AMD3100对索拉非尼治疗肝细胞肝癌的疗效影响

陆录　朱文伟　陈进宏　贾户亮

(复旦大学附属华山医院普外科，上海　200040)



·论著·

AMD3100对索拉非尼治疗肝细胞肝癌的疗效影响

陆录朱文伟陈进宏贾户亮

(复旦大学附属华山医院普外科，上海200040)

摘要目的：观察基质细胞衍生因子1-α(stromal cell derived factor 1-α, SDF1-α)受体抑制剂AMD3100联用索拉非尼对肝细胞肝癌(HCC)裸鼠的疗效。方法： 建立肝细胞肝癌原位裸鼠模型，分为对照组、索拉非尼组、索拉非尼+AMD3100 组，每组6只。索拉非尼组给予索拉非尼30 mg/(kg·d)灌胃；索拉非尼+AMD3100组在给予索拉非尼灌胃的基础上，给予AMD3100 2.5 mg/(kg·d)腹腔注射；对照组给予0.9%氯化钠液灌胃。比较三组的肿瘤肝内播散情况、肿瘤大小、肺转移情况和裸鼠的生存时间。结果：在索拉非尼治疗的基础上，联用AMD3100能够明显抑制索拉非尼引起的HCC肝内侵袭和转移，进一步减小肿瘤体积，减少肺转移，延长荷瘤裸鼠的生存期。结论：SDF1-α受体抑制剂AMD3100能够增强索拉非尼对HCC的疗效。

关键词AMD3100；索拉菲尼；肝细胞肝癌

索拉非尼(sorafenib)是目前晚期肝细胞肝癌(HCC)的一线治疗药物[1]。但是，研究[2-4]发现，索拉非尼在抑制原发瘤的同时促进了转移瘤的生长。我们的前期研究发现，应用索拉非尼后，肿瘤组织和癌旁微环境中基质细胞衍生因子1-α(stromal cell derived factor 1-α, SDF1-α)的表达明显增加。因此，本研究将SDF1-α受体抑制剂AMD3100与索拉非尼联合应用于治疗HCC裸鼠模型，探讨AMD3100协同索拉非尼治疗HCC的疗效。

1资料与方法

1.1实验动物及药物18只雄性BALB/c裸鼠，6周龄，体质量15~20 g，购自中国科学院上海药物研究所，在25℃恒温的SPF级层流室中饲养，12 h光照和12 h黑暗交替，自由进食标准颗粒饲料及饮水。索拉非尼购自拜耳(中国)有限公司， AMD3100购自德国Sigma-Aldrich公司。

1.2裸鼠原位HCC模型的建立无菌条件下将培养的1×107的人HCC LM3细胞注射到裸鼠皮下。待3~4周皮下瘤成瘤后，取瘤原，浸入冷0.9%氯化钠液中，剪成2 mm×1 mm×1 mm大小备用。以2.5 mg/kg戊巴比妥腹腔注射麻醉裸鼠，消毒手术野，在左肋缘下作长约8 mm的斜切口，将肝叶取出，斜形切开肝表面，植入备好的瘤原组织块，将肝叶放回原位，用 6-0 线缝合肝切缘，用5-0线双层缝合腹壁和皮肤。

1.3给药方法及分组将18只HCC模型裸鼠分为3组，每组6只。模型建立后2周开始给药。将索拉非尼400 mg溶于100 mL的溶剂(蓖麻油∶乙醇∶dH2O=12.5∶12.5∶75)中，AMD3100溶于0.9%氯化钠液中。索拉非尼组给予裸鼠索拉非尼30 mg/(kg·d)灌胃；索拉非尼+AMD3100组在给予索拉非尼的基础上给予裸鼠AMD3100 2.5 mg/(kg·d)腹腔注射。对照组给予0.9%氯化钠液 0.1mL/d 灌胃。

1.4观察指标给药4周后处死裸鼠，取出肿瘤，测量最长径a和最短径b，肿瘤体积=ab2/2；取双肺，石蜡包埋并连续切片，HE染色后读取每隔5张切片中的肺转移灶数目。

1.5统计学处理如连续变量呈正态分布，采用t检验(Students’t-test)；如连续变量呈非正态分布，采用秩和检验(Mann-Whitney U test)。P<0.05为差异有统计学意义。

