重组改构人肿瘤坏死因子对人胃癌细胞的药效学及其机制研究

背景:重组改构人肿瘤坏死因子(rmhTNF)与原型TNF-α相比具有高抗肿瘤活性和低毒性的特点。目的:研究注射用rmhTNF对人胃癌细胞的药效学及其机制。方法:体外培养人胃癌细胞株BGC-823和人脐静脉内皮细胞株HUVEC-12,分别以不同浓度的rmhTNF和rhTNF-α进行处理,以MTT实验、软琼脂克隆形成实验、TUNEL染色和流式细胞术分析细胞增殖、凋亡情况和细胞周期分布;以蛋白质印迹法检测BGC-823细胞凋亡相关蛋白表达;以免疫共沉淀技术检测rmhTNF与死亡受体5(DR5)间的相互作用。建立BGC-823细胞裸鼠皮下移植瘤模型,分别予rmhTNF、rhTNF-α、5-氟尿嘧啶和0.9%NaCl溶液腹腔内注射,以CD34免疫组化染色检测移植瘤组织微血管密度(MVD)。结果:rmhTNF和rhTNF-α对BGC-823和HUVEC-12细胞均有抑制增殖和诱导凋亡作用,并能使G0/G1期细胞进入S期,rmhTNF的作用强于rhTNF-α(P0.05)。rmhTNF组移植瘤组织MVD显著低于其他各组(P0.05)。rmhTNF能使BGC-823细胞procaspase-3、Bax表达上调,Bcl-2表达下调,并能与DR5相互作用。结论:rmhTNF对人胃癌细胞具有抑制增殖、诱导凋亡、影响细胞周期分布和抑制肿瘤血管形成的作用,其机制可能为rmhTNF与DR5结合,从而改变细胞内凋亡相关蛋白水平。

Pharmacodynamics and Pharmacological Mechanism of Recombinant Mutant Human Tumor Necrosis Factor in Human Gastric Cancer Cells

Background： Recombinant mutant human tumor necrosis factor （rmhTNF） is a mutant rhTNF-α with higher anti- tumor activity and lower toxicity when compared with its prototype. Aims： To investigate the pharmacodynamics and pharmacological mechanism of rmhTNF injection in human gastric cancer cells. Methods： Cultured human gastric cancer cell line BGC-823 and human umbilical vein endothelial cell line HUVEC-12 were treated with rmhTNF or rhTNF-e~ at different concentrations in vitro. The proliferation, apoptosis and cell cycle of these cells were determined by MTF assay, soft agar colony formation assay, TUNEL assay and flow cytometry. The expressions of apoptosis-related proteins in BGC- 823 cells were determined by Western blotting, and the interaction between rmhTNF and death receptor 5 （DR5） was determined by eo-immunoprecipitation. Nude mice transplanted tumor model was constructed by subcutaneous injection of BGC-823 cells, with subsequent intraperitoneal injection of rmhTNF~ rhTNF-c~, 5-fluorouraeil and normal saline, respectively. The microvessel density （MVD） of transplanted tumors was assessed by CD34 immunohistochemical staining. Results： Both rmhTNF and rhTNF-αinhibited proliferation, induced apoptosis and enhanced the entry into S phase in both BGC-823 and HUVEC-12 cells, however, rmhTNF was significantly more effective than rhTNF-α （P〈0.05）. MVD of transplanted tumors treated with rmhTNF was significantly lower than that in all the other groups （P〈0.05）. The expressions of procaspase-3 and Bax were upregulated, and Bcl-2 was downregulated in BGC-823 cells treated with rmhTNF, and an interaction between rmhTNF and DR5 was confirmed. Conclusions： rmhTNF can inhibit proliferation, induce apoptosis, affect the distribution of cell cycle, and suppress tumor angiogenesis in human gastric cancer cells. The mechanism might be due to its interaction with DR5 and the subsequent alteration of apoptosis-related proteins.