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基因工程抗体ch4E5是近年来研制出的针对CD80分子的人-鼠嵌合抗体[1],其与人B细胞淋巴瘤Raji细胞有较高的亲和力,并能抑制该类肿瘤细胞的生长增殖[2]。放射免疫治疗(radioimmunotherapy, RIT)是将单克隆抗体与放射性核素连接,注入体内后与肿瘤细胞特异性结合,并利用射线对肿瘤的辐射生物效应而达到治疗目的,对于放射线敏感的淋巴瘤的治疗有重要的理论意义和临床价值[3-4]。放射性核素131I标记技术已被广泛应用于基础和临床医学研究,但其对ch4E5的放射性核素标记尚未见报道。本研究采用 Iodogen法对ch4E5进行131I标记制备分子探针131I-ch4E5,并探索131I-ch4E5在体外对人B细胞淋巴瘤Raji细胞的亲和作用,及其在荷瘤裸鼠体内的生物学分布和对荷瘤裸鼠的抑瘤作用。
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131I-ch4E5的标记率为(84.2±2.4)%、放射化学纯度为(97.1±1.1)%、比活度为584.1 MBq/mg。
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131I-ch4E5分别在血清和PBS中第5天后的放射化学纯度均大于90%(表1),这说明标记抗体有较好的稳定性。
类别 0 d 1 d 3 d 5 d 血清 97.10±1.07 96.88±1.51 96.14±0.33 94.76±0.28 PBS 97.10±1.07 95.23±1.09 93.03±1.91 91.32±1.27 注:PBS为磷酸盐缓冲液 表 1 131I-ch4E5在血清和PBS中不同时间点的放射化学纯度(
,%)$\bar x \pm s $ Table 1. Radiochemical purity of 131I-ch4E5 at different time points in serum and PBS (
, %)$\bar x \pm s $ -
131I-ch4E5与Raji细胞的结合率如图1所示,随着Raji细胞数量的增加其结合率逐渐升高,当细胞数达到5×106以后则升高不明显,其最大结合率为(38.2±2.3)%。
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荷瘤裸鼠注射131I-ch4E5 72 h后,肿瘤与血液、心脏、肝、脾、肺、肾、胃、肠、胰腺、肌肉、骨、甲状腺、脑组织的T/NT分别为1.01±0.15、2.55±0.85、1.31±0.38、1.69±0.54、2.05±0.17、2.14±0.57、5.16±0.96、3.84±0.93、2.18±0.35、6.05±1.20、3.46±1.27、1.25±0.06、18.99±4.18,肿瘤与肌肉的T/NT最大为7.30,这表明131I-ch4E5在荷瘤裸鼠体内的分布具有靶向性。
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由图2可见,对照组肿瘤生长较快,ch4E5组肿瘤生长慢于对照组,2组比较差异有统计学意义(t=3.309,P=0.030);131I-ch4E5组肿瘤生长明显缓慢,与ch4E5组、对照组比较差异均有统计学意义(t=2.889 、6.516,P=0.044、0.003)。治疗后第15天,131I-ch4E5组肿瘤抑制率为71.7%,ch4E5组肿瘤抑制率为43.4%。
图 2 对照组、ch4E5组、131I-ch4E5组荷人B细胞淋巴瘤裸鼠的肿瘤生长曲线
Figure 2. Tumor growth curves of the human B-cell lymphoma-bearing nude mice in the control group, ch4E5 group, and 131I-ch4E5 group
各组荷瘤裸鼠肿瘤的组织病理学检查结果如图3所示,对照组肿瘤细胞增生活跃,见较多的病理性核分裂像。与对照组比较,ch4E5组病理性核分裂像相对较少,肿瘤细胞稀疏、个别核染色质浓缩、碎裂;131I-ch4E5组肿瘤细胞显著减少,大片肿瘤细胞凝固性坏死。
分子探针131I-ch4E5的制备及其动物实验研究
Preparation of molecular probe 131I-ch4E5 and its animal experiment research
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摘要:
目的 探讨靶向B细胞淋巴瘤的分子探针131I-ch4E5的制备方法及其对荷人B细胞淋巴瘤裸鼠的抑瘤作用。 方法 采用Iodogen法用131I标记抗CD80的人-鼠嵌合抗体ch4E5,采用放射性薄层色谱扫描法测定标记率和放射化学纯度。(1)体外实验:在含细胞数为1×108、1×109、1×1010、5×1010、1×1011个/L的人B细胞淋巴瘤Raji细胞的离心管中分别加入131I-ch4E5溶液,同时设置非特异结合对照管,测量每管沉淀的放射性计数,计算Raji细胞与131I-ch4E5的特异性结合率。(2)体内实验: 3只荷瘤裸鼠尾静脉注射7.4 MBq的131I-ch4E5,72 h后处死,分别取肿瘤、血等14种脏器或组织,分别计算肿瘤与正常组织的放射性比值(T/NT);将15只荷瘤裸鼠按随机数字表法分为3组,分别经尾静脉注射131I-ch4E5(131I-ch4E5组)、未标记的ch4E5(ch4E5组)及生理盐水(对照组),观察3组荷瘤裸鼠的肿瘤生长情况,给药后第15天计算肿瘤生长抑制率;之后处死荷瘤裸鼠取肿瘤组织,采用苏木精-伊红染色法做组织病理学检查。2组间的比较采用两独立样本t检验。 结果 131I-ch4E5的标记率为(84.2±2.4)%、放射化学纯度为(97.1±1.1)%。(1)体外实验:131I-ch4E5与Raji细胞的结合率随细胞数量的增加而升高,最大结合率为(38.2±2.3)%。(2)体内实验:131I-ch4E5在荷人B细胞淋巴瘤裸鼠体内的分布具有靶向性,肿瘤与肌肉的T/NT最大为7.30;与ch4E5组、对照组比较,131I-ch4E5组肿瘤生长减慢,差异均有统计学意义(t=2.889 、6.516,均P<0.05);给药后第15天,131I-ch4E5组肿瘤抑制率为71.7%、ch4E5组为43.4 %。组织病理学检查结果同样显示131I-ch4E5的治疗效果更明显。 结论 自制分子探针131I-ch4E5的标记率及放射化学纯度高,且对荷人B细胞淋巴瘤裸鼠肿瘤生长有明显的抑制作用,为进一步研究131I-ch4E5在放射免疫治疗中的应用提供了实验依据。 