Targeted adenovirus containing r-caspase-3 gene induces apoptosis of hepatocellular carcinoma cells in vitro

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Author:
WANG Wei(Department of Hepatobiliary Surgery, The First Affiliated Hospital, Guangzhou Medical College, Guangzhou 510120, China)
SHEN Hao-yuan(Department of Hepatobiliary Surgery, The First Affiliated Hospital, Guangzhou Medical College, Guangzhou 510120, China)
QIU Chang-ru(Department of Hepatobiliary Surgery, The First Affiliated Hospital, Guangzhou Medical College, Guangzhou 510120, China)
LIN Hao-ming()
CHENG Wei(Department of Hepatobiliary Surgery, The First Affiliated Hospital, Guangzhou Medical College, Guangzhou 510120, China)
Journal Title:
CHINESE JOURNAL OF BIOMEDICAL ENGINEERING
Issue:
Volume 16, Issue 04, 2010
DOI:
10.3760/cma.j.issn.1674-1927.2010.04.005
Key Word:
Adenovirus;Caspase 3;Liver heoplasms;Apoptosis;Target

Abstract: Objective To investigate the effect and specificity of adenovirus containing r-caspase-3 gene on apoptosis of hepatocellular carcinoma cells. Methods The vector α-fetoprotein enhancer-albumin promotor (pAdTrack-EAFP-PALB) was constructed and the r-caspase-3 gene was subcloned into the vector. The linearized shuttle plasmid was homogenously recombined with AdEasy-1 in BJ5183 cells. The candidate clone was analyzed by restriction endonuclease digestion and sequencing, and then pAdeasy-EAFP-PALB/r-caspase-3 vector was digested with Pac Ⅰ and transfected into AD293 cells for packaging and amplifying.Infection titer and rate of recombinant virus was monitored by green fluorescent protein (GFP) expression.The expression of r-caspase-3 was detected by RT-PCR and Western-blot. The apoptosis of HepG2, 7721, L-02 and MDA- MB- 231 cells was detected by FCM method. Results The sequence of shuttle vector pAdTrack-EAFP-PALB/r-caspase-3 was correct after digestion by restriction endonuclease. Vector pAdeasy-EAFP-PALB/r-caspase-3 was identificated by Pac Ⅰ restriction digestion and PCR. The expression of GFP was observed in the transfected AD293 cells. The expression of r-caspase-3 gene was detected in the infected HepG2 cells by RT-PCR and Western-blot. The cells were infected with recombinant r-caspase-3 after 24 hours, and their apoptotic index were as follows: HepG cells, 48.2%; 7721 cells, 17.7%; L-02 cells, 7.3%;M DA- MB- 231 cells, 0%. Conclusion The recombinant of hepatocellular carcinoma - targeting adenovirus containing r- caspase- 3 gene is constructed successfully and can induce the targeted apoptosis of hepatocellular carcinoma cells, which provides the evidences for future research in hepatocellular carcinoma.

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