重组人腺病毒P53和碘125粒子调节Bax和Bcl2对裸鼠胆管癌移植瘤增殖和凋亡的影响

和庆章 王桂良 邱 萍 徐林芳 龚敏 付云辉 韩明 文剑波

[關键词] 胆管癌;rHAd-P53;碘125;Bcl2;Bax

[中图分类号] R735.2          [文献标识码] A          [文章编号] 1673-9701（2021）29-0044-05

Impacts of recombinant human adenovirus P53 and iodine-125 particles regulating Bax and Bcl2 on proliferation and apoptosis of transplanted tumor of cholangiocarcinoma in nude mice

HE Qingzhang   WANG Guiliang   QIU Ping   XU Linfang   GONG Min   FU Yunhui   HAN Ming   WEN Jianbo

Department of Gastroenterology， Pingxiang Hospital Affiliated to Gannan Medical University， Pingxiang   337000， China

[Abstract] Objective To explore the impacts of recombinant human adenovirus P53（rHAd-P53） and iodine-125 particles regulating Bax and Bcl2 on proliferation and apoptosis of transplanted tumor of cholangiocarcinoma in nude mice. Methods Human cholangiocarcinoma cells were selected to establish human cholangiocarcinoma xenograft model in nude mice. A total of 48 nude mice were divided into the blank control group， the rHAd-P53 group， the iodine-125 particles group and the rHAd-P53 combined with iodine-125 particles group. The transplanted tumor size and weight were measured before and after treatment，the proliferation-related protein （Ki-67） was detected by immunohistochemistry， the apoptosis index was detected by TUNEL method， the expression of Bcl2 and Bax mRNA was detected by Realtime-PCR method， and the expression of Bcl2 and Bax protein was detected by Western blot method. Results After treatment， compared with the blank control group， the volume difference， tumor weight， proliferation index， Bcl2 and Bcl2/Bax mRNA， Bcl2 protein of transplanted tumor in the rHAd-P53 group， the iodine-125 particles group and the rHAd-P53 combined with iodine-125 particles group were significantly decreased， while Bax mRNA and protein were significantly increased.Compared with the rHAd-P53 group， the volume difference， tumor weight， proliferation index，significance of Bcl-2 and Bcl-2/Bax mRNA， Bcl-2 protein of transplanted tumor in the iodine-125 particles group or rHAd-P53 combined with iodine-125 particles group were all significantly decreased， while the tumor inhibition rate， Bax mRNA and protein were all significantly increased.Compared with the iodine-125 particles group， the volume difference， tumor weight， proliferation index， Bcl-2 and Bcl-2/Bax mRNA and Bcl-2 protein of transplanted tumor in rHAd-P53 combined with iodine-125 particles group were all significantly decreased， while the tumor inhibition rate， Bax mRNA and protein were all significantly increased. Conclusion RHAd-P53 and iodine-125 particles can inhibit the proliferation and promote the apoptosis of transplanted cholangiocarcinoma in nude mice by inhibiting the expression of Bcl2 and promoting the expression of Bax. rHAd-P53 can enhance the sensitivity of iodine-125 particles radiotherapy， and the effect of combining them is better than that of using them singly.

[Key words] Cholangiocarcinoma; rHAd-P53; iodine-125; Bcl2; Bax

胆管癌是一种起源于胆管上皮细胞的恶性肿瘤，其位置特殊、发病隐匿、恶性程度高、手术切除机会小、体外放疗和化疗效果均不理想[1]，碘125粒子局部放療具有创伤低、靶向效果好、并发症少、对正常组织损害小等优点，因而越来越受到国内外学者的重视[2]。P53基因是最有效的抑癌基因，能有效调节细胞周期、诱导肿瘤细胞死亡。rHAd-P53能增强肿瘤对碘125粒子放疗的敏感性[3]，现已广泛应用于临床，国内外尚没有将二者联用的研究报道，本研究应用rHAd-P53和碘125粒子单独治疗及联合治疗人裸鼠胆管癌移植瘤模型，以观察rHAd-P53对碘125粒子治疗胆管癌敏感性的影响，为基础研究及临床应用提供新的思路，现报道如下。

1 资料与方法

1.1 一般资料

BALB/c-nu裸鼠（5～6周龄，体重18～22 g，SPF环境下饲养），购自江西中医学院动物部[动物许可证号：SYXK（赣）2008-0006）];人胆管癌细胞株QBC939细胞株购自美国ATCC公司;重组人rHAd-P53（1×1012VP/支，国药准字S20040004）购自深圳赛百诺公司;Bax、Bcl2、GAPDH一抗购自美国Abcam公司;碘125粒子（粒子活度为0.6 mCi）购自北京智博高科生物技术有限公司;cDNA合成试剂盒购自美国Thermo公司;TUNEL试剂盒购自瑞士Roche公司;Trizol试剂自美国Invitrogen公司;PCR反转录试剂盒和Taq聚合酶购自日本TaKaRa公司。Real time PCR扩增基因引物序列见表1，由上海生工生物科技有限公司合成。

1.2 方法

1.2.1 裸鼠胆管癌模型制备及分组  将QBC939细胞株（1×106个）接种于裸鼠右腋窝皮下。接种后观察肿瘤生长情况，肿瘤直径约0.5 cm时即用于实验。……