Abstract:Objective To investigate the preparation method of a molecular probe 131I-ch4E5 for targeting B-cell lymphoma and its anti-tumor effect in human B-cell lymphoma-bearing nude mouse model. Methods The anti-CD80 human-mouse chimeric antibody ch4E5 was labeled with 131I through the Iodogen method, and its labeling rate and radiochemical purity were determined by radioactive thin layer chromatography scanning method. (1)In vitro experiments: 131I-ch4E5 solution was added to cytocentrifuge tubes containing 1×108, 1×109, 1×1010, 5×1010, and 1×1011 cells/L of human B-cell lymphoma cells (Raji cells) and to non-specific binding control tubes. Radioactivity counts per tube of precipitation were measured to calculate the specific binding rate of Raji cells to 131I-ch4E5. (2) In vivo experiments: 7.4 MBq of 131I-ch4E5 was injected into three tumor-bearing nude mice via the tail vein. After 72 h, the tumor-bearing nude mice were sacrificed. Fourteen organs or tissues, such as tumor and blood, were harvested and weighed, and their radioactivity was measured. The radioactivity ratio of tumor to normal tissue (T/NT) was calculated. In the treatment study, 15 tumor-bearing nude mice were divided into three groups according to the random number table method and injected with 131I-ch4E5 (131I-ch4E5 group), unlabeled ch4E5 (ch4E5 group), and normal saline (control group) through the tail vein. Tumor growth was observed in the three groups. After 15 days, the tumor inhibition rate was calculated, and then the tumor-bearing nude mice were sacrificed to harvest tumor tissues for histopathological examination by hematoxylin-eosin staining. Comparisons between the two groups were performed using two independent samples t-test. Results The labeling rate of 131I-ch4E5 was (84.2±2.4)%, and the radiochemical purity was (97.1±1.1)%. (1) In vitro experiments: the binding rate of 131I-ch4E5 to Raji cells increased with increasing cell concentration, and the maximum binding rate was (38.2±2.3)%. (2) In vivo experiments: the distribution of 131I-ch4E5 in human B-cell lymphoma-bearing nude mice was targeted, and the maximum T/NT ratio of tumor to muscle was 7.30. Compared with the ch4E5 and control groups, the 131I-ch4E5 group exhibited decreased tumor growth, and the difference was statistically significant (t=2.889, 6.516; both P<0.05). Fifteen days after the administration, the tumor inhibition rate was 71.7% in the 131I-ch4E5 group and 43.4% in the ch4E5 group. The histopathological examination results showed that tumor cell necrosis was observed in both treatment groups, and the therapeutic efficacy of the 131I-ch4E5 group was superior to that of the ch4E5 group. Conclusion The self-made molecular probe 131I-ch4E5 has high labeling rate and radiochemical purity, and has a significant inhibitory effect on the tumor growth of human B-cell lymphoma-bearing nude mice, which provides an experimental basis for further research on the application of 131I-ch4E5 in radioimmunotherapy. -
Key words:
- Molecular probes /
- Iodine radioisotopes /
- ch4E5 /
- Lymphoma, B-cell /
- Radioimmunotherapy /
- Tumor-bearing nude mice
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表 1 131I-ch4E5在血清和PBS中不同时间点的放射化学纯度(
,%)$\bar x \pm s $ Table 1. Radiochemical purity of 131I-ch4E5 at different time points in serum and PBS (
, %)$\bar x \pm s $ 类别 0 d 1 d 3 d 5 d 血清 97.10±1.07 96.88±1.51 96.14±0.33 94.76±0.28 PBS 97.10±1.07 95.23±1.09 93.03±1.91 91.32±1.27 注:PBS为磷酸盐缓冲液 -
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