肿瘤厌氧靶向双自杀基因治疗系统pH2/CD，pH2/UPRT的构建(3)
胞嘧啶脱氨酶(CD)和5-氟-胞嘧啶(5-FC)系统(CD/5-FC)是自杀基因的原型，低毒的5-FC可以被CD代谢为抗肿瘤药5-FU。全身给药的5-FC到达肿瘤，而靶向到肿瘤局部的CD基因转化5-FC至5-FU将会杀死肿瘤细胞，但对全身的毒性则大大降低。因为很多肿瘤对5-FU产生抗性，所以构建(CD/5-FC)的协同杀伤机制将可能克服其抗药性，并明显增强CD/5-FC 肿瘤自杀基因治疗系统的效力。
5-FU杀灭肿瘤细胞的机制包括被进一步代谢为5-FdUMP,5-FdUTP和5-FUTP，这些分子可以干扰细胞RNA 和DNA 合成[10-11]。UPRT 是一种来源于原核生物的酶，它可以非常有效的催化5-FU生成5-FdUMP，使得5-FdUMP，5-FdUTP,和5-FUTP大量增加，从而增强5-FC/CD系统对肿瘤细胞的杀伤作用。所以构建CD 和UPRT相结合的双自杀基因系统将可能显著增强该系统的肿瘤杀伤能力[12-16]。
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当厌氧菌被重组为携带有高效表达自杀基因的质粒时，厌氧菌的肿瘤靶向性将使得它们在肿瘤组织聚集，而原药5-FC将在肿瘤组织内部高效地被自杀基因CD/UPRT转化为细胞毒性的抗肿瘤药物。婴儿双歧杆菌是一种非致病性的厌氧菌株。本实验室在先前的研究中已经证明其对实体瘤的靶向性[1]，并构建出携带有pGEX-1LamdaT/CD和pGEX-1LamdaT/UPRT的婴儿双歧杆菌，并且表现出体外对肿瘤的杀伤作用，但由于pGEX-1LamdaT是大肠杆菌表达载体，重组至厌氧菌中其蛋白质的表达效率并不令人满意[16-17]。
质粒pH2是一个厌氧菌特异性质粒，带有一个分泌性基因表达系统，可以在乳酸菌和双歧杆菌中高效率表达分泌型蛋白质[3]。我们最终成功的构建了pH2/CD和pH2/UPRT婴儿双歧杆菌分泌性自杀基因表达系统，并已获得CD，UPRT基因mRNA水平的良好表达。在体外杀伤鼠黑色素瘤细胞的实验中，当5-FC浓度达到1 μmol/ml时，双自杀基因表达系统组中黑色素瘤B16-F10细胞的存活率几乎接近0，该自杀基因表达系统表现出非常好的抗肿瘤效果，而该系统在鼠体内的抑瘤效果，以何种剂量，何种方式给药能达到最好的抑瘤效果，以及此系统的临床实用价值是我们将来所要解决的问题，有待我们进一步的实验验证。
, http://www.100md.com
参考文献
[1] 吴瑜，易成，王树人,等.婴儿双歧杆菌对小鼠黑色素瘤模型肿瘤组织的靶向性.四川大学学报(医学版),2003,34(3):435-438.
[2] Kazuyuki Yazawa,Minoru Fujimori,Jun Amano,et al.Bifidobacterium longum as a delivery system for cancer gene therapy:selective localization and growth in hypoxic tumors.Cancer Gene Ther,2000,7:269-274.
[3] O’sullivan DJ,Klaenhammer TR.High-and low-copy-number Lactococcus shuttle cloning vectors with features for clone screening.Gene,1993,137:227-231.
, http://www.100md.com
[4] Alessandra Argnani,Rob J.Leer,Nicole van Luijk,et al.A convenient and reproducible method to genetically transform bacteria of the genus Bifidobacterium.Microbiology,1996,142:109-114.
[5] Pinedo HM,Peters GFJ.Fluorouracil:biochemistry and pharmacology.J Clin Oncol,1988,6:1644-1653.
[6] Breitman ML,Clapoff S,Rossant J,et al.Genetic ablation:targeted expression of a toxin gene causes microphthalmia in transgenic mice.Science,1987,238(4833):1563-1565.
