Construction and Expression of The Recombinant of Hepatocellular Carcinoma-Targeting Adenovirus Containing r-Caspase-3 Gene_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

WANG Wei , CHENG Wei , QIU Changru

Department of Hepato-Biliary Surgery, The First Affiliated Hospital, Guangzhou Medical College, Guangzhou 510120, ChinaCorresponding Author： WANG Wei, E-mail: wwsir8@sohu.com;

Corresponding author：

Keywords：

Adenovirus; r-Caspase-3 gene; Target; Hepatocellular carcinoma

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Objective 　To construct the recombinant of hepatocellular carcinoma-targeting adenovirus containing r-Caspase-3 gene and provide the gene therapic strategy for hepatocellular carcinoma.
Methods 　The 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 PacⅠand transfected into AD293 cells for packaging and amplifying, recombinant virus was constructed successfully. Infection titer and efficiency of recombinant virus were monitored by green fluorescent protein (GFP) expression. The expression of r-Caspase-3 in infected HepG2 cells was detected by RT-PCR and Western blot. The apoptosis of HepG2 cells was detected by SRB dyeing method.
Results 　Shuttle vector pAdTrack-EAFP-PALB/r-Caspase-3 was correct after identification by restriction endonuclease analysis and sequencing. By PCR and PacⅠ restriction endonuclease analysis， the homologous recombinant of pAdEasy-EAFP-PALB/r-Caspase-3 was successful. The expression of GFP was observed when linearized pAdEasy-EAFP-PALB/r-Caspase-3 was transfected into AD293 cells. AD293 cells could be infected repeatedly by recombinant adenovirus. The expression of r-Caspase-3 gene on HepG2 cells was detected by RT-PCR and Western-blot methods respectively, which confirmed that the Ad-EAFP-PALB/r-Caspase-3 was constructed successfully. The specificity of Ad-EAFP-PALB/r-caspase-3 which targeting induced hepatocellular carcinoma cells was founded by SRB dyeing test.
Conclusion 　The Recombinant of hepatocellular carcinoma-targeting adenovirus containing r-Caspase-3 gene was constructed successfully and which established the foundation of r-Caspase-3 gene therapy in future research to hepatocellular carcinoma.

Citation： WANG Wei,CHENG Wei,QIU Changru. Construction and Expression of The Recombinant of Hepatocellular Carcinoma-Targeting Adenovirus Containing r-Caspase-3 Gene. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2008, 15(8): 589-594. doi: Copy

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10.	沈鼎明，黄爱龙，肖彧君，等. NF-κB 超抑制物κIBαM 重组腺病毒的构建 [Ｊ]. 免疫学杂志， 2004； 20(1)∶65.
11.	Yang LQ, Fang DC, Wang RQ, et al. Effect of NF-kappaB, survivin, Bcl-2 and Caspase3 on apoptosis of gastric cancer cells induced by tumor necrosis factor related apoptosis inducing ligand [Ｊ]. World J Gastroenterol, 2004； 10(1)∶22.
12.	Meggiato T, Calabrese F, De Cesare CM, et al. C-JUN and CPP32 (CASPASE 3) in human pancreatic cancer: relation to cell proliferation and death [Ｊ]. Pancreas, 2003; 26(1)∶65.
13.	Condorelli G, Roncarati R, Ross J Jr, et al. Heart-targeted overexpression of caspase3 in mice increases infarct size and depresses cardiac function [Ｊ]. Proc Natl Acad Sci USA, 2001; 98 (17)∶ 9977.
14.	Manekeller S, Sioutis M, Hirner A, et al. Influence of neoadjuvant chemotherapy on liver integrity and ischemic tolerance [Ｊ]. Z Gastroenterol, 2008； 46(1)∶17.
15.	Niu J, Li XN, Qian H, et al. siRNA mediated the type 1 insulin-like growth factor receptor and epidermal growth factor receptor silencing induces chemosensitization of liver cancer cells [Ｊ]. J Cancer Res Clin Oncol, 2008; 134(4)∶503.
16.	Park HJ, Shin DH, Chung WJ, et al. Epigallocatechin gallate reduces hypoxia-induced apoptosis in human hepatoma cells [Ｊ]. Life Sci, 2006; 78(24)∶2826.
17.	Nakanishi H, Yasui K, Ikehara Y, et al. Establishment and characterization of three novel human gastric cancer cell lines with differentiated intestinal phenotype derived from liver metastasis [Ｊ]. Clin Exp Metastasis, 2005; 22(2)∶ 137.
18.	Dotti G, Savoldo B, Pule M, et al. Human cytotoxic T lymphocytes with reduced sensitivity to Fas-induced apoptosis [Ｊ]. Blood, 2005; 105(12)∶4677.

