CAJ | 학술논문

目的评估长的反义RNA干扰片段在培养细胞株中对HBV复制的抑制效应.方法将HBV基因组S区的全部核苷酸序列插入至pTARGETTM载体中,并将重组载体转染入HepG2.2.15细胞中.用酶联免疫吸附法检测HBsAg与HBeAg水平,用荧光定量PCR法检测HBVDNA水平.对数据采用多个独立样本Kruskal-Wallis检验与两两比较的Mann-Whitney U检验.结果经过处理后,HepG2.2.15细胞上清液中HBsAg表达量(A值)在HBS2组(携带长片段反义RNA)为0.621±0.027,在HBS4组(携带正义RNA)为3.399±0.018,对照组为2.232±0.187;HBeAg表达量(A值)在HBS2组、HBS4组和对照组分别为0.749±0.019、1.548±0.025和1.570±0.044; HBV DNA水平(×104拷贝/ml)在HBS2组、HBS4组、对照组分别为1.597±0.082、3.381±0.297和3.610±0.063.与对照组相比,HBS2组HBsAg、HBeAg和HBV DNA表达量均降低,统计量Z值均为-2.309,P值均＜0.05; HBS4组HBsAg表达量增高(Z=-2.309,P＜0.05),而HBeAg和HBV DNA表达量无明显差异,统计量Z值分别为-0.866、-1.155,P值均＞0.05.结论长片段反义RNA能抑制HBV基因的表达和病毒复制.
목적 평고장적반의RNA간우편단재배양세포주중대HBV복제적억제효응.방법장HBV기인조S구적전부핵감산서렬삽입지pTARGETTM재체중,병장중조재체전염입HepG2.2.15세포중.용매련면역흡부법검측HBsAg여HBeAg수평,용형광정량PCR법검측HBVDNA수평.대수거채용다개독립양본Kruskal-Wallis검험여량량비교적Mann-Whitney U검험.결과 경과처리후,HepG2.2.15세포상청액중HBsAg표체량(A치)재HBS2조(휴대장편단반의RNA)위0.621±0.027,재HBS4조(휴대정의RNA)위3.399±0.018,대조조위2.232±0.187;HBeAg표체량(A치)재HBS2조、HBS4조화대조조분별위0.749±0.019、1.548±0.025화1.570±0.044; HBV DNA수평(×104고패/ml)재HBS2조、HBS4조、대조조분별위1.597±0.082、3.381±0.297화3.610±0.063.여대조조상비,HBS2조HBsAg、HBeAg화HBV DNA표체량균강저,통계량Z치균위-2.309,P치균＜0.05; HBS4조HBsAg표체량증고(Z=-2.309,P＜0.05),이HBeAg화HBV DNA표체량무명현차이,통계량Z치분별위-0.866、-1.155,P치균＞0.05.결론 장편단반의RNA능억제HBV기인적표체화병독복제.
Objective To evaluate the inhibitory effects of long antisense RNA on HBV replication in HepG2.2.15 cells. Methods The coding region of HBV S gene was cloned into pTARGET vector in sense and antisense orientations and the recombinant plasmids were transfected into HepG2.2.15 cells which were divided into HBS2 (antisense RNA) group, HBS4 (sense RNA) group and control group. HBsAg and HBeAg in the culture supernant were detected by ELISA. The HBV DNA in the supernant was quantified by real-time PCR. Results After treatment, the levels of HBsAg in HepG2.2.15 cell supernatants of three groups were 0.621 ± 0.027, 3.399 ± 0.018 and 2.232 ± 0.187 respectively; the levels of HBeAg were 0.749 ± 0.019,1.548 ± 0.025 and 1.570 ± 0.044 respectively and the levels of HBV DNA were 1.597 ± 0.082, 3.381 ± 0.297 and 3.610 ± 0.063 respectively. The expressions of HBsAg and HBeAg and the HBV DNA level in HBS2 group were remarkably reduced as compared to the control (Z = -2.309, P ＜ 0.05); whereas the sense plasmid transfection (HBS4) did not affect HBeAg (Z= -0.866) and HBV DNA (Z = -1.155) levels in the culture supernant but slightly increased the HBsAg level (Z = -2.309). Conclusion Antisense RNA might be a useful tool to repress HBV replication.