2结果

2.1AMD3100对肿瘤体积及侵袭转移能力的影响对照组肿瘤平均体积为4.4 cm3。索拉非尼组肿瘤平均体积为2.7 cm3，明显小于对照组(P=0.02)，但肿瘤向肝内播散。与索拉非尼组比较，AMD3100+索拉非尼组肿瘤体积更小，平均为1.3 cm3，差异有统计学意义(P=0.04)；肿瘤边界清晰，肝内播散被明显抑制。见图1、2A。

图1　HCCLM3裸鼠原位瘤模型大体标本

2.2AMD3100对肿瘤肺转移的影响索拉非尼组裸鼠的肺转移灶数目较对照组明显减少(10个比18个，P=0.01)，索拉非尼+AMD3100组肺转移灶的数目进一步减少(4个，P=0.02)，见图2B。

图2A：裸鼠原位肝细胞肝癌模型肿瘤体积比较；B：裸鼠原位肝细胞肝癌模型肺转移数目的比较；C：原位肝细胞肝癌模型裸鼠生存时间的比较

2.3AMD3100对裸鼠生存期的影响索拉非尼组的裸鼠平均生存时间为76 d，长于对照组(69 d)；索拉非尼+AMD3100组裸鼠的平均生存时间达 91 d。见图2C。

3讨论

抗血管生成在HCC的治疗中非常重要[5-6]。索拉非尼是目前临床上治疗晚期HCC的一线用药，它主要通过抑制血管内皮生长因子(VEGF)发挥抗血管生成的作用[7]。但是两个大型的临床研究[8-9]显示，其对于HCC的治疗效果不佳，对患者的中位生存期延长不足3个月。

SDF1又称为CXCL12，是一种趋化因子，主要由成纤维细胞、炎性细胞、内皮细胞等分泌[10]。SDF1-α通过与其受体CXCR4结合，促进肿瘤的生长[10]。有文献[11]报道，AMD3100作为CXCR4的特异性抑制剂，能够增强前列腺癌对于放疗的敏感性。AMD3100能够抑制囊腺癌的嗜神经侵袭[12]。然而，AMD3100应用于肝癌的报道比较少见。本研究将CXCR4的特异性抑制剂AMD3100与索拉非尼联用，发现AMD3100能够明显抑制索拉非尼引起的肿瘤肝内播散，并可进一步减小肿瘤体积、抑制肿瘤肺转移、延长荷瘤裸鼠的生存时间，说明AMD3100能够增强索拉非尼的疗效。

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The Effect of AMD3100 on the Efficacy of Sorafenib in the Treatment of Hepatocellular Carcinoma

LULuZHUWenweiCHENJinhongJIAHuliang

DepartmentofSurgery，HuaShanHospital,FudanUniversity,Shanghai200040,China

AbstractObjective：To investigate the effect of AMD3100, an inhibitor of receptor of stromal cell derived factor 1-α (SDF1-α), combined with sorafenib on nude mice with hepatocellular carcinoma (HCC). Methods:Nude mice models of HCC in situ was established and the mice were divided into control group, sorafenib group and sorafenib combined with AMD3100 group, with six mice in each group. In sorafenib group, sorafenib 30 mg/(kg·d) was given by garage. In sorafenib combined with AMD3100 group, AMD3100 2.5 mg/(kg·d) was administrated by intraperitoneal injection based on the method of sorafenib group. In the control group, 0.9% sodium chloride solution was given by garage. The intrahepatic invasion, the tumor volume, the situation of pulmonary metastasis and the survival time of nude mice, were compared among the three groups. Results: When sorafenib was combined with AMD3100, the intrahepatic invasion and metastasis of HCC caused by sorafenib could be significantly inhibited. Thus the tumor volume was decreased and the pulmonary metastasis could be reduced. The survival time was prolonged. Conclusions: AMD3100, a SDF1-α receptor inhibitor, can enhance the efficacy of sorafenib.

Key WordsAMD3100；Sorafenib；Hepatocellular carcinoma

中图分类号R735.7

文献标识码A

通讯作者贾户亮，E-mail：jbl-1@163.com

基本项目：国家自然科学基金项目(编号：81101564)