明显抑制人肝癌细胞SMMC-7721的增殖和祼鼠SMMC-7721移植瘤的生长，呈现出较好的剂量依赖关系，具有较强的体内外抗肿瘤活性。维A酸注射液体内抗肿瘤活性的机制可能与促进caspase-3的活化，诱导肿瘤组织细胞凋亡有关。关键词：维A酸注