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目的探讨超声辐照维拉帕米(verpamil,VER)微泡逆转肿瘤多药耐药的作用及机制。方法制备包裹维拉帕米的乳酸-羟基乙酸共聚物(VER-PLGA)超声微泡造影剂;通过长春新碱(VCR)多次小剂量递增诱导法建立神经母细胞瘤耐药株(SK-N-SH/VCR),并检测其耐药性;根据处理因素不同分为6组:A:空白对照组(SK-N-SH/VCR培养组),B:SK-N-SH/VCR+VER(50μg/ml)组,C:SK-N-SH/VCR+VER(250μg/ml)组,D:SK-N-SH/VCR+超声辐照组,E:SK-N-SH/VCR+PLGA微泡+超声辐照组,F:SK-N-SH/VCR+VER-PLGA微泡(VER50μg/ml)+超声辐照组。48h后,利用RT-PCR和免疫组织化学染色法(ICC)检测各组细胞中MDR1基因及P-gp的表达水平,评估VER在不同处理组中逆转肿瘤细胞耐药的效果。结果①成功制备包裹维拉帕米的VER-PLGA微泡造影剂,包封率为32%;②成功建立神经母细胞瘤耐药株SK-N-SH/VCR,MTT法证实其对不同化疗药物的耐药倍数是野生株的2.3～5.4倍;③ICC证实耐药株高表达MDR1基因;④RT-PCR及免疫组化法检测发现MDR1基因在F组中的光密度值及P-gp表达水平较其他组明显降低(P<0.05)。结论超声辐照包裹小剂量维拉帕米的微泡可有效逆转肿瘤多药耐药。
Objective To investigate the effect and mechanism of ultrasound-induced microbubbles reversion of multidrug resistance of tumor cells by verpamil (VER) microbubbles. Methods VER-PLGA ultrasound microbubble contrast agent was prepared by wrapping verapamil. The neuroblastoma multi-drug resistant strain SK- N-SH / VCR), and tested for drug resistance; divided into 6 groups according to different treatment factors: A: blank control group, SK-N-SH / VCR culture group, V: SK-N-SH / VCR + VER group, C: SK-N-SH / VCR + VER group at 250μg / PLGA microbubbles + ultrasound irradiation group, F: SK-N-SH / VCR + VER-PLGA microbubbles (VER50μg / ml) + ultrasound irradiation group. After 48h, the expression of MDR1 gene and P-gp in each group were detected by RT-PCR and immunohistochemical staining (ICC), and the effect of VER reversing the drug resistance in different treatment groups was evaluated. Results ① We successfully prepared VER-PLGA microbubbles contrast agent encapsulated verapamil with the entrapment efficiency of 32%. ② The SK-N-SH / VCR neuroblastoma cell line was established successfully by MTT assay, MDR1 gene was highly expressed in resistant strains by ICC; ④RT-PCR and immunohistochemistry showed that MDR1 gene’s optical density value and P-gp expression level in group F Than other groups was significantly lower (P <0.05). Conclusion Microbubbles coated with verapamil under ultrasound irradiation can effectively reverse multidrug resistance of tumor.