双特异性单克隆抗体介导的单核－巨噬细胞抗肝癌作用

目的研究双特异性单克隆抗体介导的单核－巨噬细胞抗肝癌作用。方法采用化学交联法，用异型化学交联剂Ｎ－琥珀酰亚氨基－３－（２－二硫吡啶）丙酸酯（ＳＰＤＰ），将具有高亲和性的抗肝癌单抗ＨＡｂ１８Ｆ（ａｂ′）２与抗单核－巨噬细胞的单抗ＭＡｂ７Ｆ（ａｂ′）２联结成双特性单抗ＨＡｂ１８Ｆ（ａｂ′）２－ＭＡｂ７Ｆ（ａｂ′）２，经ＦＰＬＣＳｅｐｈａｃｒｙ１Ｓ－２００纯化。用间接免疫荧光法鉴定其特性。结果ＨＡｂ１８Ｆ（ａｂ′）２及ＨＡｂ１８Ｆ（ａｂ′）２－ＭＡｂ７Ｆ（ａｂ′）２对肝癌细胞间接免疫荧光染色阳性，ＭＡｂ７Ｆ（ａｂ′）２及ＨＡｂ１８Ｆ（ａｂ′）２－ＭＡｂ７Ｆ（ａｂ′）２对单个核细胞染色为阳性。而这些抗体对肺腺癌细胞均呈阴性；单纯ＨＡｂ１８Ｆ（ａｂ′）２无导向单核－巨噬细胞杀伤肝癌细胞作用，ＨＡｂ１８Ｆ（ａｂ′）２－ＭＡｂ７Ｆ（ａｂ′）２及ＭＡｂ７Ｆ（ａｂ′）２均可介导单核－巨噬细胞对肝癌细胞起杀伤作用，并且前者强于后者。结论ＨＡｂ１８Ｆ（ａｂ′）２－ＭＡｂ７Ｆ（ａｂ′）是一种具有双特异性的单克隆抗体，可以提高人单核－巨噬细胞杀伤肝癌细胞的作用。     

双特异性单克隆抗体介导的单核—巨噬细胞抗肝癌作用

研究双特异性单克隆抗体介导的单核－巨噬细胞抗肝癌作用。方法采用化学交联法，用异型化学交联剂Ｎ－琥珀酰亚氨基－３－，将具有高亲和性的抗肝癌单抗ＨＡｂ１８Ｆ２与抗单核－巨噬细胞的单抗ＭＡｂ７Ｆ２联结双特异性单抗ＨＡｂ１８Ｆ２－Ｍａｂ７Ｆ２，经ＦＰＬＣ Ｓｅｐｈａｃｒｙ１ Ｓ－２００纯化。     

双特异单克隆抗体介导的人单核—巨噬细胞对肝癌细胞的杀伤作用

证实双特异性单克隆抗体在肝癌导向治疗，尤其是细胞免疫治疗方面的作用。方法：用化学交联剂ＳＰＤＰ将抗肝癌单克隆抗体ＨＡｂ１８与抗单核－巨噬细胞单克隆抗体ＭＡｂ７联结成双特异抗体ＨＡｂ１８－ＭＡｂ７，用流式细胞仪证实其双特异性；软琼脂培养法证明其对肝癌细胞的导向杀伤作用。结果ＨＡｂ１８－ＭＡｂ７具有特异性地结合肝癌细胞及单核－巨噬细胞的双重特性。软琼脂培养结果表明：ＨＡｂ１８－ＭＡｂ７实验组较对照组克     

双特异单克隆抗体介导的人单核－巨噬细胞对肝癌细胞的杀伤作用

目的：证实双特异性单克隆抗体在肝癌导向治疗，尤其是细胞免疫治疗方面的作用．方法：用化学交联剂ＳＰＤＰ将抗肝癌单克隆抗体ＨＡｂ１８与抗单核－巨噬细胞单克隆抗体ＭＡｂ７联结成双特异抗体ＨＡｂ１８－ＭＡｂ７；用流式细胞仪证实其双特异性；软琼脂培养法证明其对肝癌细胞的导向杀伤作用．结果：ＨＡｂ１８－ＭＡｂ７具有特异性地结合肝癌细胞及单核－巨噬细胞的双重特性；软琼脂培养结果表明：ＨＡｂ１８－ＭＡｂ７实验组较对照组克隆形成率明显降低（Ｐ＜０．０１）．结论：ＨＡｂ１８－ＭＡｂ７可明显提高单核巨噬细胞对肝癌细胞的杀伤作用．     