, 百拇医药
[7] Moulder JE,Rockwell S.Hypoxic fractions of solid tumors:experimental techniques,methods of analysis,and survey of existing data.Int J Radiat Oncol Biol Phys,1984,10:695-712.
[8] Vaupel PW,Hockel M.Oxygenation status of human tumors:a reappraisal using computerized pO2 histography.Tumor Oxygenation,1994:219-232.
[9] Vaupel PW.Oxygenation of solid tumors in drug resistance in oncology.Marcel Dekker,1993:53-85.
, 百拇医药
[10] Palmiter RD,Behringer RR,Quaife CJ,et al.Cell lineage ablation in transgenic mice by cell-specific expression of a toxin gene.Cell,1987,50(3):435-443.
[11] Van der Wilt CL,Pinedo HM,Smid K,et al.Effect of folinic acid on fluorouracil activity and expression of thymidine synthase.Semin Oncol,1992,19:16-25.
[12] Tiraby M,Cazaux C,Baron M,et al.Concomitant expression of E.coli cytosine deaminase and uracil phosphoribosyltransferase improves the cytotoxicity of 5-fluorocytosine.FEMS Microbiol Lett,1998,167(1):41-49.
, 百拇医药
[13] Adachi Y,Tamiya T,Ichikawa T,et al.Experimental gene therapy for brain tumors using adenovirus-mediated transfer of cytosine deaminase gene and uracil phosphoribosyltransferase gene with 5-fluorocytosine.Hum Gene Ther,2000,11(1):77-89.
[14] Koyama F,Sawada H,Hirao T,et al.Combined suicide gene therapy for human colon cancer cells using adenovirus-mediated transfer of escherichia coli cytosine deaminase gene and Escherichia coli uracil phosphoribosyltransferase gene with 5-fluorocytosine.Cancer Gene Ther,2000,7(7):1015-1022.
, 百拇医药
[15] Miyagi T,Koshida K,Hori O,et al.Gene therapy for prostate cancer using the cytosine deaminase/uracil phosphoribosyltransferase suicide system.J Gene Med,2003,5(1):30-37.
[16] 安丽娜，李著华，岳扬，等.婴儿双歧杆菌介导的CD和UPRT联合5-FC基因疗法对黑色素瘤的体外治疗实验研究.四川大学学报(医学版),2007，38(1):27-30,63.
[17] 易成，郭志英，黄英，等.婴儿双歧杆菌介导的CD/5-FC自杀基因系统对黑色素瘤的抑瘤实验.四川大学学报(医学版)，2005，36(2)：165-168., 百拇医药(叶 鹏 李著华 杨彦彪 王树人)
5-FU杀灭肿瘤细胞的机制包括被进一步代谢为5-FdUMP,5-FdUTP和5-FUTP，这些分子可以干扰细胞RNA 和DNA 合成[10-11]。UPRT 是一种来源于原核生物的酶，它可以非常有效的催化5-FU生成5-FdUMP，使得5-FdUMP，5-FdUTP,和5-FUTP大量增加，从而增强5-FC/CD系统对肿瘤细胞的杀伤作用。所以构建CD 和UPRT相结合的双自杀基因系统将可能显著增强该系统的肿瘤杀伤能力[12-16]。
, http://www.100md.com
当厌氧菌被重组为携带有高效表达自杀基因的质粒时，厌氧菌的肿瘤靶向性将使得它们在肿瘤组织聚集，而原药5-FC将在肿瘤组织内部高效地被自杀基因CD/UPRT转化为细胞毒性的抗肿瘤药物。婴儿双歧杆菌是一种非致病性的厌氧菌株。本实验室在先前的研究中已经证明其对实体瘤的靶向性[1]，并构建出携带有pGEX-1LamdaT/CD和pGEX-1LamdaT/UPRT的婴儿双歧杆菌，并且表现出体外对肿瘤的杀伤作用，但由于pGEX-1LamdaT是大肠杆菌表达载体，重组至厌氧菌中其蛋白质的表达效率并不令人满意[16-17]。
质粒pH2是一个厌氧菌特异性质粒，带有一个分泌性基因表达系统，可以在乳酸菌和双歧杆菌中高效率表达分泌型蛋白质[3]。我们最终成功的构建了pH2/CD和pH2/UPRT婴儿双歧杆菌分泌性自杀基因表达系统，并已获得CD，UPRT基因mRNA水平的良好表达。在体外杀伤鼠黑色素瘤细胞的实验中，当5-FC浓度达到1 μmol/ml时，双自杀基因表达系统组中黑色素瘤B16-F10细胞的存活率几乎接近0，该自杀基因表达系统表现出非常好的抗肿瘤效果，而该系统在鼠体内的抑瘤效果，以何种剂量，何种方式给药能达到最好的抑瘤效果，以及此系统的临床实用价值是我们将来所要解决的问题，有待我们进一步的实验验证。
, http://www.100md.com
参考文献
[1] 吴瑜，易成，王树人,等.婴儿双歧杆菌对小鼠黑色素瘤模型肿瘤组织的靶向性.四川大学学报(医学版),2003,34(3):435-438.
