中华肝脏病杂志
中華肝髒病雜誌
중화간장병잡지
CHINESE JOURNAL OF HEPATOLOGY
2014年
3期
213-218
,共6页
倪艳%蒋凤%徐华%曾腾%雷宇%陈压西%周智%任红
倪豔%蔣鳳%徐華%曾騰%雷宇%陳壓西%週智%任紅
예염%장봉%서화%증등%뢰우%진압서%주지%임홍
肝炎病毒,乙型%细胞因子%调控表达%抑制作用
肝炎病毒,乙型%細胞因子%調控錶達%抑製作用
간염병독,을형%세포인자%조공표체%억제작용
Hepatitis B virus%Cytokines%Regularly expression%Inhibition
目的 研究四环素调控表达的肿瘤坏死因子α (TNFα)抑制HBV复制的作用. 方法 以pcDNA4TM/四环素操纵子序列(TO)载体为模板,用PCR法扩增含四环素调控表达序列片段TO,插入pUC118载体中测序,选择测序正确的序列取代pVITRO3载体中相应的片段,构建成pVITRO3-TO.用相同的方法以pcDNA6/四环素阻遏蛋白(TR)(C)载体为模板扩增TR基因,经测序证实后插入pVITRO3-TO载体的一个多克隆位点,构建成pVITRO3-TO-TR.在另一个多克隆位点插入TNF α 基因,构建pVITRO3-TO-TR-TNF α .将重组质粒瞬时转染HepG2细胞,研究四环素调控表达TNF α 的有效性.以HepG2.2.15细胞为HBV复制模型,转染pVITRO3-TO-TR-TNF α 后,经潮霉素抗性筛选及有限稀释,选择高效表达TNF α 的细胞建立细胞系,观察经四环素调控表达后,TNF α 抑制HBV复制的作用.多组间数据比较使用方差分析. 结果 经过改建的双表达载体能够有效表达TNF α,并可以用不同剂量的四环素进行调控,四环素的最佳工作浓度为1.0μ g/ml.诱导1、3、5d后,转染pVITRO3-TOTR-TNF α 组上清液中的HBsAg抑制率分别为14.8％、11.5％、28.44％,转染pVITRO3-TOTR组分别为1.2％、1.1％、0,诱导5d后的HBsAg抑制率差异有统计学意义(F=13.65,P＜0.05);转染pVITRO3-TOTR-TNF α 组的HBeAg的抑制率分别为50％、26.67％、47.85％,转染pVITRO3-TOTR组分别为0.3％、1.3％、0,诱导1、3、5d时的HBeAg抑制率差异均有统计学意义(F值分别为27.31、14.06和43.76,P值均＜0.05).荧光定量PCR结果显示,诱导5d后,TNF α 对细胞内和培养上清液的HBV DNA有明显的抑制作用,抑制率分别为70.26％和79.86％,与对照组相比,差异均有统计学意义(F值分别为96.7和62.72,P＜0.05).进一步的研究结果表明,TNF α对HBV S基因的mRNA转录有明显抑制作用.结论 成功构建了四环素调控表达的TNF α 载体,瞬时和稳定转染细胞后均能有效表达.在细胞模型中对HBV DNA的抑制作用最好,其次是对HBeAg的抑制,对HBsAg的抑制作用最弱.TNF α对病毒转录水平的抑制可能是其发挥抗HBV作用的一个重要环节.
目的 研究四環素調控錶達的腫瘤壞死因子α (TNFα)抑製HBV複製的作用. 方法 以pcDNA4TM/四環素操縱子序列(TO)載體為模闆,用PCR法擴增含四環素調控錶達序列片段TO,插入pUC118載體中測序,選擇測序正確的序列取代pVITRO3載體中相應的片段,構建成pVITRO3-TO.用相同的方法以pcDNA6/四環素阻遏蛋白(TR)(C)載體為模闆擴增TR基因,經測序證實後插入pVITRO3-TO載體的一箇多剋隆位點,構建成pVITRO3-TO-TR.在另一箇多剋隆位點插入TNF α 基因,構建pVITRO3-TO-TR-TNF α .將重組質粒瞬時轉染HepG2細胞,研究四環素調控錶達TNF α 的有效性.以HepG2.2.15細胞為HBV複製模型,轉染pVITRO3-TO-TR-TNF α 後,經潮黴素抗性篩選及有限稀釋,選擇高效錶達TNF α 的細胞建立細胞繫,觀察經四環素調控錶達後,TNF α 抑製HBV複製的作用.多組間數據比較使用方差分析. 結果 經過改建的雙錶達載體能夠有效錶達TNF α,併可以用不同劑量的四環素進行調控,四環素的最佳工作濃度為1.0μ g/ml.誘導1、3、5d後,轉染pVITRO3-TOTR-TNF α 組上清液中的HBsAg抑製率分彆為14.8％、11.5％、28.44％,轉染pVITRO3-TOTR組分彆為1.2％、1.1％、0,誘導5d後的HBsAg抑製率差異有統計學意義(F=13.65,P＜0.05);轉染pVITRO3-TOTR-TNF α 組的HBeAg的抑製率分彆為50％、26.67％、47.85％,轉染pVITRO3-TOTR組分彆為0.3％、1.3％、0,誘導1、3、5d時的HBeAg抑製率差異均有統計學意義(F值分彆為27.31、14.06和43.76,P值均＜0.05).熒光定量PCR結果顯示,誘導5d後,TNF α 對細胞內和培養上清液的HBV DNA有明顯的抑製作用,抑製率分彆為70.26％和79.86％,與對照組相比,差異均有統計學意義(F值分彆為96.7和62.72,P＜0.05).進一步的研究結果錶明,TNF α對HBV S基因的mRNA轉錄有明顯抑製作用.結論 成功構建瞭四環素調控錶達的TNF α 載體,瞬時和穩定轉染細胞後均能有效錶達.在細胞模型中對HBV DNA的抑製作用最好,其次是對HBeAg的抑製,對HBsAg的抑製作用最弱.TNF α對病毒轉錄水平的抑製可能是其髮揮抗HBV作用的一箇重要環節.