131碘—肝癌单克隆抗体Fab片段偶合物体内放射免疫显像研究

目的研究１３１Ⅰ标记肝癌单克隆抗体ＨＡｂ１８Ｆａｂ片段体内放射免疫显像效果。方法以木瓜蛋白酶切割肝癌单克隆抗体ＨＡｂ１８制备ＨＡｂ１８Ｆａｂ,应用高效碘标记法将１３１Ⅰ标记于ＨＡｂ１８Ｆａｂ,再以１３１Ⅰ－ＨＡｂ１８Ｆａｂ偶合物进行荷肝癌裸鼠及肝癌病人体内放射免疫显像。结果１０只荷肝癌裸鼠全部明确显像,其中２只鼠４．５小时即出现瘤区放射性浓聚,８只鼠在８～１２小时清晰显像,对照组鼠瘤区未出现放射性浓聚。１３１Ⅰ－ＨＡｂ１８Ｆａｂ的瘤／肝比值２４小时为２１．０７±０．０５,明显高于１３１Ⅰ－ＨＡｂ１８组最佳显像１６８小时的５．８３±０．０５。肝癌病人１３１Ⅰ－ＨＡｂ１８Ｆａｂ的最佳显像时间为１６～２４小时。结论１３１Ⅰ－ＨＡｂ１８Ｆａｂ放免显像集中肝癌定位诊断、定性诊断和及时诊断三方面优点于一体,可能成为肝癌诊断的一项重要技术。     

肝癌单克隆抗体F（ab‘）2段与葡萄球菌肠毒素A结合物的制备及 …

制备眼抗原葡萄球菌肠毒素与抗人肝细胞癌单克隆抗体（ＭｃＡｂ）ＨＡｂ１８Ｆ（ａｂ’）２段的结合物，并对其活行鉴定。方法：提取ＭｃＡｂＨＡｂ１８并用木反蛋白酶切取其（ａｂ’）－ＳＥＡ结合物，     

抗人肝癌单克隆抗体和Fab片段在裸鼠体内分布及药物偶…

大鼠抗人肝癌单克隆抗体（ＭｃＡｂ）３Ａ５（ＩｇＧ１）腹水经亲和层析柱纯化后，用木瓜蛋白酶消化得到Ｆａｂ片段，经ＥＬＩＳＡ检测均具有良好的免疫活性。将＾１２５Ｉ－Ｆａｂ和＾１２５Ｉ－３Ａ５注入移植人肝癌裸鼠体内Ｆａｂ片段分布在主要脏器的每克组织注射剂量百分比（ＩＤ％／ｇ）的持续时间和数值均低于完整ＭｃＡｂ，但肿瘤与非肿瘤组织（Ｔ／ＮＴ）的比值则高于完整ＭｃＡｂ，说明Ｆａｂ片段的分布对肿瘤组织具有较高     

木瓜蛋白酶制备抗人肝癌单抗F（ab‘）2及Fab片段

为了制备肝癌ＭｃＡｂ ＨＡｂ１８Ｆ（ａｂ’）２及Ｆａｂ片段。用激活的木瓜酶分别在偏酸性和偏碱性条件下消化１８ｈ和１ｈ，得到Ｆ（ａｂ’）２和Ｆａｂ片段，经ＤＥＡＥ－４０ＨＲ阴离子交换色谱柱在Ｗａｔｅｒｓ６５０Ｅ快速蛋白液相色谱系统（ＥＰＬＣ）上建立相应的纯化方法。该法制备Ｆ（ａｂ’）２得率为４３％－６２％，制备Ｆａｂ得率４２％，其活性用免疫荧光法测定均为１．３６×１０＾－９ｍｏｌ／Ｌ。本法操作简单，     

单克隆抗体MAb7及其相应抗原在单核—巨噬细胞系统及中性粒细…

建立一株抗人单核－巨噬细胞及中性粒细胞的杂交瘤ＭＡｂ７。用人肝癌组织细胞悬液免疫ＢＡＬＢ／ｃ小鼠，取脾细胞与ＳＰ２／０细胞融合，ＡＢＣ染色法筛选单克隆抗体，ＡＰＡＡＰ法鉴定骨细胞特征。ＭＡｂ７在研究单核－巨噬细胞的分化、免疫功能及病理分型等方面有一定的意义和价值。     

