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目的研究白喉毒素的无毒突变体交叉反应物质197(cross-reacting material 197,CRM197)转运大分子物质通过血肿瘤屏障的效果和机制。方法制备CRM197-HRP偶联物,建立体外血肿瘤屏障模型,给予CRM197-HRP作用2 h后,通过TMB显色法检测transwell下室中HRP活性的变化;给予CRM197作用1 h后,采用免疫组化法检测体外血肿瘤屏障内皮细胞中p-Akt的表达变化;Western blot法检测p-Akt、p-FOXO1A和质膜微囊蛋白caveolin-1的表达变化。结果和对照组相比,CRM197-HRP能有效通过血肿瘤屏障,并且其转运水平呈时间依赖性增加;p-Akt主要分布在内皮细胞的胞质和胞核中,CRM197作用1 h后p-Akt的表达水平下降;同时伴有内皮细胞中p-FOXO1A表达下降和质膜微囊蛋白caveolin-1的表达上调。结论 CRM197可能通过抑制内皮细胞中p-Akt的表达,减少转录因子FOXO1A的磷酸化水平,进一步上调质膜微囊蛋白caveolin-1的表达,增加血肿瘤屏障的通透性。
Objective To study the effect and mechanism of cross-reacting material 197 (CRM197), a non-toxic mutant of diphtheria toxin, to transport macromolecules through the blood-tumor barrier. Methods The CRM197-HRP conjugate was prepared and the in vitro hematopoietic tumor barrier model was established. After treated with CRM197-HRP for 2 h, the change of HRP activity in the transwell lower chamber was detected by TMB colorimetric assay. After treated with CRM197 for 1 h, The expression of p-Akt in endothelial cells was detected by Western blot. The expressions of p-Akt, p-FOXO1A and plasma membrane caveolin-1 were detected by Western blot. Results Compared with the control group, CRM197-HRP could effectively pass through the blood-cancer barrier and its transport level increased in a time-dependent manner. P-Akt was mainly distributed in the cytoplasm and nucleus of endothelial cells. After 1 h of CRM197 treatment, p- Akt expression decreased; accompanied by decreased expression of p-FOXO1A in endothelial cells and plasma membrane caveolin-1 expression increased. Conclusion CRM197 may inhibit the expression of p-Akt in endothelial cells, decrease the phosphorylation level of FOXO1A and further upregulate the expression of caveolin-1, and increase the permeability of blood-tumor barrier.