肝癌 Open Access
Copyright ©The Author(s) 2003. Published by Baishideng Publishing Group Inc. All rights reserved.
世界华人消化杂志. 2003-04-15; 11(4): 404-407
在线出版日期: 2003-04-15. doi: 10.11569/wcjd.v11.i4.404
单克隆抗体3A5-复方中药安迪偶联物的肝癌导向治疗
梁军, 孙纪元, 谢艳华, 栗燕, 闫露, 王四旺
梁军, 中国人民解放军第四军医大学唐都医院放疗科陕西省西安市710038
梁军, 男, 1964-06-24生, 吉林省吉林市人, 汉族, 医学硕士, 主治医师, 讲师.
孙纪元, 谢艳华, 王四旺, 中国人民解放军第四军医大学药物研究所陕西省西安市710032
栗燕, 闫露, 中国人民解放军第四军医大学西京医院综合诊疗科陕西省西安市710032
基金项目: 陕西省中医药基金课题, No. 00531.
通讯作者: 王四旺, 710032, 陕西省西安市长乐西路17号, 中国人民解放军第四军医大学药物研究所. wangsiw@fmmu.edu.cn
电话: 029-3377181传真: 029-3377181
收稿日期: 2002-12-08
修回日期: 2002-12-15
接受日期: 2002-12-22
在线出版日期: 2003-04-15

目的

探讨单克隆抗体3A5-复方中药安迪粉针剂偶联物(3A5-Andi)的肝癌导向治疗作用.

方法

用单克隆抗体3A5与安迪粉针剂(Andi)制备偶联物, ELISA检测3A5-Andi偶联物保持对人肝癌BEL-7402细胞的免疫反应性；采用小鼠/裸鼠移植性肿瘤模型, 5-FU为阳性对照组, 观察12、24和50mg/kg的导向治疗作用.

结果

克隆生成法显示, 3A5-Andi具有比Andi更强的细胞毒性, 二者的IC50分别为2.8 mg/L和5.1 mg/L(P<0.01). 24 mg/kg和50 mg/kg3A5-Andiip对小鼠移植性腹水型H22肝癌的生命延长率(LPR)分别为133%和167%(P<0. 05); 而12 mg/kg和24 mg/kgAndi的LPR分别为112%和138%(P<0.05). 24 mg/kg3A5-Andi和12 mg/kgAndiiv对裸鼠移植性人肝癌BEL-7402细胞瘤的瘤重抑制率分别为63.5%和52.2%(P＞0.05). 24 mg/kg3A5-Andi和12 mg/kgAndipt对BEL-7402人肝癌的抑制率分别为75. 4%和61.0%(P＞0.05).

结论

3A5-Andi偶联物具有一定的肿瘤导向治疗作用；3A5-Andi与Andi比较, 3A5-Andi对裸鼠移植人肝癌BEL-7402具有较强的治疗作用；pt方式3A5-Andi比iv疗效更好.

关键词: N/A
引文著录: 梁军, 孙纪元, 谢艳华, 栗燕, 闫露, 王四旺. 单克隆抗体3A5-复方中药安迪偶联物的肝癌导向治疗. 世界华人消化杂志 2003; 11(4): 404-407
Effect of monoclonal antibody 3A5 coupled with Chinese medicine compound Andi in targeted treatment of hepatocellular carcinoma
Jun Liang, Ji-Yuan Sun, Yan-Hua Xie, Yan Li, Lu Yan, Si-Wang Wang
Jun Liang, Department of Radio therapy, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, Shaanxi Province, China
Ji-Yuan Sun, Yan-Hua Xie, Si-Wang Wang, Institute of Pharmaceuticals, Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China
Yan Li, Lu Yan, Department of Comprehensive Diagnosis and Therapy, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China
Supported by: the Fund for Traditional Chinese Medicine Research of Shaanxi Province, No. 00531.
Received: December 8, 2002
Revised: December 15, 2002
Accepted: December 22, 2002
Published online: April 15, 2003

AIM

To explore the anti-tumor effect of monoclonal antibody 3A5 coupled with Chinese medicine compound Andi injection in targeted treatment of hepatocellular carcinoma(HCC).

