腺病毒介导hCTR1转染用于治疗顺铂耐受宫颈癌的基础研究

Basic Research of the Adenovirus-mediated hCTR1 Transfection on the Treatment of Cisplatin Resistant Cervical Cancer

目的诱导培养对顺铂耐药的人宫颈癌细胞株,并进一步研究针对宫颈癌细胞耐药的机制和相应的治疗方法。方法采用浓度梯度递增法使用顺铂处理人宫颈癌细胞C-33A,诱导出顺铂耐药细胞株C-33A/cis后,通过Western blot检测铜离子转运蛋白(copper transporter 1,CTR1)的表达改变,使用顺铂耐药细胞株C-33A/cis建立肿瘤皮下荷瘤模型,使用转染有hCTR1基因的腺病毒注射液(ad-hCTR1)并联合顺铂给药,观测肿瘤体积的改变,并在最后一次注射10d后处死小鼠,通过免疫组化方法检测肿瘤组织中CTR1的表达。结果浓度梯度递增法诱导出的耐药细胞株C-33A/cis较亲代细胞C-33A对顺铂引起的细胞毒性敏感性下降,顺铂的半致死剂量浓度(IC50)由(1.86±0.08)μmol/L提升至(8.11±0.21)μmol/L。Western blot结果显示耐药细胞C-33A/cis中CTR1的表达降低。利用顺铂耐药细胞株C-33A/cis建立的皮下瘤模型,在给予ad-hCTR1后,肿瘤组织内可检测到CTR1表达增高;对荷瘤鼠给予腺病毒转染CTR1联合顺铂给药后,肿瘤体积增长受到抑制。结论腺病毒转染CTR1联合顺铂给药可能提高耐药性宫颈癌的治疗效果。

Objective To induce eisplatin-resistant cervical squamous carcinoma cell line and investigate the drug resistant mechanisms and adenovirus trans-gene therapeutical treatment. Methods The cisplatin-resistant subline, designated C-33A/cis, was originated by growing parental C-33A cells with gradually increasing doses of cisplatin. The cytotoxicity of cisplatin was determined by CCK-8 assay, and the CTR1 expression was measured by Western blot. Subcutaneous xenograft cervical tumor model was established by cisplatin-resistant C-33A/cis cell line. Recombinant adenovirus ad-hCTR1 was transfected into tumor by intratumoral injection and combined with cisplatin chemotherapy. The changes in the volume of tumor were observed and the mice were executed at 10th day after the last injection, and the expression of CTR1 in tumor tissues was detected by immunohistochemistry. Results Cisplatin-resistant cervical carcinoma C-33A/cis cells were successfully induced by gradually increased concentration of cisptatin. The cytotoxic IC50 value of cisplatin on C-33A/cis had been upgraded from （1.86±0.08） to （8. 11±0.21）μmol/L, while the CTR1 was found decreased by Western blot assay. Immunohistochemical analysis indicated that CTR1 expression was increased by intratumoral injection of adenovirus ad-hCTR1, and the tumor growth of C-33A/cis drug-resistant cervical carcinoma xenograft was inhibited by ad-hCTR1 transfection combined with cisplatin. Conclusion The combination therapy of ad-hCTR1 transfection and cisplatin was effective to inhibit the growth of drug-resistant C-33A/cis tumor.