中华肿瘤杂志
中華腫瘤雜誌
중화종류잡지
CHINESE JOURNAL OF ONCOLOGY
2012年
7期
486-491
,共6页
汪颖%吴志远%任书荣%魏勇%张昆%曲春枫
汪穎%吳誌遠%任書榮%魏勇%張昆%麯春楓
왕영%오지원%임서영%위용%장곤%곡춘풍
肝脏肿瘤%肿瘤种植%模型,动物%肝炎,乙型
肝髒腫瘤%腫瘤種植%模型,動物%肝炎,乙型
간장종류%종류충식%모형,동물%간염,을형
Liver neoplasms%Neoplasm seeding%Models,animal%Hepatitis B
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,2×104个细胞在24h内的HBsAg表达量可达到110 ng,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62.18 mm2.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.
目的 構建持續穩定錶達乙型肝炎病毒(HBV)抗原的腫瘤細胞,建立持續錶達HBV抗原的種植性肝髒腫瘤動物模型.方法 以含有2倍體HBV的逆轉錄病毒載體質粒轉染黑色素瘤細胞繫B16,穫得穩定錶達HBV錶麵抗原(HBsAg)和覈心抗原(HBcAg)的B16細胞(B16/HBV),分彆按每隻小鼠100、1000和5000箇細胞接種到具有相同遺傳揹景的C57BL/6J小鼠肝髒被膜下,每組5隻小鼠.註射後每週採血,進行血清HBsAg、乙型肝炎錶麵抗體(抗HBs)檢測.採用小鼠腹部超聲觀測其成瘤情況,計算存活時間.對死亡小鼠肝髒進行病理分析,併檢測腫瘤組織中HBsAg和HBcAg的錶達.結果 轉染HBV病毒的腫瘤細胞B16/HBV傳代20次以上後,細胞上清中有持續穩定的分泌型HBsAg錶達,2×104箇細胞在24h內的HBsAg錶達量可達到110 ng,併隨時間延長而逐漸纍積.未能檢測到細胞上清中有HBeAg的錶達.免疫組化結果顯示,轉染細胞中存在穩定的HBcAg錶達,主要分佈在細胞覈中.每隻小鼠註射100箇B16/HBV細胞,小鼠肝髒成瘤率為80.0％；註射1000和5000箇B16/HBV細胞,成瘤率為100％.註射後第4週,註射1000箇B16/HBV細胞小鼠的腹部超聲檢測結果提示,肝髒內有異常迴聲,不均質迴聲麵積為62.18 mm2.剖腹檢查顯示,在肝髒中所形成的腫瘤大體呈圓形,血運豐富,瘤組織質軟,併有多箇轉移竈.在成瘤小鼠血清中,可檢測到持續錶達的HBsAg和抗HBs；所形成的腫瘤組織細胞中有均勻一緻的HBsAg和HBcAg錶達.結論 HBV逆轉錄病毒載體質粒轉染的B16/HBV細胞可穩定錶達HBsAg和HBcAg.通過在肝髒被膜下註射B16/HBV細胞,建立瞭能持續分泌錶達HBV抗原的種植性肝髒腫瘤小鼠模型.
목적 구건지속은정표체을형간염병독(HBV)항원적종류세포,건립지속표체HBV항원적충식성간장종류동물모형.방법 이함유2배체HBV적역전록병독재체질립전염흑색소류세포계B16,획득은정표체HBV표면항원(HBsAg)화핵심항원(HBcAg)적B16세포(B16/HBV),분별안매지소서100、1000화5000개세포접충도구유상동유전배경적C57BL/6J소서간장피막하,매조5지소서.주사후매주채혈,진행혈청HBsAg、을형간염표면항체(항HBs)검측.채용소서복부초성관측기성류정황,계산존활시간.대사망소서간장진행병리분석,병검측종류조직중HBsAg화HBcAg적표체.결과 전염HBV병독적종류세포B16/HBV전대20차이상후,세포상청중유지속은정적분비형HBsAg표체,2×104개세포재24h내적HBsAg표체량가체도110 ng,병수시간연장이축점루적.미능검측도세포상청중유HBeAg적표체.면역조화결과현시,전염세포중존재은정적HBcAg표체,주요분포재세포핵중.매지소서주사100개B16/HBV세포,소서간장성류솔위80.0％；주사1000화5000개B16/HBV세포,성류솔위100％.주사후제4주,주사1000개B16/HBV세포소서적복부초성검측결과제시,간장내유이상회성,불균질회성면적위62.18 mm2.부복검사현시,재간장중소형성적종류대체정원형,혈운봉부,류조직질연,병유다개전이조.재성류소서혈청중,가검측도지속표체적HBsAg화항HBs；소형성적종류조직세포중유균균일치적HBsAg화HBcAg표체.결론 HBV역전록병독재체질립전염적B16/HBV세포가은정표체HBsAg화HBcAg.통과재간장피막하주사B16/HBV세포,건립료능지속분비표체HBV항원적충식성간장종류소서모형.
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 pLXSN2HBV.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 5μl 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 receiving 100 B16/HBV cells.HBsAg and anti-HBs were detectable in their sera from 2 weeks after tumor cell inoculation.The mice receiving 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 HBcAg.Conclusions 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.