肝癌导向治疗中三种^131Ⅰ标记抗体的药代动力学观察

作者对＾１３１Ⅰ标记铁蛋白多克隆抗体，抗人肝癌单克隆抗体和乙型肝炎病毒Ｘ蛋白单克隆抗体的药代动力学进行研究。结果：（１）裸鼠人肝癌模型＾１３１Ⅰ－ＦｔＡｂ分别经静脉，腹腔途径给药，其药代动力学模式有很大差异，相同剂量的ＦｔＡｂ与ＨＢｘＭｃＡｂ经ｉｐ给药Ｔｍａｘ分别为８．１ｈ８．４ｈ，Ｔ１／２分别为４０．５ｈ和４２．２ｈ；不同剂量Ｔ的＾１３１Ⅰ－ＨＢｘＭｃＡｂ，低剂量组与高剂量组Ｔｍａｘ分别为１４．     

Radiolocalization of human hepatoma with anti-human hepatoma monoclonal antibodies and its F(ab')2 in tumor-bearing nude mice

This paper reports the results and procedures for the radiolocalization of human hepatoma by murine MAb (HAb 18) and its F (ab')2 in animal model. 131I-labelled HAb 18 IgG and 125I-labelled its F(ab')2 were intravenously injected respectively to human hepatoma bearing nude mice. Clear tumor imaging manifested 48 hrs. after injection of 125I-labeled F(ab')2 and 120 hrs. after 131I-labeled intact MAb. The animals were killed 48, 72, 120, and or 168 hrs. after injection respectively. Bound radioactivity was counted in a variety of solid organs. Tumor: liver ratios for intact MAb or its F(ab')2 were greater than those for unrelated MAb at each time points, the differences being greatest after 168 hrs. (ratio intact MAb 5.15 +/- 0.64, ratio unrelated MAb 0.93 +/- 0.16) and after 48 hrs. (ratio F(ab')2 14.47 +/- 2.35, mean +/- sd). The amount of I-MAb bound was greater in the tumor than in any other solid organ. The clearance rate of F(ab')2 was faster then that of intact MAb. It was shown that HAb 18 MAb may be of value as an immunodiagnostic agent for hepatoma.     

Preparation and in vitro killing effect of adriamycin-loaded immunonanosphere against hepatoma led by F (ab')2 Fragment of monoclonal antibodies]

OBJECTIVE: To study the preparation method and in vitro killing effect of adriamycin (ADR)-loaded human serum albumin (HSA) immunonanosphere (HAb18 F(ab')2-ADR-HSA-NP) against hepatoma led by F(ab')2 fragment of human hepatoma specific monoclonal antibody HAb18. METHODS: After ADR loaded HSA nanosphere (ADR-HSA-NP) was prepared in the emulsifying high temperature solidifying way, HAb18 F(ab')2-ADR-HSA-NP was prepared using the modified N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) method. In vitro binding characters of HAb18 F(ab')2-ADR-HSA-NP and ADR-HSA-NP and hepatoma cell SMMC-7721 were observed under optical microscopy and electronic microscopy. In vitro effects of killing hepatoma cell SMMC-7721 of two microspheres were determined using the method of 3H-TdR. RESULTS: The surfaces of HAb18 F(ab')2-ADR-HSA-NP gave out bright yellow-green fluorescence after it was dyed with fluorescent agent, whereas ADR-HSA-NP did not give out fluorescence. HAb18 F(ab')2-ADR-HSA-NP could integrate with hepatoma cell SMMC-7721 and effectively killed hepatoma cell SMMC-7721 with dose dependence, but ADR-HSA-NP could not obviously integrate and kill SMMC-7721. Neither of the two microspheres could bind and kill human large intestine cancer cell SW1116. CONCLUSION: HAb18 F(ab')2-ADR-HSA-NP has a good character for in vitro specific targeting to bind and kill human hepatoma cell.     