목적 연구사배소조공표체적종류배사인자α (TNFα)억제HBV복제적작용. 방법 이pcDNA4TM/사배소조종자서렬(TO)재체위모판,용PCR법확증함사배소조공표체서렬편단TO,삽입pUC118재체중측서,선택측서정학적서렬취대pVITRO3재체중상응적편단,구건성pVITRO3-TO.용상동적방법이pcDNA6/사배소조알단백(TR)(C)재체위모판확증TR기인,경측서증실후삽입pVITRO3-TO재체적일개다극륭위점,구건성pVITRO3-TO-TR.재령일개다극륭위점삽입TNF α 기인,구건pVITRO3-TO-TR-TNF α .장중조질립순시전염HepG2세포,연구사배소조공표체TNF α 적유효성.이HepG2.2.15세포위HBV복제모형,전염pVITRO3-TO-TR-TNF α 후,경조매소항성사선급유한희석,선택고효표체TNF α 적세포건립세포계,관찰경사배소조공표체후,TNF α 억제HBV복제적작용.다조간수거비교사용방차분석. 결과 경과개건적쌍표체재체능구유효표체TNF α,병가이용불동제량적사배소진행조공,사배소적최가공작농도위1.0μ g/ml.유도1、3、5d후,전염pVITRO3-TOTR-TNF α 조상청액중적HBsAg억제솔분별위14.8％、11.5％、28.44％,전염pVITRO3-TOTR조분별위1.2％、1.1％、0,유도5d후적HBsAg억제솔차이유통계학의의(F=13.65,P＜0.05);전염pVITRO3-TOTR-TNF α 조적HBeAg적억제솔분별위50％、26.67％、47.85％,전염pVITRO3-TOTR조분별위0.3％、1.3％、0,유도1、3、5d시적HBeAg억제솔차이균유통계학의의(F치분별위27.31、14.06화43.76,P치균＜0.05).형광정량PCR결과현시,유도5d후,TNF α 대세포내화배양상청액적HBV DNA유명현적억제작용,억제솔분별위70.26％화79.86％,여대조조상비,차이균유통계학의의(F치분별위96.7화62.72,P＜0.05).진일보적연구결과표명,TNF α대HBV S기인적mRNA전록유명현억제작용.결론 성공구건료사배소조공표체적TNF α 재체,순시화은정전염세포후균능유효표체.재세포모형중대HBV DNA적억제작용최호,기차시대HBeAg적억제,대HBsAg적억제작용최약.TNF α대병독전록수평적억제가능시기발휘항HBV작용적일개중요배절.
Objective To study the role of tumor necrosis factor-alpha (TNFα) in the anti-replication effects of tetracycline (Tet) on hepatitis B virus (HBV).Methods The Tet-dependent regulatory fragment (TO) was PCR amplified from the pcDNA4TM/TO vector,inserted into the pUC 118 cloning vector,and verified by sequencing.The counterpart fragment in the pVITRO3 expression vector,which contains two multiple cloning sites (MCSs),was replaced with the confirmed TO to generate a pVITRO3-TO vector.The Tet repressor (TR) gene from the PeDNA6/TR regulatory vector was incorporated into one MCS of pVITRO3-TO and the TNFα gene was subsequently incorporated into the other MCS.The resultant vector,pVITRO3-TOTR-TNFα,was transiently transfected into HepG2 cells.TNFα expression from the vector was induced by exposure to various concentrations of Tet and measured by enzyme-linked immunosorbent assay to determine the appropriate Tet concentration for experimentation.To investigate whether Tet inhibits TNFα expression as a mechanism of its anti-replication activity against HBV,the HepG2.2.15 cell line stably transfected with pVITRO3-TOTR-TNFα was used as an HBV replication model.Levels of hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) were detected by immunoassay.HBV DNA level was detected by fluorescence quantitative PCR.Results The TNFα expression from the newly constructed pVITRO3-TOTR-TNFα vector was Tet-controllable in the eukaryotic cells examined.The optimal concentration of Tet for the experimental system was 1.0 μg/ml.HBsAg and HBeAg expression was down-regulated in the HepG2.2.15 cells stably transfected with the pVITRO3-TO-TR-TNFα vector.After incubation with Tet for 1,3 and 5 days,the inhibition rate of HBsAg was 2％,1.1％ and 0,compared to 14.8％,11.5％ and 28.4％ in the non-Tet control group.The corresponding inhibition rates of HBeAg were 50.0％,26.7％ and 47.9％,compared to 0.3％,1.6％ and 0.0％,in the control group.HBV DNA levels in the cells and the cell culture supematants exposed to Tet were decreased by 70.3％ and 79.9％,respectively.TNFα inhibited production of HBsAg mRNA.Conclusion A Tet-dependent regulatory fragment double-expressing TNFα single vector system was constructed successfully,achieving controllable TNFα expression in both transiently transfected eukaryotic cells and stable cell lines.In this HBV cell model system,Tet-induced overexpression of human TNFα inhibited HBV DNA replication and reduced HBsAg and HBeAg expression.Inhibition of HBV transcription may be a key role of TNFα against HBV replication.
