• 廣州醫(yī)學(xué)院第一附屬醫(yī)院肝膽外科（廣州 510120）;

目的 　構(gòu)建肝細(xì)胞肝癌特異性表達(dá)反向半胱氨酸蛋白酶3（r-Caspase-3）重組腺病毒，為肝細(xì)胞肝癌的基因治療提供新策略。
方法 　構(gòu)建甲胎蛋白（AFP）增強(qiáng)子和白蛋白 (ALB) 啟動(dòng)子腺病毒載體（pAdTrack-EAFP-PALB），然后將目的基因r-Caspase-3亞克隆到載體pAdTrack-EAFP-PALB上， 獲得重組腺病毒穿梭載體pAdTrack-EAFP-PALB /r-Caspase-3, 經(jīng)PmeⅠ酶切線性化后與pAdEasy-1同源重組，獲得重組腺病毒骨架pAdEasy-EAFP-PALB /r-Caspase-3； 鑒定正確的pAdEasy-EAFP-PALB/r-Caspase-3 經(jīng)PacⅠ酶切線性化后脂質(zhì)體轉(zhuǎn)染AD293 細(xì)胞進(jìn)行包裝、擴(kuò)增，獲得病毒。綠色熒光蛋白(green fluorescent protein，GFP)監(jiān)測(cè)病毒滴度和感染效率； RT-PCR和Western blot 法檢測(cè)r-Caspase-3在HepG2細(xì)胞中的表達(dá)； SRB染色法評(píng)估重組腺病毒對(duì)HepG2細(xì)胞的抑制作用，初步觀察HepG2細(xì)胞凋亡狀況。
結(jié)果 　穿梭載體pAdTrack-EAFP-PALB/r-Caspase-3酶切、測(cè)序正確。穿梭載體、pAdEasy-1載體同源重組后PCR 及PacⅠ酶切鑒定結(jié)果表明pAdEasy-EAFP-PALB/r-Caspase-3 重組成功； 經(jīng)PacⅠ酶切線性化后，pAdEasy-EAFP-PALB/r-Caspase-3 轉(zhuǎn)染AD293 細(xì)胞即可觀察到GFP 的表達(dá)； 回收病毒可重復(fù)感染AD293 細(xì)胞，RT-PCR和Western blot 均可檢測(cè)到r-Caspase-3的表達(dá)，證實(shí)Ad-EAFP-PALB/r-Caspase-3病毒顆粒包裝成功； SRB染色檢測(cè)發(fā)現(xiàn)Ad-EAFP-PALB/r-Caspase-3具有凋亡誘導(dǎo)特異性。
結(jié)論 　靶向性Ad-EAFP-PALB/r-Caspase-3重組腺病毒構(gòu)建成功，并具有凋亡誘導(dǎo)靶向性，為進(jìn)一步研究靶向性r-Caspase-3基因治療肝細(xì)胞肝癌及其生物學(xué)功能奠定了基礎(chǔ)。

引用本文： 王煒,程衛(wèi),丘昶儒. 肝細(xì)胞肝癌靶向性r-Caspase-3重組腺病毒的構(gòu)建與表達(dá). 中國(guó)普外基礎(chǔ)與臨床雜志, 2008, 15(8): 589-594. doi: 復(fù)制

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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. 　王煒， 秦兆寅， 王劍利， 等. 重組的r-Caspase-3促凋亡基因體內(nèi)外治療胰腺癌的試驗(yàn)研究 [Ｊ]. 中華肝膽外科雜志， 2003； 9(5)∶303.
7. 　王煒， 秦兆寅， 劉志國(guó). 重組型Caspase-3基因的構(gòu)建及其在胰腺癌細(xì)胞中凋亡活性的觀察 [Ｊ]. 中國(guó)普外基礎(chǔ)與臨床雜志， 2002； 9(4)∶249.
8. 　鄭仲承. 寡核苷酸的優(yōu)化設(shè)計(jì) [Ｊ]. 生命的化學(xué), 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， 黃愛(ài)龍， 肖彧君， 等. NF-κB 超抑制物κIBαM 重組腺病毒的構(gòu)建 [Ｊ]. 免疫學(xué)雜志， 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. 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. 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. 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. 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. 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. 6. 　王煒， 秦兆寅， 王劍利， 等. 重組的r-Caspase-3促凋亡基因體內(nèi)外治療胰腺癌的試驗(yàn)研究 [Ｊ]. 中華肝膽外科雜志， 2003； 9(5)∶303.
  7. 7. 　王煒， 秦兆寅， 劉志國(guó). 重組型Caspase-3基因的構(gòu)建及其在胰腺癌細(xì)胞中凋亡活性的觀察 [Ｊ]. 中國(guó)普外基礎(chǔ)與臨床雜志， 2002； 9(4)∶249.
  8. 8. 　鄭仲承. 寡核苷酸的優(yōu)化設(shè)計(jì) [Ｊ]. 生命的化學(xué), 2001; 21(3)∶ 254.
  9. 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. 10. 　沈鼎明， 黃愛(ài)龍， 肖彧君， 等. NF-κB 超抑制物κIBαM 重組腺病毒的構(gòu)建 [Ｊ]. 免疫學(xué)雜志， 2004； 20(1)∶65.
  11. 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. 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. 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. 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. 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. 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. 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. 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.