188Re-HAb18 F(ab')2在荷瘤鼠体内的药代动力学

目的　研究 1 88Re标记的肝癌单抗片段 HAb18F(ab ) 2 在荷肝癌裸鼠体内的药代动力学 ,求取药代动力学参数 ,建立药代动力学模型 .方法　 5组裸鼠经尾静脉注入1.85 MBq1 88Re- HAb18F(ab ) 2 ,在不同时间点处死裸鼠 ,测量血及各脏器放射性计数 .用 3P97药代动力学软件处理 1 88Re- HAb18F(ab ) 2 给药后荷瘤鼠各时间点的血液放射性浓度及各主要脏器的放射性浓度 ,计算药代参数 .结果　 1 88Re-HAb18F(ab ) 2 在裸鼠体内药代动力学参数分别为 T1 /2 2 .2 9h,Vd1.49L,AUC2 0 .49× 10 9Bq· h· L- 1 ,CL 0 .45×10 - 3L· h- 1 .1 88Re- HAb18F(ab ) 2 特异性聚集于肿瘤中 ,在瘤体中以较高水平维持较长时间 ,在正常组织中代谢较快 ,主要从肾脏中排泄 .结论　 1 88Re- HAb18F(ab ) 2 在荷肝癌裸鼠体内药代动力学符合线性一室模型     

抗人肝癌免疫毫微球的制备及其抗癌效果观察

目的 制备抗人肝癌免疫毫微球,观察其活性及抗癌效果.方法 抗人肝癌单克隆抗体HAb18通过异型双功能交联剂SPDP与载阿霉素(ADR)人血清白蛋白毫微球[HSA(ADR)-NS]偶联,制成抗人肝癌免疫毫微球HAb18-HSA(ADR)-NS,使用凝集试验及免疫荧光检测其活性,光镜和电镜下观察其与人肝癌细胞株SMMC-7721特异性结合.MTT法检测该免疫毫微球的体外杀伤性.于人肝癌裸鼠模型上分别使用HAbl8-HSA(ADM)-NS、HSA(ADM)-NS及ADM,检测3者的肿瘤抑制率.结果 HAb18-HSA(ADM)-NS具有单抗活性,能与肝癌细胞特异结合;其体外杀伤SMMC-7721细胞IC50值为44.6μg/ml,与HSA(ADM)-NS(345.5μg/ml)及ADM(365.5μg/ml)相比,明显降低;体内肿瘤抑制率比HSA(ADM)-NS及ADM明显增强(P＜0.001).结论 HAb18-HSA(ADM)-NS具有免疫活性,对肝癌细胞有主动靶向性,体内外均具有比HSA(ADM)-NS及ADM更强的抗癌效果.     

Targeting therapy with adriamycin-monoclonal antibody (HAb18) conjugate in nude mice with human hepatocellular carcinoma xenografts]

Adriamycin (ADM) was linked covalently to the murine monoclonal antibody (McAb) HAb18 against human hepatocellular carcinoma via a dextran T-40 bridge. The molar ratio of HAb18: DEX: ADM was 1:2.4:56 in the conjugate. It was confirmed by immunochemical staining and ELISA that the antigen binding capacity of HAb18 in the conjugate was well preserved. In vitro study indicated that the conjugate cytotoxicity to tumor target cell lines was selective. The radio-immunoimaging and distribution of the conjugate labelled with 125I in the nude mice bearing human hepatocellular carcinoma showed that 125I-HAb 18-DEX-ADM conjugate was localized in the tumor site. The HAb18-DEX-ADM targeting therapy in nude mice bearing human hepatocellular carcinoma showed the following results: decreased tumor size and partly necrosed cancer tissue in contrast to the rapidly growing tumor in the control group (P < 0.05). Our study suggested that targeting therapy with HAb18-DEX-ADM conjugate may be useful in the treatment of patients with hepatocellular carcinoma.     

