负载人microRNA-149的复制缺陷型腺病毒对人肝癌细胞株增殖的影响

Replication-deficient Adenovirus Carrying hsa-micro RNA-149 Inhibits Proliferation of Human Hepatocellular Carcinoma Cell Lines

目的包装负载人micro RNA-149的复制缺陷型腺病毒(Ad-mi R149),并检测其对人肝癌细胞株增殖的影响。方法全基因合成micro RNA-149前体核苷酸序列,插入腺病毒穿梭质粒的多克隆位点,随后与包装质粒共转染人胚肾293细胞,包装负载micro RNA-149的复制缺陷型腺病毒;采用MMT方法检测Ad-mi R149对人肝癌细胞株增殖的影响。结果成功包装负载人micro RNA-149的复制缺陷型腺病毒,采用倍比稀释法检测病毒滴度为5×109 pfu/ml。采用电子显微镜技术,观察到HEK293细胞中大量包装病毒颗粒。采用PCR扩增,Ad-mi R149可获得腺病毒及micro RNA-149特异片段,而对照AdLac Z只能扩增出腺病毒特异片段;采用MTT方法发现,Ad-mi R149可显著抑制肝癌细胞株增殖。结论成功包装负载人micro RNA-149的复制缺陷型腺病毒并初步证实其对肝癌细胞增殖的抑制作用。

Objective To construct a replication-deficient recombinant adenovirus carrying micro RNA-149(Ad-mi R149) and to investigate its effect on the proliferation of human hepatocellular carcinoma cell lines.Methods The pre-micro RNA-149 sequence was synthesized and inserted into multi-clone site in the adenoviral shuttle plasmid p DC315 in sense direction,and then the recombinant plasmid was co-transfected into human embryo kidney 293(HEK293) cells together with packaging plasmid p BHGlox(delta E1,3).The recombinant adenovirus carrying hsa-micro RNA-149 was selected and amplified.The effect of Ad-mi R149 on the proliferation of human hepatocellular carcinoma cell lines was observed by MTT method.Results The replication-deficient recombinant adenovirus carrying micro RNA-149 was packaged successfully.After virus amplification,the titer of Ad-mi R149 was achieved to 5×109 pfu/ml.Virus replication was observed through the transmission electron microscope.Both adenovirus and micro RNA-149 special fragment could be amplified from Ad-mi R149 by PCR amplification.Compared with the negative control,only adenovirus special fragment could be amplified from Ad-Lac Z.Ad-mi R149 suppressed the proliferation of human hepatocellular carcinoma cell lines significantly detected by MTT.Conclusion The recombinant adenovirus carrying hsa-micro RNA-149 is constructed successfully and its inhibiting effect on human hepatocellular carcinoma cell lines is confirmed preliminarily.