Basic Research
Copyright ©2007 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Aug 21, 2007; 13(31): 4214-4218
Published online Aug 21, 2007. doi: 10.3748/wjg.v13.i31.4214
Construction of targeted plasmid vector pcDNA3.1-Egr.1p-p16 and its expression in pancreatic cancer JF305 cells induced by radiation in vitro
Hong-Bing Ma, Xi-Jing Wang, Zheng-Li Di, Hui Xia, Zheng Li, Jie Liu, Jie Ma, Hua-Fen Kang, Cong-Mei Wu, Ming-Hua Bai
Hong-Bing Ma, Xi-Jing Wang, Hua-Fen Kang, Ming-Hua Bai, Department of Oncology, The Second Affiliated Hospital, Medical college of Xi’an Jiaotong University , Xi’an 710004, Shaanxi Province, China
Zheng-Li Di, Medical Department, Xi’an Central Hospital , Xi’an 710003, Shaanxi Province, China
Jie Ma, Hui Xia, Zheng Li, Jie Liu, National Laboratory of Molecular Oncology, Cancer Institute(Hospital), Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China
Cong-Mei Wu, Research Cencer of Reproductive Medicine, Medical college of Shantou University, Shantou 515031, Guangdong Province, China
Author contributions: All authors contributed equally to the work.
Supported by the Technology Project Entry Foundation of Shanxi Province, No. 2002K10-G3
Correspondence to: Dr. Hong-Bing Ma, Department of Oncology, Second Hospital, Medical college of Xi’an Jiaotong University, Xi’an 710004, Shaanxi Province, China. mahongbingxa@126.com
Telephone: +86-29-87679789
Received: February 21, 2007
Revised: March 15, 2007
Accepted: March 21, 2007
Published online: August 21, 2007
Abstract

AIM: To construct pcDNA3.1-Egr.1p-p16 recombinant plasmid and investigate the expression of p16 in pancreatic cancer JF305 cells induced by radiation and the feasibility of gene radiotherapy for pancreatic carcinoma.

METHODS: Human p16 cDNA was ligated to the downstream of Egr-1 promotor to construct pcDNA3.1-Egr.1p-p16 plasmid by restriction enzyme digested. The recombined plasmids were transfected into pancreatic cancer JF305 cells with lipofectamine. p16 mRNA level was detected by RT-PCR. The expression of p16 after different doses of X-ray radiation was detected by Western blot technique. Cell survival was assessed by clonogenic assays and cell viability was analysed by trypen blue exclusion. Flow cytometry was performed to study the apoptosis of JF305 cells.

RESULTS: Restriction enzyme digestion showed the correctly constructed pcDNA3.1-Egr.1p-p16. The p16 expression in cells transfected with pcDNA3.1-Egr.1p-p16 induced by different doses of radiation was higher than that in the control group (P < 0.05). Eight hours after 2 Gy X-ray radiation, the expression reached its peak (87.00 ng/L), and was significantly higher than that in the control group (P < 0.0.5). Clonogenic analysis and trypan blue extraction test showed that the pcDNA3.1-Egr.1p-p16 transfer enhanced radiation-induced cell killing in p16-null JF305 cell lines. The induction of apoptosis was lower in combined transfection and irradiation group than that in irradiation alone.

CONCLUSION: X-ray can induce the recombinant plasmid pcDNA3.1-Egr.1p-p16 expression in JF305 cells. The detection of dose and time provides an experimental basis for in vivo study in future.

Keywords: pcDNA3.1-Egr.1p-p16; Reconstruction plasmid; X-ray radiation