[2] Kazuyuki Yazawa,Minoru Fujimori,Jun Amano,et al.Bifidobacterium longum as a delivery system for cancer gene therapy:selective localization and growth in hypoxic tumors.Cancer Gene Ther,2000,7:269-274.
[3] O’sullivan DJ,Klaenhammer TR.High-and low-copy-number Lactococcus shuttle cloning vectors with features for clone screening.Gene,1993,137:227-231.
, http://www.100md.com
[4] Alessandra Argnani,Rob J.Leer,Nicole van Luijk,et al.A convenient and reproducible method to genetically transform bacteria of the genus Bifidobacterium.Microbiology,1996,142:109-114.
[5] Pinedo HM,Peters GFJ.Fluorouracil:biochemistry and pharmacology.J Clin Oncol,1988,6:1644-1653.
[6] Breitman ML,Clapoff S,Rossant J,et al.Genetic ablation:targeted expression of a toxin gene causes microphthalmia in transgenic mice.Science,1987,238(4833):1563-1565.
, 百拇医药
[7] Moulder JE,Rockwell S.Hypoxic fractions of solid tumors:experimental techniques,methods of analysis,and survey of existing data.Int J Radiat Oncol Biol Phys,1984,10:695-712.
[8] Vaupel PW,Hockel M.Oxygenation status of human tumors:a reappraisal using computerized pO2 histography.Tumor Oxygenation,1994:219-232.
[9] Vaupel PW.Oxygenation of solid tumors in drug resistance in oncology.Marcel Dekker,1993:53-85.
, 百拇医药
[10] Palmiter RD,Behringer RR,Quaife CJ,et al.Cell lineage ablation in transgenic mice by cell-specific expression of a toxin gene.Cell,1987,50(3):435-443.
[11] Van der Wilt CL,Pinedo HM,Smid K,et al.Effect of folinic acid on fluorouracil activity and expression of thymidine synthase.Semin Oncol,1992,19:16-25.
[12] Tiraby M,Cazaux C,Baron M,et al.Concomitant expression of E.coli cytosine deaminase and uracil phosphoribosyltransferase improves the cytotoxicity of 5-fluorocytosine.FEMS Microbiol Lett,1998,167(1):41-49.
, 百拇医药
[13] Adachi Y,Tamiya T,Ichikawa T,et al.Experimental gene therapy for brain tumors using adenovirus-mediated transfer of cytosine deaminase gene and uracil phosphoribosyltransferase gene with 5-fluorocytosine.Hum Gene Ther,2000,11(1):77-89.
[14] Koyama F,Sawada H,Hirao T,et al.Combined suicide gene therapy for human colon cancer cells using adenovirus-mediated transfer of escherichia coli cytosine deaminase gene and Escherichia coli uracil phosphoribosyltransferase gene with 5-fluorocytosine.Cancer Gene Ther,2000,7(7):1015-1022.
, 百拇医药
[15] Miyagi T,Koshida K,Hori O,et al.Gene therapy for prostate cancer using the cytosine deaminase/uracil phosphoribosyltransferase suicide system.J Gene Med,2003,5(1):30-37.
[16] 安丽娜，李著华，岳扬，等.婴儿双歧杆菌介导的CD和UPRT联合5-FC基因疗法对黑色素瘤的体外治疗实验研究.四川大学学报(医学版),2007，38(1):27-30,63.
[17] 易成，郭志英，黄英，等.婴儿双歧杆菌介导的CD/5-FC自杀基因系统对黑色素瘤的抑瘤实验.四川大学学报(医学版)，2005，36(2)：165-168., 百拇医药(叶 鹏 李著华 杨彦彪 王树人)