METHODS

Monoclonal antibody 3A5 was used to prepare conjugates with Andi powder. The immunoreactivity of the conjugates on human HCC BEL-7402 cell was detected by ELISA. The targeted therapeutic effects of 12, 24 and 50 mg/kg conjugates were observed in mice/nude mice transplantation tumor models. The positive control group was treated with 5-FU.

RESULTS

The detection of clone production indicated that 3A5-Andi has more cytotoxicity than Andi, and their IC50 were 2.8 mg/L and 5.1 mg/L respectively (P < 0.01). The survival rates of 24 mg/kg and 50 mg/kg of 3A5-Andi ip on mice transplantation ascites H22 HCC were 133% and 167% respectively (P < 0.05), but those of 12 mg/kg and 24 mg/kg of Andi only were 112% and 138% respectively (P < 0.05). The inhibitory rates of 24 mg/kg of 3A5-Andi and 12 mg/kg of Andi iv on nude mice transplantated human HCC BEL-7 402 cell tumor were 63.5% and 52.5% respectively (P > 0.05). The inhibitory rates of 24 mg/kg 3A5-Andi and 12 mg/kg Andi pt on human HCC BEL-7402 cell were 75.4% and 61.0% respectively (P > 0.05).

CONCLUSION

3A5-Andi conjugates have a tumor targeted therapeutic effect. This effect is stronger than that of Andi only on nude mice transplanted human HCC BEL-7402, and the administration of pt is better than that of iv.

Key Words: N/A
0 引言

我们以往的研究已经证实复方中药安迪粉针剂(Andi)具有明显抗肿瘤活性, 而毒副作用较低. 目前, 随着单克隆抗体产品, 如Herceptin、Rituxan及Panorex等在美国及欧洲的批准上市, 使得以单抗为载体的抗肿瘤导向药物研究再度引起人们的关注. 肝癌在我国常见. 所以, 我们采用抗人肝癌大鼠型单克隆抗体3A5与Andi制成偶联物, 并就偶联物的肿瘤导向治疗作用进行了实验研究.

1 材料和方法
1.1 材料

人肝癌BEL-7402细胞, 由本校病理学教研室惠赠, 引株采用含100 ml/L小牛血清-RPMI1640培养液, 于50 mL/LCO2, 37 ℃, 100%湿度的HEL-LAB型CO2培养箱(美国生产)内培养, 每周传代2次；Balb/c(nu/nu)裸鼠, ♀；昆明小鼠, 雌雄兼用； 6-7 wk, 18.5-20.6 g；动物购自第四军医大学实验动物中心；抗肝癌大鼠单克隆抗体3A5为IgG型单抗, 与人肝癌细胞BEL-7402呈强阳性反应. Andi为本校药物研究所生产(蟾酥和人参组成), 按文献[1]方法制备. 批号: 20000318. 碳二亚胺(EDC)、N-羟基琥珀酰亚胺均购自Sigma公司.

