| 激动性CD40抗体通过调节Th1/Th2平衡增强抗肿瘤作用的机制研究 |
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| 引用本文： | 郑凯源,黄飞,陆礼,张骜,付蔚华. 激动性CD40抗体通过调节Th1/Th2平衡增强抗肿瘤作用的机制研究[J]. 中国肿瘤临床, 2018, 45(4): 163-166. DOI: 10.3969/j.issn.1000-8179.2018.04.190 |
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| 作者姓名： | 郑凯源 黄飞 陆礼 张骜 付蔚华 |
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| 作者单位： | 天津医科大学总医院普通外科研究所（天津市300052） |
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| 摘 要： | 目的 探讨激动性CD40抗体中单链抗体（CD40ScFv）和单克隆抗体（CD40mAb）抑制小鼠乳腺癌细胞生长情况及对癌组织中Th1和Th2平衡的改变，分析通过调节Th1/Th2平衡达到抗肿瘤作用的机制。 方法 将含CD40ScFv基因片段的重组质粒转化至Rosetta大肠杆菌中，诱导表达并经纯化后获得有功能的CD40ScFv蛋白，行SDS-PAGE和Western blot检测。体外培养小鼠乳腺癌4T1细胞，在Balb/C小鼠皮下成瘤建立模型，分为CD40ScFv组、CD40mAb组和生理盐水组（NS组），分别给予CD40ScFv、CD40mAb和生理盐水，观察各组小鼠肿瘤体积的变化。取出瘤体组织，ELISA法检测肿瘤组织上清中IL-12的浓度。提取肿瘤组织浸润淋巴细胞（TILs），应用流式细胞技术检测TILs中Th1与Th2的比例。 结果 诱导表达的CD40ScFv鉴定表达成功，分子大小约为27 KDa，蛋白纯化后经BCA测定浓度为1.12 mg/mL。CD40ScFv组与CD40mAb组肿瘤大小分别为（3.044±0.239）cm3与（2.749±0.261）cm 3，均小于NS组的（3.933±0.326）cm3，差异具有统计学意义（P <0.05）。CD40ScFv组、CD40mAb组肿瘤组织中IL-12浓度分别为（396.27±48.13）pg/mL、（457.63±58.37）pg/mL，均显著高于NS组的（79.51±14.97）pg/mL，差异具有统计学意义（P <0.05）。CD40ScFv组与CD40mAb组的Th1/Th2细胞比值分别为6.32±0.87与5.54±0.71，均显著高于NS组的1.79±0.38，差异具有统计学意义（P < 0.05）。 结论 激动性CD40抗体在体内通过调节Th1与Th2的比例，发挥抑制肿瘤生长的作用，其中通过促进DC活化后分泌IL-12是影响肿瘤微环境中Th1/Th2平衡的重要机制之一。
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| 关 键 词： | 激动性CD40抗体 肿瘤微环境 辅助性T淋巴细胞 |
| 收稿时间： | 2017-10-30 |
Antitumor effect of agonistic CD40 antibody via the regulation of Th1/Th2 balance in the tumor microenvironment |
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| Affiliation: | General Hospital Affiliated to Tianjin Medical University General Surgery Research Institute, Tianjin 300052, China |
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| Abstract: | Objective To explore the agonistic CD40 antibody (CD40ScFv and CD40mAb)-mediated suppression of mouse breast cancer cell growth and change in tumor tissue Th1/Th2 balance, as well as the mechanism underlying its antitumor effect. Methods The recombinant plasmid containing the CD40ScFv gene fragment was transformed into the Rosetta strain of Escherichia coli to express and purify the recombinant functional CD40ScFv protein. The 4T1 mouse breast cancer cells were cultured in vitro. Balb/C tumor model mice were divided into CD40ScFv, CD40mAb agonist, and saline (NS group) groups, which were administered CD40ScFv, CD40mAb agonist, and saline, respectively, to observe the change in tumor volume. The tumor tissues were removed and enzyme-linked immunosorbent assay (ELISA) was used to determine IL-12 concentration in the tumor tissue supernatants. The tumor infiltrating lymphocytes (TILs) were extracted by enzymatic digestion. The proportion of Th1 and Th2 cells in TILs was determined by flow cytometry. Results The CD40ScFv protein was successfully identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot. The molecular size and concentration of the purified protein were 27kDa and 1.12 mg/mL, respectively. The tumor sizes of the CD40ScFv and CD40mAb groups were (3.044±0.239) cm3 and (2.749±0.261) cm3, respectively, which were significantly smaller (P <0.05) than that of the NS group (3.933±0.326) cm3. The tumor IL-12 concentration (determined by ELISA) in the CD40ScFv group (396.27±48.13) pg/ mL and the CD40mAb agonist group (457.63±58.37) pg/mL were significantly higher (P <0.05) than that of the NS group (79.51±14.97) pg/mL. The results of flow cytometry showed that the excited Th1/Th2 cell ratios were 6.32±0.87 and 5.54±0.71 for the CD40ScFv and CD40mAb groups, respectively, which were significantly higher (P <0.05) than that of the NS group (1.79±0.38). Conclusions The agonistic CD40 antibody inhibited tumor growth by regulating the Th1/Th2 ratio and IL-12 secretion via promotion of DC activation, which is one of the important mechanisms affecting Th1/Th2 balance in the tumor microenvironment |
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