稳定表达肝癌相关抗原HAb18G/CD147成纤维细胞株的建立及其意义

目的:探讨将HAb18G全长cDNA转染成纤维细胞NIH/3T3的可行性及其意义.方法:将含有肝癌相关抗原HAb18G全长cDNA的真核表达载体pcDNA3.0/HAb18G利用脂质体介导法转染小鼠成纤维细胞NIH/3T3,G418筛选建立稳定表达阳性克隆细胞株t3T3;利用流式细胞仪、间接免疫荧光染色方法鉴定目的蛋白转染情况;RT-PCR琼脂糖电泳、明胶酶谱分别从mRNA和蛋白水平检测转染前后HAb18G刺激3T3细胞分泌基质金属蛋白酶(MMP-2、MMP-9)的情况.结果:利用脂质体介导法将HAb18G全长cDNA转染入成纤维细胞NIH/3T3,G418筛选,流式细胞仪分析、间接免疫荧光染色证实成功建立了稳定表达HAb18G的阳性克隆株;RT-PCR琼脂糖电泳证实t3T3细胞MMP-2、MMP-9 mRNA转录水平较未转染前增多;明胶酶谱表明t3T3分泌MMPs能力较未转染前增强.结论:HAb18G全长cDNA可以稳定转染成纤维细胞,并能从核酸和蛋白水平诱导基质金属蛋白酶高表达,具有其独特的生物学意义.     

Localization of hepatocellular carcinoma with monoclonal antibodies]

We prepared monoclonal antibodies (MAbs) against hepatocellular carcinoma using cell suspensions isolated from surgical fresh hepatoma specimens as antigen. Totally we got 6 strains of hybridoma cell lines stably secreting MAbs for more than 2 years. Immunocytochemically they stained positively most of the paraffin embedded hepatoma tissues (63.1 to 91.1%) without reaction to the normal liver tissues. Localization of human hepatoma with 125I or 131I labelled MAbs in nude mice was done by IV injection, which showed clear tumor image by ECT radioimmunodetection and autoradiography of tissues. The T/N ratios of different MAbs were 3.1, 3.6, 5.15 and that of HAb 18-F (ab')2 was 14.4. Among 15 patients suspected to have hepatoma and given the labelled MAb, 13 proved pathologically to be hepatocellular carcinoma.     

黄荆子乙酸乙酯提取物抑制人乳腺癌MCF-7裸鼠移植瘤生长

目的:观察黄荆子乙酸乙酯提取物(EVn-50)对人乳腺癌(MCF-7)裸鼠移植瘤生长的影响。方法:MTT法检测6种黄荆子提取物对人乳腺癌MCF-7细胞增殖的影响;获得目标抗肿瘤有效组分即EVn-50,人乳腺癌(MCF-7)裸鼠移植瘤模型治疗实验,评价EVn-50治疗人乳腺癌的有效性。结果:不同提取方式获得的6种黄荆子提取物对人乳腺癌(MCF-7)细胞的增殖活性具有不同程度的抑制作用,以EVn-50作用最强,其IC50值为0.89μg/mL。EVn-50对人乳腺癌(MCF-7)裸鼠移植瘤生长具有抑制作用,呈剂量依赖性。结论:EVn-50具有抑制人乳腺癌MCF-7细胞增殖和人乳腺癌(MCF-7)裸鼠移植瘤生长作用。     

表皮生长因子受体信号通路在人子宫内膜癌移植瘤孕激素耐药机制中的作用

目的建立人子宫内膜癌皮下移植瘤孕激素耐药模型,探讨表皮生长因子受体(EGFR)下游信号通路在子宫内膜癌孕激素耐药机制中的作用。方法分别利用人子宫内膜癌孕激素敏感型Ishikawa细胞株和孕激素不敏感型Ishikawa-pLWERNL细胞株制备裸鼠皮下移植瘤模型(每组18只);成瘤后,分别将接种Ishikawa细胞株和接种Ishikawa-pLWERNL细胞株的裸鼠各自分成3组(每组6只),分别给予甲羟孕酮(MPA组)、吉非替尼(吉非替尼组)、生理盐水(对照组)。疗程结束后称取各组肿瘤湿重,采用Western blotting法检测肿瘤组织中EGFR、孕激素受体B(PR-B)、ERK1/2、p-ERK1/2、AKT及p-AKT蛋白的表达。结果在接种Ishikawa细胞株的裸鼠中,MPA组、吉非替尼组移植瘤湿重与对照组相比,分别减轻43.0%和31.5%,差异有统计学意义(P均<0.05)。在接种Ishikawa-pLWERNL细胞株的裸鼠中,吉非替尼组移植瘤湿重与对照组相比,减轻35.7%,差异有统计学意义(P<0.05);而MPA组移植瘤湿重与对照组相比,减轻2.9%,差异无统计学意义(P>0.05)。接种I...