表达乙型肝炎病毒抗原的种植性肝脏肿瘤动物模型的建立  

Establishment of a syngeneic mouse model of liver tumor stably expressing hepatitis B virus antigens

作　　者：汪颖[1] 吴志远[1] 任书荣[1] 魏勇[1] 张昆[1] 曲春枫[1] 

机构地区：[1]中国医学科学院北京协和医学院肿瘤医院肿瘤研究所分子肿瘤学国家重点实验室,100021

出　　处：《中华肿瘤杂志》2012年第7期486-491,共6页Chinese Journal of Oncology

基　　金：国家自然科学基金（30973387）;高等学校博士学科点专项科研基金（200800231092）

摘　　要：目的构建持续稳定表达乙型肝炎病毒（HBV）抗原的肿瘤细胞，建立持续表达HBV抗原的种植性肝脏肿瘤动物模型。方法以含有2倍体HBV的逆转录病毒载体质粒转染黑色素瘤细胞系B16，获得稳定表达HBV表面抗原（HBsAg）和核心抗原（HBcAg）的B16细胞（B16／HBV），分别按每只小鼠100、1000和5000个细胞接种到具有相同遗传背景的C57BL／6J小鼠肝脏被膜下，每组5只小鼠。注射后每周采血，进行血清HBsAg、乙型肝炎表面抗体（抗HBs）检测。采用小鼠腹部超声观测其成瘤情况，计算存活时间。对死亡小鼠肝脏进行病理分析，并检测肿瘤组织中HBsAg和HBcAg的表达。结果转染HBV病毒的肿瘤细胞B16／HBV传代20次以上后，细胞上清中有持续稳定的分泌型HBsAg表达，2×10^4个细胞在24h内的HBsAg表达量可达到110ng，并随时间延长而逐渐累积。未能检测到细胞上清中有HBeAg的表达。免疫组化结果显示，转染细胞中存在稳定的HBcAg表达，主要分布在细胞核中。每只小鼠注射100个B16／HBV细胞，小鼠肝脏成瘤率为80．0％；注射1000和5000个B16／HBV细胞，成瘤率为100％。注射后第4周，注射1000个B16／HBV细胞小鼠的腹部超声检测结果提示，肝脏内有异常回声，不均质回声面积为62．18min2。剖腹检查显示，在肝脏中所形成的肿瘤大体呈圆形，血运丰富，瘤组织质软，并有多个转移灶。在成瘤小鼠血清中，可检测到持续表达的HBsAg和抗HBs；所形成的肿瘤组织细胞中有均匀一致的HBsAg和HBcAg表达。结论HBV逆转录病毒载体质粒转染的B16／HBV细胞可稳定表达HBsAg和HBcAg。通过在肝脏被膜下注射B16／HBV细胞，建立了能持续分泌表达HBV抗原的种植性肝脏肿瘤小鼠模型。Objective To establish a syngeneic mouse model of liver tumor stably expressing hepatitis B virus （HBV） antigens. Methods Melanoma cell line B16 cells were transfected with pLXSN- 2HBV. Cells （named B16/HBV） stably and persistently expressing HBV surface （HBsAg） and core （HBcAg） antigens were identified. The cells were injected into the hepatic subcapsular space of fifteen C57BL/6J mice. The mice were divided into 3 groups, receiving 100, 1000 or 5000 cells in a total volume of 5 μl per mouse, respectively, five mice in each group. Two weeks after the tumor cell inoculation, serum samples from the mice were collected weekly and the serum concentration of HBsAg and anti-HBs was quantified by ELISA. The tumor growth in the mouse liver was monitored by a high-resolution ultrasound system. Expression of HBsAg and HBcAg in the tumor tissues was determined by immunohistochemistry. Results Liver tumors were formed in all the mice receiving 1000 and 5000 B16/HBV cells per mouse, and in 80% of the mice reeeiving 100 B16/HBV cells. HBsAg and anti-HBs were detectable in their sera from 2 weeks after tumor cell inoculation. The mice reeeiving 100 cells per mouse began to die 4 weeks, those receiving 1000 cells per mouse began to die 3-4 weeks and those receiving 5000 cells began to die 2-3 weeks after the cell inoculation. All the tumor cells expressed HBsAg and HBeAg. Conelusions The B16/HBV cells stably and persistently express HBV antigens both in vitro and in vivo. A mouse model of transplanted liver tumor stably expressing HBV antigens has been successfully established by inoculation of those cells into the hepatic subcapsular space.

关 键 词：肝脏肿瘤 肿瘤种植 模型 动物 肝炎 乙型 

分 类 号：R735[医药卫生—肿瘤] R51[医药卫生—临床医学] R-332