中华实验和临床感染病杂志(电子版)
中華實驗和臨床感染病雜誌(電子版)
중화실험화림상감염병잡지(전자판)
Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Version)
2015年
5期
708-713
,共6页
赵杨静%李国力%张恒辉%谢兴旺%贺改霞%闫琳琳%李明珍%魏来%曾辉
趙楊靜%李國力%張恆輝%謝興旺%賀改霞%閆琳琳%李明珍%魏來%曾輝
조양정%리국력%장항휘%사흥왕%하개하%염림림%리명진%위래%증휘
肝炎病毒,乙型%肝癌细胞株%病毒整合
肝炎病毒,乙型%肝癌細胞株%病毒整閤
간염병독,을형%간암세포주%병독정합
Hepatitis B virus%Hepatocellular carcinoma cell%Virus integration
目的 探讨常见肝癌细胞株中整合的乙型肝炎病毒(HBV)序列和整合位点.方法 分别将来源于HBV感染者的肝癌细胞株(MHCC97H、MHCC97L、MHCCLM3和PLC/PRF/5),稳定转染HBV的肝癌细胞株(HepG2.2.15、HepAD38和DE19),和无HBV感染者来源的肝癌细胞株(HepG2、HuH-7)培养72 h,检测培养上清中乙型肝炎病毒表面抗原(HBsAg)、乙型肝炎病毒E抗原(HBeAg)及HBV DNA滴度.采用HBV-Alu-PCR方法,扩增肝癌细胞株中整合的HBV基因X/C/S片段及其侧翼的人基因组DNA片段,对扩增片段进行测序确定HBV整合在人类染色体的精确位置,并用生物信息学分析确定其上下游基因.结果 MHCC97H、MHCC97L和MHCCLM3未检出HBsAg和HBeAg,但HBV DNA滴度为2.00 × 105~4.00 × 105IU/ml;HepG2.2.15、HepAD38、DE19和PLC/PRF/5 HBsAg阳性,且HBV DNA滴度> 5.00 ×105IU/ml,其中HepG2.2.15、HepAD38HBeAg阳性;HepG2、HuH-7 HBsAg、HBeAg和HBV DNA均低于检测下限.除HepG2和HuH-7未检测到整合外,其余细胞株中可检测到1个或多个整合的不同HBV亚基因组片段:HepG2.2.15检测到5个HBx和HBc整合片段,HepAD38和PLC/PRF/5各检测到2个HBx整合片段,DE19检测到一个HBc整合片段.3个MHCC97的衍生细胞株:MHCC97L(低转移潜能肝癌细胞)、MHCC97H(高转移潜能肝癌细胞)和MHCCLM3(MHCC97H肺转移肝癌细胞)检测到完全相同的HBc片段整合在16q13上IRX3和IRX5基因之间的非编码区.此外,各细胞株HBV整合位点上下游的基因主要包括肿瘤相关基因,核糖体蛋白编码基因和钙信号相关基因.结论 HBV感染者来源和稳定转染HBV的肝癌细胞株存在HBV整合;HBV基因X/C片段比S片段有更高的整合频率;同一个克隆来源的肝癌细胞株的HBV整合位点稳定,提示HBV整合分析可能对肝细胞癌原发灶和转移灶癌细胞克隆来源的鉴定提供参考.
目的 探討常見肝癌細胞株中整閤的乙型肝炎病毒(HBV)序列和整閤位點.方法 分彆將來源于HBV感染者的肝癌細胞株(MHCC97H、MHCC97L、MHCCLM3和PLC/PRF/5),穩定轉染HBV的肝癌細胞株(HepG2.2.15、HepAD38和DE19),和無HBV感染者來源的肝癌細胞株(HepG2、HuH-7)培養72 h,檢測培養上清中乙型肝炎病毒錶麵抗原(HBsAg)、乙型肝炎病毒E抗原(HBeAg)及HBV DNA滴度.採用HBV-Alu-PCR方法,擴增肝癌細胞株中整閤的HBV基因X/C/S片段及其側翼的人基因組DNA片段,對擴增片段進行測序確定HBV整閤在人類染色體的精確位置,併用生物信息學分析確定其上下遊基因.結果 MHCC97H、MHCC97L和MHCCLM3未檢齣HBsAg和HBeAg,但HBV DNA滴度為2.00 × 105~4.00 × 105IU/ml;HepG2.2.15、HepAD38、DE19和PLC/PRF/5 HBsAg暘性,且HBV DNA滴度> 5.00 ×105IU/ml,其中HepG2.2.15、HepAD38HBeAg暘性;HepG2、HuH-7 HBsAg、HBeAg和HBV DNA均低于檢測下限.除HepG2和HuH-7未檢測到整閤外,其餘細胞株中可檢測到1箇或多箇整閤的不同HBV亞基因組片段:HepG2.2.15檢測到5箇HBx和HBc整閤片段,HepAD38和PLC/PRF/5各檢測到2箇HBx整閤片段,DE19檢測到一箇HBc整閤片段.3箇MHCC97的衍生細胞株:MHCC97L(低轉移潛能肝癌細胞)、MHCC97H(高轉移潛能肝癌細胞)和MHCCLM3(MHCC97H肺轉移肝癌細胞)檢測到完全相同的HBc片段整閤在16q13上IRX3和IRX5基因之間的非編碼區.此外,各細胞株HBV整閤位點上下遊的基因主要包括腫瘤相關基因,覈糖體蛋白編碼基因和鈣信號相關基因.結論 HBV感染者來源和穩定轉染HBV的肝癌細胞株存在HBV整閤;HBV基因X/C片段比S片段有更高的整閤頻率;同一箇剋隆來源的肝癌細胞株的HBV整閤位點穩定,提示HBV整閤分析可能對肝細胞癌原髮竈和轉移竈癌細胞剋隆來源的鑒定提供參攷.
