中华检验医学杂志
中華檢驗醫學雜誌
중화검험의학잡지
CHINESE JOURNAL OF LABORATORY MEDICINE
2011年
3期
265-270
,共6页
管世鹤%杨凯%卢银平%杨东亮
管世鶴%楊凱%盧銀平%楊東亮
관세학%양개%로은평%양동량
细胞系,肿瘤%肝炎病毒,乙型%病毒复制%拉米夫定%干扰素α
細胞繫,腫瘤%肝炎病毒,乙型%病毒複製%拉米伕定%榦擾素α
세포계,종류%간염병독,을형%병독복제%랍미부정%간우소α
Cell line,tumor%Hepatitis B virus%Virus replication%Lamivudine%Interferonalpha
目的 研究LAM单独和与IFN-序贯处理对人肝胚瘤细胞株HepG2.2.15细胞的HBV复制的影响,探讨LAM与IFN-α在体外抑制HBV复制的差异.方法 以未处理的HepG2.2.15细胞作为对照组;以1 000 IU/ml浓度IFN-α连续处理HepG2.2.15细胞10 d为单独IFN-α处理组;以0.2、1、5、20、100 μmol/L的LAM处理HepG2.2.15细胞为单独LAM处理组;序贯处理组则以浓度为0.04、0.2、5、25、125、200μmoL/L的LAM连续处理HepG2.2.15细胞7 d,再补充1 000 IU/ml IFN-α与LAM联合处理3 d,然后停止LAM处理,再单独以1 000 IU/ml IFN-α连续处理细胞10 d;分别用ELISA法、点杂交和Southern杂交分析不同处理时期、不同处理组HepG2.2.15细胞分泌的HBV抗原、细胞外HBV DNA、细胞内HBV复制中间体DNA以判断HepG2.2.15细胞内HBV复制情况.结果 LAM连续处理至10 d时,单独LAM处理组HepG2.2.15细胞分泌的HBsAg分别是1.77±0.22、1.65±0.25、1.95±0.19、1.34±0.11、1.07±0.05,分泌的HBeAg是1.41±0.13、1.37±0.09、1.63±0.07、1.26±0.12、1.05 ±0.09,对照组分泌的HBsAg和HBeAg分别是3.34±0.15和3.33±0.05,单独LAM处理组与对照组相比分泌的HBsAg和HBeAg下降,差异有统计学意义(HBsAg的t值为10.21、10.04、9.94、18.62、24.86,HBeAg的t值为23.87、32.97、34.22、27.57和38.35,P均＜0.05).点杂交、Southern杂交分析显示LAM连续处理10 d后,单独LAM处理组的细胞外HBV DNA和细胞内HBV复制中间体DNA不能被检测到.停止LAM处理并代之以1 000 IU/ml IFN-α序贯处理10 d,序贯处理组均有细胞内HBV复制中间体DNA的出现,且细胞外HBV抗原和HBV DNA恢复表达;即HBV颗粒在HepG2.2.15细胞内又重新恢复复制状态并分泌至细胞外.结论 LAM与IFN-α在体外细胞模型中具有不同的抗病毒效应,导致HepG2.2.15细胞内HBV复制的差异性.
目的 研究LAM單獨和與IFN-序貫處理對人肝胚瘤細胞株HepG2.2.15細胞的HBV複製的影響,探討LAM與IFN-α在體外抑製HBV複製的差異.方法 以未處理的HepG2.2.15細胞作為對照組;以1 000 IU/ml濃度IFN-α連續處理HepG2.2.15細胞10 d為單獨IFN-α處理組;以0.2、1、5、20、100 μmol/L的LAM處理HepG2.2.15細胞為單獨LAM處理組;序貫處理組則以濃度為0.04、0.2、5、25、125、200μmoL/L的LAM連續處理HepG2.2.15細胞7 d,再補充1 000 IU/ml IFN-α與LAM聯閤處理3 d,然後停止LAM處理,再單獨以1 000 IU/ml IFN-α連續處理細胞10 d;分彆用ELISA法、點雜交和Southern雜交分析不同處理時期、不同處理組HepG2.2.15細胞分泌的HBV抗原、細胞外HBV DNA、細胞內HBV複製中間體DNA以判斷HepG2.2.15細胞內HBV複製情況.結果 LAM連續處理至10 d時,單獨LAM處理組HepG2.2.15細胞分泌的HBsAg分彆是1.77±0.22、1.65±0.25、1.95±0.19、1.34±0.11、1.07±0.05,分泌的HBeAg是1.41±0.13、1.37±0.09、1.63±0.07、1.26±0.12、1.05 ±0.09,對照組分泌的HBsAg和HBeAg分彆是3.34±0.15和3.33±0.05,單獨LAM處理組與對照組相比分泌的HBsAg和HBeAg下降,差異有統計學意義(HBsAg的t值為10.21、10.04、9.94、18.62、24.86,HBeAg的t值為23.87、32.97、34.22、27.57和38.35,P均＜0.05).點雜交、Southern雜交分析顯示LAM連續處理10 d後,單獨LAM處理組的細胞外HBV DNA和細胞內HBV複製中間體DNA不能被檢測到.停止LAM處理併代之以1 000 IU/ml IFN-α序貫處理10 d,序貫處理組均有細胞內HBV複製中間體DNA的齣現,且細胞外HBV抗原和HBV DNA恢複錶達;即HBV顆粒在HepG2.2.15細胞內又重新恢複複製狀態併分泌至細胞外.結論 LAM與IFN-α在體外細胞模型中具有不同的抗病毒效應,導緻HepG2.2.15細胞內HBV複製的差異性.