1. 13　Fujikawa K, Shiraki K, Sugimoto K, et al. Reduced expression of ICE/caspase1 and CPP32/caspase3 in human hepatocellular carcinoma [Ｊ]. Anticancer Res, 2000; 20(3B)∶1927 14　Hoshi T, Sasano H, Kato K, et al. Immunohistochemistry of Caspase3/CPP32 in human stomach and its correlation with cell proliferation and apoptosis [Ｊ]. Anticancer Res, 1998; 18(6A)∶4347 15　Srinivasula SM, Ahmad M, MacFarlane M, et al. Generation of constitutively active recombinant caspases-3 and-6 by rearrangement of their subunits [Ｊ]. J Biol Chem， 1998； 273(17)∶10107.
2. 　Lam PY, Sia KC, Khong JH, et al. An efficient and safe herpes simplex virus type 1 amplicon vector for transcriptionally targeted therapy of human hepatocellular carcinomas [Ｊ]. Mol Ther, 2007; 15(6)∶1129.
3. 　Pin RH, Reinblatt M, Fong Y. Utilizing alpha-fetoprotein expression to enhance oncolytic viral therapy in hepatocellular carcinoma [Ｊ]. Ann Surg, 2004; 240(4)∶659.
4. 　He P, Tang ZY, Ye SL, et al. The targeted expression of interleukin-2 in human hepatocellular carcinoma cells [Ｊ]. J Exp Clin Cancer Res, 2000; 19(2)∶183.
5. 　He P, Tang ZY, Liu BB, et al. The targeted expression of the human interleukin-2/interferon alpha2b fused gene in alpha-fetoprotein-expressing hepatocellular carcinoma cells [Ｊ]. J Cancer Res Clin Oncol, 1999; 125(2)∶77.
6. 　王炜，秦兆寅，王剑利，等. 重组的r-Caspase-3促凋亡基因体内外治疗胰腺癌的试验研究 [Ｊ]. 中华肝胆外科杂志， 2003； 9(5)∶303.
7. 　王炜，秦兆寅，刘志国. 重组型Caspase-3基因的构建及其在胰腺癌细胞中凋亡活性的观察 [Ｊ]. 中国普外基础与临床杂志， 2002； 9(4)∶249.
8. 　郑仲承. 寡核苷酸的优化设计 [Ｊ]. 生命的化学, 2001; 21(3)∶ 254.
9. 　He TC, Zhou S, da Costa LT, et al. A simplified system for generating recombinant adenoviruses [Ｊ]. Proc Natl Acad Sci USA， 1998； 95(5)∶2509.
10. 　沈鼎明，黄爱龙，肖彧君，等. NF-κB 超抑制物κIBαM 重组腺病毒的构建 [Ｊ]. 免疫学杂志， 2004； 20(1)∶65.
11. 　Yang LQ, Fang DC, Wang RQ, et al. Effect of NF-kappaB, survivin, Bcl-2 and Caspase3 on apoptosis of gastric cancer cells induced by tumor necrosis factor related apoptosis inducing ligand [Ｊ]. World J Gastroenterol, 2004； 10(1)∶22.
12. 　Meggiato T, Calabrese F, De Cesare CM, et al. C-JUN and CPP32 (CASPASE 3) in human pancreatic cancer: relation to cell proliferation and death [Ｊ]. Pancreas, 2003; 26(1)∶65.
13. 　Condorelli G, Roncarati R, Ross J Jr, et al. Heart-targeted overexpression of caspase3 in mice increases infarct size and depresses cardiac function [Ｊ]. Proc Natl Acad Sci USA, 2001; 98 (17)∶ 9977.
14. 　Manekeller S, Sioutis M, Hirner A, et al. Influence of neoadjuvant chemotherapy on liver integrity and ischemic tolerance [Ｊ]. Z Gastroenterol, 2008； 46(1)∶17.
15. 　Niu J, Li XN, Qian H, et al. siRNA mediated the type 1 insulin-like growth factor receptor and epidermal growth factor receptor silencing induces chemosensitization of liver cancer cells [Ｊ]. J Cancer Res Clin Oncol, 2008; 134(4)∶503.
16. 　Park HJ, Shin DH, Chung WJ, et al. Epigallocatechin gallate reduces hypoxia-induced apoptosis in human hepatoma cells [Ｊ]. Life Sci, 2006; 78(24)∶2826.
17. 　Nakanishi H, Yasui K, Ikehara Y, et al. Establishment and characterization of three novel human gastric cancer cell lines with differentiated intestinal phenotype derived from liver metastasis [Ｊ]. Clin Exp Metastasis, 2005; 22(2)∶ 137.
18. 　Dotti G, Savoldo B, Pule M, et al. Human cytotoxic T lymphocytes with reduced sensitivity to Fas-induced apoptosis [Ｊ]. Blood, 2005; 105(12)∶4677.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Construction and Expression of The Recombinant of Hepatocellular Carcinoma-Targeting Adenovirus Containing r-Caspase-3 Gene

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