목적 탐토상견간암세포주중정합적을형간염병독(HBV)서렬화정합위점.방법 분별장래원우HBV감염자적간암세포주(MHCC97H、MHCC97L、MHCCLM3화PLC/PRF/5),은정전염HBV적간암세포주(HepG2.2.15、HepAD38화DE19),화무HBV감염자래원적간암세포주(HepG2、HuH-7)배양72 h,검측배양상청중을형간염병독표면항원(HBsAg)、을형간염병독E항원(HBeAg)급HBV DNA적도.채용HBV-Alu-PCR방법,확증간암세포주중정합적HBV기인X/C/S편단급기측익적인기인조DNA편단,대확증편단진행측서학정HBV정합재인류염색체적정학위치,병용생물신식학분석학정기상하유기인.결과 MHCC97H、MHCC97L화MHCCLM3미검출HBsAg화HBeAg,단HBV DNA적도위2.00 × 105~4.00 × 105IU/ml;HepG2.2.15、HepAD38、DE19화PLC/PRF/5 HBsAg양성,차HBV DNA적도> 5.00 ×105IU/ml,기중HepG2.2.15、HepAD38HBeAg양성;HepG2、HuH-7 HBsAg、HBeAg화HBV DNA균저우검측하한.제HepG2화HuH-7미검측도정합외,기여세포주중가검측도1개혹다개정합적불동HBV아기인조편단:HepG2.2.15검측도5개HBx화HBc정합편단,HepAD38화PLC/PRF/5각검측도2개HBx정합편단,DE19검측도일개HBc정합편단.3개MHCC97적연생세포주:MHCC97L(저전이잠능간암세포)、MHCC97H(고전이잠능간암세포)화MHCCLM3(MHCC97H폐전이간암세포)검측도완전상동적HBc편단정합재16q13상IRX3화IRX5기인지간적비편마구.차외,각세포주HBV정합위점상하유적기인주요포괄종류상관기인,핵당체단백편마기인화개신호상관기인.결론 HBV감염자래원화은정전염HBV적간암세포주존재HBV정합;HBV기인X/C편단비S편단유경고적정합빈솔;동일개극륭래원적간암세포주적HBV정합위점은정,제시HBV정합분석가능대간세포암원발조화전이조암세포극륭래원적감정제공삼고.
Objective To investigate the integration of hepatitis B virus (HBV) in human hepatocellular carcinoma (HCC) cell lines.Methods HCC cell lines, which established from patients with HBV infection (MHCC97H, MHCC97L, MHCCLM3 and PLC/PRF/5), stably transfected with HBV (HepG2.2.15, HepAD38 and DE19) and without HBV infection (HepG2 and HuH-7), were cultured for 72 h and cell supernatants were harvested for HBsAg, HBeAg and HBV DNA quantiifcation. HBV-Alu-PCR was used to amplify the integrated HBV X/C/S genes and their lfanking sequences in human genomic DNA of the HCC cell lines. The integrated HBV fragments and integration sites were determined by sequencing the PCR products. The upstream and downstream human genes adjacent to the integration sites were identiifed by bioinformatics analysis.Results MHCC97H, MHCC97L and MHCCLM3 were negative for HBsAg andHBeAg, but positive for HBV DNA with a titer ranged between 2.00× 105to 4.00× 105IU/ml. HepG2.2.15, HepAD38, DE19 and PLC/PRF/5 were positive for HBsAg with a HBV DNA titer higher than 5.00× 105 IU/ml. HepG2.2.15 and HepAD38 were positive for HBeAg. HepG2 and HuH-7 were negative for HBsAg,HBeAg and HBV DNA. One or more integrated HBV sub-genome fragments were detected in cell lines except for HepG2 and HuH-7. Among them, up to five integration sites were detected in HepG2.2.15 (HBx/HBc), two in HepAD38 and PLC/PRF/5 (HBx), one in DE19 (HBc). Interestingly, three HCC cells (MHCC97H, MHCC97L and MHCCLM3), which had the same clone origin from MHCC97, contained exactly the same HBC fragment integrated into the same site. Furthermore, HBV integration mainly targeted genes belonging to distinct pathways: cancer related genes, calcium signaling related genes, ribosomal protein encoding genes. Conclusions HBV integration occurs in HCC cell lines from patients with HBV infection and HCC cell lines stably transfected with HBV virus genome. HBx and HBc are the main HBV fragments integrated into the genomic DNA of HCC cells. HCC cells from the same clone origin contain the same HBV integration site, which may be used to trace the clone origin of primary or metastatic HCC cells.