SL7207/pBud-VP3-IL-24的肿瘤靶向性及其对胃癌细胞免疫作用研究

Tumor-targeting of SL7207/pBud-VP3-IL-24 and its immune effect on gastric cancer cells

目的探讨基于白细胞介素(IL)-24的减毒沙门菌载体SL7207/pBud-VP3-IL-24对胃癌细胞的靶向性及肿瘤免疫作用。方法建立小鼠胃癌细胞荷瘤模型,将SL7207/pBud-VP3-IL-24(A组)、SL7207/pBud(B组)、生理盐水(C组)经口服饲喂胃癌荷瘤小鼠,分别采用组织匀浆菌落计数、RT-PCR分析及荧光蛋白定位检测减毒沙门菌株SL7207、apoptin-EGFP融合绿色荧光蛋白及IL-24基因在小鼠体内的分布,苏木精-伊红(HE)染色观察肿瘤组织病理变化,通过四甲基偶氮唑盐(MTT)法、乳酸脱氢酶(LDH)释放法及中性红试验检测淋巴细胞增殖反应、自然杀伤细胞(NK细胞)毒作用及腹腔巨噬细胞吞噬活性。结果在A组治疗后7d,小鼠肿瘤组织发现有减毒沙门菌株SL7207;随着时间推移,肿瘤组织中细菌数量逐渐增多,第21天,肿瘤组织中细菌数量比其他组织明显增多(P<0.05)。病理切片提示胃癌组织细胞溶解坏死。同时,IL-24基因和apoptin-EGFP蛋白在肿瘤组织有所表达,RT-PCR定量显示IL-24基因在肿瘤组织较其他组织多(P<0.05)。MTT和LDH实验显示,A组淋巴细胞增殖(0.75±0.11)和NK细胞活性(0.38±0.06)%明显高于B、C组,差异有统计学意义(P<0.05)。中性红实验结果提示,A组小鼠腹腔巨噬细胞吞噬功能增强(0.49±0.10),差异有统计学意义(P<0.05)。结论 SL7207/pBud-VP3-IL-24对胃癌组织具有靶向性,能在肿瘤组织优先表达其携带基因及蛋白,并通过增强机体非特异性免疫功能来调节抗肿瘤免疫。

Objective To investigate the targeting of IL-24-based attenuated Salmonella carrier SL7207/pBud-VP3-IL-24 on gastric cells and its tumor immune effect.Methods The mice model bearing gastric cancer cells was established and was orally administered with pBud-VP3-IL-24.The mouse in vivo distribution status of attenuated Salmonella SL7207,apoptin-EGFP fusion green fluorescent protein and IL-24 gene was detected by tissue homogenate colony count,MTT assay and RT-PCR.The tumor histopathological change was observed by HE statining.The lymphocyte proliferation reaction and natural killer cells(NK)toxicity and phagocytic activity of peritoneal macrophage were detected by MTT,lactate dehydrogenase(LDH)release and neutral red test.Results On 7 d after SL7207/pBud-VP3-IL-24 treatment,attenuated Salmonella was found in mouse tumor tissue.The number of bacteria in tumor tissue gradually increased with the passing of time.On 21 d,the number of bacteria in tumor tissue was significantly higher than that in other tissues(P<0.05).The pathological section indicated that gastric cancer tissues cells were lysed and necrosed,meanwhile IL-24 gene and apoptin-EGFP protein had some expression in tumor tissue.The RT-PCR quantitation analysis showed that the expression level of IL-24 gene in tumor tissue was higher than that in other tissues(P<0.05).MTT and LDH showed that SL7207/pBud-VP3-IL-24 could promote lymphocyte proliferation and enhance NK cell killing activity(P<0.05).The neutral red test results showed that the phagocytic activity of mouse peritoneal macrophages was enhanced(P<0.05).Conclusion SL7207/pBud-VP3-IL-24 shows gastric cancer tissue targeting effect,can preferentially express its carrying genes and proteins in tumor tissue,and regulates anti-tumor immunity by enhancing body non-specific immune function.