染料木黄酮对MKN45胃癌细胞及其皮下移植瘤抗血管生成作用的研究

Genistein’s anti-angiogenic effect on MKN 45 gastric cancer cells and its subcutaneous xeno-transplanted tumors

摘要:

目的：验证染料木黄酮（Genistein）对人胃癌细胞MKN45及其皮下移植瘤模型的抗血管生成作用，并建立以光学分子影像对其抗血管生成作用进行监测的实验方法。方法：MTT法检测Genistein对MKN45人胃癌细胞的增殖抑制作用；血管内皮生长因子（vascular endothelial growth factor，VEGF）-ELISA验证Genistein对MKN45细胞及MKN45皮下移植瘤VEGF表达的抑制作用。免疫组化检测Genistein治疗后肿瘤VEGF、CD31及Ki67表达水平的变化；将近红外荧光染料分子Dylight 680-NHS与所制备的F（ab′）2段贝伐单抗（Bevacizumab）偶联并纯化制得靶向VEGF的荧光分子探针：Dylight 680-Bevacizumab-F（ab′）2，并将其应用活体荧光显像检测Genistein对肿瘤VEGF的抑制效果。结果：MTT及Ki67检测结果表明Genistein对MKN45人胃癌细胞及其皮下移植瘤无明显的增殖抑制效果（MTT检测结果中P=0.118）；VEGF-ELISA检测结果表明Genistein能够明显降低MKN45细胞（P=0.002）及其皮下移植瘤（P=0.000）VEGF的表达水平。Genistein对MKN45皮下移植瘤的治疗实验结果表明：Genistein具有一定的抑瘤疗效（P=0.034），通过免疫组化分析可知肿瘤VEGF及CD31表达水平明显降低；同时，荧光分子探针Dylight 680-Bevacizumab-F（ab′）2应用于活体荧光成像能够有效地检测皮下肿瘤VEGF表达水平的变化。结论：Genistein主要通过抑制MKN45肿瘤（细胞）VEGF的表达产生抗血管生成作用而非直接抑制肿瘤（细胞）的增殖达到抑瘤效果。荧光分子探针Dylight 680-Bevacizumab-F（ab′）2应用于活体显像能够对Genistein抗肿瘤血管生成的疗效进行早期预测，具有良好的临床转化前景。

Abstract:

Objective：To verify genistein’s anti-angiogenic effect on MKN 45 gastric cancer cells and its subcutaneous xeno-transplanted tumors and to develop an optical imaging method for monitoring this anti-angiogenesis. Methods：MTT assay and VEGF-ELISA were performed to test the inhibitory effect of genistein on proliferation of MKN45 human gastric cancer cells and VEGF expression. IHC was applied to detect VEGF，CD31 and Ki67 expression after MKN45 subcutaneous xeno-transplanted tumors being treated with genistein. The optical molecular probe targeting VEGF：Dylight 680-Bevacizumab-F（ab′）2 was firstly prepared by conjugating near-infrared fluorescent dye Dylight 680-NHS and bevacizumab’s F（ab′）2 fragments. Then，it was applied in in vivo optical imaging to verify tumor VEGF down regulation induced by genistein. Results：Results of MTT（P=0.118） and Ki67 IHC showed that genistein had no significant inhibitory effect on the proliferation of MKN45 human gastric cancer cells and its subcutaneous xeno-trans-planted tumors. VEGF-ELISA results showed geni-stein could significantly down regulate VEGF expression of MKN45 cells and its xeno-transplanted tumors（P=0.000）. Re-sults of genistein treatment indicated that genistein had a certain anti-tumor effect（P=0.034）. IHC results showed tumor VEGF and CD31 expressions were both reduced after genistein treat－ment. In addition，Dylight 680-Bevacizumab-F（ab′）2 applied to in vivo optical imaging could effectively detect reduced VEGF ex－pression. Conclusion：The inhibitory effect of genistein is mainly realized by genistein’s anti-angiogenesis，which significantly inhibit tumor VEGF expression，rather than genistein’s direct tumor proliferation inhibition. Optical molecular probes Dylight 680-Beva－cizumab-F（ab′）2 applied in vivo optical imaging，with favorable clinical prospect，could predict anti-angiogenic effect of genistein at early stage.