CAJ | 학술논문

目的：研究靶向HBV X（ HBx）基因的长发夹RNA （ lhRNA）表达载体对HBV基因复制和表达的影响。方法以HBx为靶点，合成四条小干扰RNA（ siRNA）寡核苷酸并构建lhRNA表达载体。 siRNA寡核苷酸序列（4个转染组）或长发夹 RNA （ lhRNA ）表达载体（2个转染组）转染HepG2．2．15细胞后，通过时间分辨免疫荧光法、荧光定量PCR及反转录PCR分别检测细胞培养上清液中的乙型肝炎病毒表面抗原（ HBsAg）、HBV DNA及细胞内的HBx mRNA水平。以转染阴性序列或空载体为阴性对照组。采用独立样本t检验评估siRNA对HBV基因复制和表达的抑制效果。结果与阴性对照组相比， siRNA-1及 siRNA-4组以较高浓度（60 nmol／L 或90 nmol／L ）转染入HepG2．2．15细胞后，培养上清液中的HBsAg、HBV DNA及HBx mRNA水平均显著降低（P＜0．05），其中，siRNA-1组在转染后不同时间点（24，48，72 h）细胞培养上清液中的HBsAg和HBV DNA载量均较对照组显著降低（ P＜0．05或P＜0．01）。构建成功两个lhRNA表达载体：pMD-HBxlh1和pMD-HBxlh4，其转染入细胞后，培养上清液中的 HBsAg 及 HBV DNA 水平显著低于阴性对照组（ P ＜0.05）。结论新证实一个HBx基因的有效干扰靶点即 siRNA-1。靶向HBx的lhRNA 表达载体pMD-HBxlh1和pMD-HBxlh4可有效抑制HBV基因的复制和表达。
목적：연구파향HBV X（ HBx）기인적장발협RNA （ lhRNA）표체재체대HBV기인복제화표체적영향。방법이HBx위파점，합성사조소간우RNA（ siRNA）과핵감산병구건lhRNA표체재체。 siRNA과핵감산서렬（4개전염조）혹장발협 RNA （ lhRNA ）표체재체（2개전염조）전염HepG2．2．15세포후，통과시간분변면역형광법、형광정량PCR급반전록PCR분별검측세포배양상청액중적을형간염병독표면항원（ HBsAg）、HBV DNA급세포내적HBx mRNA수평。이전염음성서렬혹공재체위음성대조조。채용독립양본t검험평고siRNA대HBV기인복제화표체적억제효과。결과여음성대조조상비， siRNA-1급 siRNA-4조이교고농도（60 nmol／L 혹90 nmol／L ）전염입HepG2．2．15세포후，배양상청액중적HBsAg、HBV DNA급HBx mRNA수평균현저강저（P＜0．05），기중，siRNA-1조재전염후불동시간점（24，48，72 h）세포배양상청액중적HBsAg화HBV DNA재량균교대조조현저강저（ P＜0．05혹P＜0．01）。구건성공량개lhRNA표체재체：pMD-HBxlh1화pMD-HBxlh4，기전염입세포후，배양상청액중적 HBsAg 급 HBV DNA 수평현저저우음성대조조（ P ＜0.05）。결론신증실일개HBx기인적유효간우파점즉 siRNA-1。파향HBx적lhRNA 표체재체pMD-HBxlh1화pMD-HBxlh4가유효억제HBV기인적복제화표체。
Objective To investigate the effect of long hairpin RNA ( lhRNA) expression vector targeting HBV X gene ( HBx) on replication of hepatitis B virus ( HBV) and gene expression.Methods Four kinds of small interference RNAs ( siRNAs) were synthesized and lhRNA expression vectors targeting HBx were constructed.Four siRNA oligonucleotides and two lhRNA expression vectors were transfected into HepG2.2.15 cells.HBsAg, HBV DNA in culture supernatants and HBx mRNA in HepG2.2.15 cells were detected by time-resolved immunofluorometric assay, real-time quantitative PCR, and reverse transcription PCR, respectively.Negative sequence group or empty vector group was taken as the control.Independent-samples t test was performed to evaluate the inhibition effect on replication of HBV and gene expression. Results Compared with the negative control, HBsAg, HBV DNA level in culture supernatants and HBx mRNA in HepG2.2.15 cells were significantly decreased after siRNA-1 and siRNA-4 transfected at high concentrations (60 nmol/L or 90 nmol/L) (P<0.05), especially the HBsAg and HBV DNA levels in the siRNA-1 transfection group, which were significantly decreased at 24, 48 and 72 h after transfection ( P<0.05 or P <0.01 ) . Two lhRNA expression vectors ( pMD-HBxlh1 and pMD-HBxlh4 ) were successfully constructed and transfected into HepG2.2.15 cells, HBsAg and HBV DNA level in transfected cells was significantly lower than those in negative control (P<0.05).Conclusion The novel siRNA-1 is confirmed to target HBx gene and lhRNA expression vector targeting HBx can effectively inhibit the replication of HBV and expression of HBV gene.