목적 연구LAM단독화여IFN-서관처리대인간배류세포주HepG2.2.15세포적HBV복제적영향,탐토LAM여IFN-α재체외억제HBV복제적차이.방법 이미처리적HepG2.2.15세포작위대조조;이1 000 IU/ml농도IFN-α련속처리HepG2.2.15세포10 d위단독IFN-α처리조;이0.2、1、5、20、100 μmol/L적LAM처리HepG2.2.15세포위단독LAM처리조;서관처리조칙이농도위0.04、0.2、5、25、125、200μmoL/L적LAM련속처리HepG2.2.15세포7 d,재보충1 000 IU/ml IFN-α여LAM연합처리3 d,연후정지LAM처리,재단독이1 000 IU/ml IFN-α련속처리세포10 d;분별용ELISA법、점잡교화Southern잡교분석불동처리시기、불동처리조HepG2.2.15세포분비적HBV항원、세포외HBV DNA、세포내HBV복제중간체DNA이판단HepG2.2.15세포내HBV복제정황.결과 LAM련속처리지10 d시,단독LAM처리조HepG2.2.15세포분비적HBsAg분별시1.77±0.22、1.65±0.25、1.95±0.19、1.34±0.11、1.07±0.05,분비적HBeAg시1.41±0.13、1.37±0.09、1.63±0.07、1.26±0.12、1.05 ±0.09,대조조분비적HBsAg화HBeAg분별시3.34±0.15화3.33±0.05,단독LAM처리조여대조조상비분비적HBsAg화HBeAg하강,차이유통계학의의(HBsAg적t치위10.21、10.04、9.94、18.62、24.86,HBeAg적t치위23.87、32.97、34.22、27.57화38.35,P균＜0.05).점잡교、Southern잡교분석현시LAM련속처리10 d후,단독LAM처리조적세포외HBV DNA화세포내HBV복제중간체DNA불능피검측도.정지LAM처리병대지이1 000 IU/ml IFN-α서관처리10 d,서관처리조균유세포내HBV복제중간체DNA적출현,차세포외HBV항원화HBV DNA회복표체;즉HBV과립재HepG2.2.15세포내우중신회복복제상태병분비지세포외.결론 LAM여IFN-α재체외세포모형중구유불동적항병독효응,도치HepG2.2.15세포내HBV복제적차이성.
Objective To investigate the characteristics of HBV replication in HepG2.2. 15 cells treated with LAM alone or sequentially treated with LAM and IFN-α, and to further explore the different suppressive effect on HBV replication by LAM and IFN-α in vitro. Methods Untreated HepG2. 2. 15 cells were used as control group, HepG2. 2. 15 cells treated with 1 000 IU/ml of IFN-α alone for 10 d were served as IFN-α group. The HepG2.2. 15 cells treated with 0.2,1,5,20,100 μmol/L of LAM were used as LAM groups. HepG2. 2. 15 cells treated with 0. 04,0. 2,5,25,125,200 μmol/L of LAM for 7 d, then combined with 1 000 IU/ml of IFN-α for another 3 d. Afterwards, the cells were treated with 1 000 IU/ml of IFN-α only for another 10 d. These cells were served as LAM/IFN-α sequential treatment group. The ELISA was used for analyzing the secreted HBV antigens, while the Dot blot, Southern blot were applied for analyzing the extracellular HBV DNA and intracellular HBV replicative intermediate DNA in HepG2. 2. 15 cells of different treatment groups. Results The secreted HBsAg in the LAM group were 1. 77 ± 0. 22, 1.65 ±0.25, 1.95 ±0. 19, 1.34 ±0. 11, 1.07 ±0.05, respectively, and the secretion of HBeAg were 1.41 ±0. 13, 1.37 ± 0. 09, 1.63 ± 0. 07, 1.26 ± 0. 12, 1.05 ± 0. 09. The secreted HBsAg and HBeAg in control group were 3. 34 ± 0. 15 and 3.33 ± 0. 05. Statistical analysis showed that HBsAg and HBeAg secretion in the LAM group were significantly reduced by treatment with LAM. The t values of HBsAg were 10. 21,10.04, 9.94, 18.62, 24.86, and the t values of HBeAg were 23.87, 32.97, 34.22, 27.57, 38.35,respectively, all P values were ＜ 0.05. Dot blot, Northern blot hybridization analysis indicated that the extracellular HBV DNA and intracellular HBV replicative intermediate DNA could not be detected in the LAM group after cells treated by LAM for 10 days. When LAM was replaced with treatment of 1 000 IU/ml of IFN-α alone, it could not suppress the HBV replication effectively. Moreover, the intracellular HBV replicative intermediate DNA still existed in almost all groups, accompanied with the recovered expression of HBV antigens as well as extracellular HBV DNA, which suggested that the HBV particles restored replication again and secreted extracellular in HepG2. 2. 15 cells, although the sequential treatment lasted for 10 days.Conclusion The effect of viral suppression by LAM and IFN-α in vitro were different, which attributed to the different HBV replicative characteristcs in HepG2. 2. 15 cells.