1.2 方法

单克隆抗体3A5的制备与纯化按文献[J Immumol Methods 1984; 71: 9]提供的方法进行, 取杂交瘤腹水用MARK 3-Sepharose 4B亲和层析柱纯化, 依次用PBS, PBS+2.5 mol/L NaCl和0.1 mol/L甘氨酸-盐酸缓冲液洗脱, 将第2蛋白峰用0.3 mol/L Tris-HCl(pH 8.0)缓冲液中和后, 再用PBS透析、浓缩备用. 单抗3A5与Andi偶联物的制备按文献[Methods Enzymol 1983; 93: 168]活泼酯法制备, 滴定透析外液, 测定其中Andi量, 推算偶联物中Andi的含量并计算单抗与药物的浓度比. 免疫反应性检测采用ELISA法测定3A5对人肝癌BEL-7402细胞的免疫反应性[ Methods Enzymol 1983; 92: 168]. 采用克隆生成测定法观察3A5-Andi对BEL-7402细胞杀伤作用, 取对数生长的肿瘤细胞接种于96孔培养板中, 每孔50个细胞, 培养24 h. 加入待测样品, 每一药物浓度设4个平行孔, 各药物均用无血清RPMI1640培养液稀释, 对照组(CN)只加无血清RPMI 1 640培养液, 每孔加液50 mL.药物与细胞接触24 h, 吸去培养液, 用无血清RPMI 1 640培养液洗2次, 然后加入含血清的RPMI 1 640新鲜培养液, 继续培养. 接种7 d后在倒置显微镜下数细胞集落数(细胞数超过30个以上), 以CN组细胞集落数为100 %, 求得药物不同浓度下的集落生成率.对小鼠H22腹水型肝癌的影响实验: 18-22 g昆明小鼠腹腔接种H22细胞6×109/ L.次日ip 12, 24, 50 mg/kg 3A5-Andi和6, 12, 24 mg/kg Andi, 1次/d, 共18次; CN组 ip NS. 记录动物存活时间, 按公式(t1/t2-1)×100 %计算生命延长率[increase life span ILS; t1治疗组动物生存时间, t2 CN组动物生存时间]; 实验过程中每5 d测量动物体质量1次. 对人肝癌裸鼠移植瘤的影响: 将直径2 mm的BEL-7 402人肝癌瘤块接种于balb/c (nu/nu)裸鼠右腋皮下. 于接种后3 d iv 和瘤体周围注射(pt) 3A5-Andi和Andi, 3A5-Andi的剂量为24 mg/kg, Andi的剂量为12 mg/kg, 2d /次, 共6次. CN组动物iv和pt生理盐水(NS). 各组动物均为8只. 治疗期间每5 d测量肿瘤长径a (cm)以及与其相垂直的短径b (cm) 1次并按下式计算瘤质量: m (瘤)/g = (a×b2)×0.5. 于30 d后处死动物, 剥离瘤块称质量, 并计算抑瘤率.

统计学处理 采用多样本秩和检验方法(Kruskal-Wallis法)检验ILS；单因素方差分析法检验瘤质量差异.

2 结果

纯化3A5抗体对BEL-7402细胞呈强阳性反应. 经测定偶联物中的3A5与Andi浓度比约为1: 68. 将3A5单抗与3A5-Andi分别以PBS倍比稀释成一系列浓度, 用ELISA法测定他们对靶细胞BEL-7402的反应性. 结果表明偶联物中3A5反应性未见明显改变(图1).

图1
图1 3A5-Andi及3A5单抗对人肝癌BEL-7402细胞的免疫反应性.
2.1 对人肝癌细胞的杀伤作用

克隆形成测定结果, 药物与细胞作用24 h, 3A5-Andi和Andi对人肝癌BEL-7402细胞的半数抑制浓度(IC50)分别为2.8mg/L和5.1 mg/L. 3A5-Andi对靶细胞的杀伤力强于Andi(图2).

图2
图2 3A5-Andi和Andi对人肝癌BEL-7402细胞的抑制作用.
2.2 对腹水型H22肝癌荷瘤小鼠的影响

3A5-Andi对腹水型H22肝癌荷瘤小鼠的治疗作用明显强于Andi组(P＜0.01). 50mg/kg3A5-Andi组观察60d尚存活3只动物, 解剖发现腹腔内无腹水和肿瘤(表1).

