【摘要】 背景与目的:有研究发现P53氨基端15肽与穿膜肽(antennapedia,Ant)的融合肽能迅速穿透细胞膜,直接、快速引起乳腺癌、胰腺癌细胞坏死而非凋亡,但对正常细胞几乎没有毒性。本研究构建缺陷型腺病毒载体表达NT4p53(N15)Ant融合基因,研究其体外抑瘤作用。方法:应用Ad-EasyTM系统,在大肠杆菌同源重组,构建NT4p53(N15)Ant腺病毒表达载体,在HEK-293细胞内成功包装并鉴定后,感染肝癌细胞株HepG2,用倒置相差显微镜、透射电镜观察细胞形态学变化、四甲基偶氮唑蓝(MTT)比色实验及培养基中的乳酸脱氢酶(lactatedehydrogenase,LDH)测定研究其体外抑瘤作用。结果:Ad-NT4p53(N15)Ant对肝癌细胞株HepG2有明显抑制作用,48、72、96h细胞存活率分别为36.67%、20.47%、17.82%,与空病毒处理组比较差异有统计学意义(P<0.05);电镜观察到感染的HepG2细胞胞膜、核膜破坏,胞浆内出现囊泡状物质,染色质聚集。Ad-NT4p53(N15)Ant处理HepG2细胞24、48、72、96h时培养液中LDH释放分别为94、236、267、313U/L,较空病毒处理组明显增加,其差异有统计学意义(P<0.05)。结论:构建的腺病毒载体Ad-NT4p53(N15)Ant感染HepG2细胞后能抑制细胞增殖。更多还原

【Abstract】 BACKGROUND & OBJECTIVE: Peptide p53(N15)Ant from the amino-terminal of p53 fused with cell penetrating peptide antennapedia (Ant) can induce rapid cell death resembling necrosis in breast cancer and pancreatic cancer, whereas it is low cytotoxic to normal cells. This study was to construct an recombinant adenovirus vector containing NT4p53(N15)Ant gene and investigate its antitumor effects. METHODS: Adenovirus packaging system was used to construct Ad-NT4p53(N15)Ant by recombinant technique in vitro. The recombinant adenovirus was packaged in HEK-293 cells and identified by reverse transcription-polymerase chain reaction (RT-PCR). Human hepatocellular carcinoma (HCC) cell line HepG2 were infected with Ad-NT4p53(N15)Ant or parallel control recombinant adenovirus Ad-GFP. After infection of Ad-NT4p53(N15)Ant, cell morphology was observed under inverted phase contrast microscope and transmission electron microscope. The effect of Ad-NT4p53(N15)Ant on HepG2 cells was detected by MTT assay and lactate dehydrogenase (LDH) release assay. RESULTS: NT4p53(N15)Ant significantly inhibited the proliferation of HepG2 cells. The survival rates of HepG2 cells in NT4p53(N15)Ant group were 36.67% at 48 h, 20.47% at 72 h and 17.82% at 96 h after infection, which were significantly lower than those in Ad-GFP group (P<0.05). Distinct cell membrane disruption, pore-like structures in cytoplasm, and chromatin condensation were observed in Ad-NT4p53(N15)Ant group. LDH release in supernatant of HepG2 cells were 94 U/L at 24 h, 236.3 U/L at 48 h, 267 U/L at 72 h and 313 U/L at 96 h in Ad-NT4p53(N15)Ant group, which were significantly higher than those in Ad-GFP group (P<0.05). CONCLUSION: NT4p53(N15)Ant could significantly inhibit the proliferation of HepG2 cells through necrosis pathway.更多还原