温郁金醇提物对人耐长春新碱胃腺癌细胞SGC-7901皮下移植瘤的逆转作用及对P糖蛋白表达的影响

Reversal Effect of Curcuma Wenyujin Extract on SGC-7901 / VCR Induced Subcutaneous Transplanted Tumor in Nude Mice and Its Effect on the Expression of P-glycoprotein

目的通过建立人耐长春新碱胃腺癌细胞SGC-7901（vincristine-resistant gastric cancer SGC-7901 cells,SGC-7901/VCR）裸鼠皮下移植瘤模型探讨温郁金醇提物逆转人胃癌多药耐药的作用及可能机制。方法运用四甲基偶氮唑蓝法（methyl thiazolyl tetrazolium,MTT）法对SGC-7901/VCR耐药性进行鉴定,50只BALB/c裸小鼠,采用组织块法建立人胃癌SGC-7901/VCR皮下移植瘤模型,2-3周后选取瘤体大小相近裸鼠36只,按随机数字表法分为模型组,长春新碱（vincristine,VCR）组,温郁金低、高剂量组及温郁金低、高剂量联合VCR组,共6组,每组6只。模型组给予生理盐水腹腔注射10m L/kg,2天1次;VCR组给予腹腔注射VCR 0.28 mg/kg,2天1次;温郁金低、高剂量组分别给予灌胃相应药物1.4、2.8 g/kg,1天1次,1次0.2 m L;温郁金低、高剂量联合VCR化疗组分别给予低、高剂量温郁金（1.4、2.8 g/kg）联合VCR（0.28 mg/kg,2天1次）;各组均为14天。观察各组瘤体生长情况,计算抑瘤率,同时采用免疫组化及蛋白印迹法（Western blot）检测瘤体P糖蛋白（P-glycoprotein,P-gp）的定位及表达。结果 SGC-7901/VCR对VCR、阿霉素（ADM）、5-氟尿嘧啶（5-FU）及顺铂（DDP）均有一定耐药性,其中对VCR耐药性最强。SGC-7901/VCR脱药培养后增殖活性明显增强,各组荷瘤裸鼠随着时间的延长,肿瘤体积逐渐增大,以温郁金高剂量联合VCR组体积最小。与模型组、VCR组比较,温郁金高剂量联合VCR组较瘤体明显缩小（P〈0.05）,其抑瘤率（51.56%）明显增高,差异亦有统计学意义（P〈0.05）。Western blot检测显示：与模型组比较,VCR组灰度值升高（P〈0.05）,温郁金高剂量组、温郁金低、高剂量联合VCR组中P-gp的相对表达量均明显减低（P〈0.05）。与VCR组比较,温郁金低、高剂量组及温郁金低、高剂量联合VCR组P-gp的灰度值均降低（P〈0.05）。免疫组化显示：与模型组比较,温郁金高剂量组、温郁金低、高剂量联合VCR组P-gp的表达积分均降低（P〈0.05）;与VCR组比较,温郁金低、高剂量组及温郁金低、高剂量联合VCR组P-gp的表达积分均明显降低（P〈0.05）。结论温郁金可逆转SGC-7901/VCR皮下移植瘤对VCR的耐药性,其作用机制可能与下调P-gp的表达有关,提示温郁金可能作为一种基于P-gp的多药耐药逆转剂。

Objective To explore the reversal effect of multidrug resistance of Curcuma Wenyujin（ CW） and its possible mechanism by establishing Vincristine-resistant gastric cancer SGC-7901 cells（ SGC-7901 / VCR） induced subcutaneous transplanted tumor in nude mice. Methods First we identified the resistance of SGC-7901 / VCR by using methyl thiazolyl tetrazolium（ MTT）. The SGC-7901 / VCR induced subcutaneous transplanted tumor model was established in 50 BALB / c nude mice by tissue block method. After 2- 3 weeks 36 mice with similar tumor size were selected and divided into6 groups by random digit table,i. e.,the model group,the Vincristine（ VCR） group,the low dose CW group,the high dose CW group,the low dose CW combined VCR group,and the high dose CW combined VCR group,6 in each group.Normal saline was intraperitoneally injected to mice in the model group at 10 m L / kg,once per 2 days. VCR was intraperitoneally injected to mice in the VCR group at 0. 28 mg / kg once per 2 days. CW at 1. 4 and 2. 8 g / kg was administered to mice in the low and high dose CW groups by gastrogavage,0. 2 m L each time,once daily. CW at 1. 4 and 2. 8 g / kg was administered by gastrogavage and VCR was intraperitoneally injected at 0. 28 mg / kg,once per 2 days to mice in the low dose CW combined VCR group and the high dose CW combined VCR group. All medication lasted for 14 days. The tumor growth was observed. The inhibition rate was calculated. Meanwhile,the positioning and expression of P-glycoprotein（ P-gp） were detected by immunohistochemistry and Western blot. Results SGC-7901 / VCR had strong resistance to VCR,Adramycin（ ADM）,fluorouracil（ 5-FU）,and Cisplatin（ DDP）,especially to VCR. Proliferation activities of SGC-7901 / VCR were significantly enhanced after drug elution. The tumor volume gradually increased as time went by.The tumor volume was the minimum in the high dose CW combined VCR group. The tumor volume was obviously reduced in the high dose CW combined VCR group with obviously reduced with increased inhibition rate of 51. 56 %,when compared with that of the model group and the VCR group（ P 〈0. 05）. Western blot test showed that,when compared with the model group,the gray level of P-gp in the VCR group increased（ P 〈0. 05）,and the relative expression of P-gp in the high dose CW group,the low dose CW combined VCR group,and the high dose CW combined VCR group significantly decreased（ P 〈0. 05）. Compared with the VCR group,the gray level of the P-gp decreased in the low dose CW group,the high dose CW group,the low dose CW combined VCR group,and the high dose CW combined VCR group（ P 〈0. 05）.Results of immunohistochemistry showed that,when compared with the model group,expression scores of P-gp in the high dose CW group,the low dose CW combined VCR group,and the high dose CW combined VCR group decreased with statistical difference（ P 〈0. 05）. Compared with the VCR group,expression scores of P-gp were obviously lowered in the low dose CW group,the high dose CW group,the low dose CW combined VCR group,and the high dose CW combined VCR group（ P 〈0. 05）. Conclusions CW could reverse the drug resistance of SGC-7901 / VCR subcutaneous transplanted tumor. And its mechanism might be related to down-regulating the expression of P-gp,suggesting that CW could be used as a kind of multidrug resistance reversal agent based on P-gp.