表1 Andi及3A5-Andi对腹水型H22肝癌荷瘤小鼠的LPR (n=20).
分组给药量/mg/kgt (中位生存)/dLPR/%存活动物数60 d
CN10(H2O)13.5
Andi2432.1b1382
Andi1228.6b1121
Andi625.5b890
3A5-Andi5036.1b1673
3A5-Andi2431.5b1332
3A5-Andi1229.0b1151
bP<0.01 vs CN.
2.3 对裸鼠人肝癌移植性肿瘤的影响

3A5-Andi对移植人肝癌BEL-7402肿瘤的生长, 在接种后15d内不明显, 15d后CN组肿瘤生长速度明显加快, 而3A5-Andi和Andi治疗组则显著抑制, 其抑制程度3A5-Andi强于Andi组, 且随时间延长而抑制作用更趋显著. 12 mg/kgAndiiv组抑瘤率为52.2%；24 mg/kg3A5-Andiiv组肿瘤抑制率为63.5%, 但二者比较无显著性差异(P>0.05, 表2); pt给药方式, 二者的抑瘤率分别为61.0%和75. 4%(P>0.05).

表2 Andi及3A5-Andi对裸鼠移植人肝癌BEL-7 402的治疗作用(n=8).
分组剂量/mg/kg给药途径m(裸鼠)/g*m(瘤)/g抑瘤率/%
CN10pt4. 01±1. 061. 95±1. 21
Andi12pt5. 63±1. 380. 76±0. 49b61. 0
3A5-Andi24pt6. 11±1. 400. 48±0. 22b75. 4
CN10iv3. 88±0. 971. 86±0. 92
Andi12iv5. 57±1. 240. 89±0. 52d52. 2
3A5-Andi24iv5. 35±1. 260. 67±0. 25d63. 5
*为实验末与实验前裸鼠的体质量差值.
bP<0.01 vs CN(pt);
dP<0.05 vs CN(iv).
3 讨论

近年来, 对中草药抗肝癌的作用和机制进行了广泛研究[2-13]. Andi抑制癌细胞生长的途径是多方面的. 梁军et al[1]的研究表明, Andi对HL－60细胞具有显著的诱导分化作用. 在孙纪元et al[14]的研究中, 已发现Andi具有促进60Co-射线照后小鼠骨髓造血干细胞(CFU-S)向粒-单核系祖细胞(CFU-GM)分化的作用, 并使白细胞升高. 单抗和单抗导向药物经区域性给药后(包括动脉灌注、腔内给药、瘤内或瘤旁给药), 由于其具有大分子的药代动力学性质, 使得偶联物在肿瘤部位滞留时间延长, 局部浓度高, 且减少了非肿瘤组织对导向药物的摄取, 从而具有独特的应用价值, 在临床应用中已取得一定的治疗效果, 成为导向治疗中值得重视的方法[15-31]. 安迪粉针剂与抗肝癌大鼠单抗3A5偶联对肿瘤的治疗作用还未见报道.

我们将安迪粉针剂与抗肝癌大鼠单抗3A5偶联, 观察其经静脉给药和肿瘤局部给药对肿瘤的治疗作用. 结果表明, 3A5-Andi保持3A5单抗对BEL-7402细胞的免疫反应性, 与3A5单抗相比活性相当. 克隆生成法测定结果, 3A5-Andi保持了Andi的细胞毒活性. 对小鼠腹水型H22肝癌的治疗结果表明：3A5-Andi比Andi具有较强的治疗作用, 其中ip 50mg/kg3A5-Andi观察60 d时尚存活3只动物, 经解剖发现腹腔内无腹水及肿瘤. 对裸鼠人肝癌BEL-7402细胞移植瘤的治疗结果表明, 3A5-Andi抑瘤作用强于Andi; pt的治疗效果优于iv方式.

总之, 实验结果提示: (1)3A5-Andi偶联物具有一定的肿瘤导向治疗作用. (2)3A5-Andi与Andi比较, 3A5-Andi对裸鼠移植人肝癌BEL-7402具有较强的治疗作用；(3)pt方式3A5-Andi比iv疗效更好.

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