小鼠角质化细胞生长因子及其受体的重组腺病毒表达载体的构建
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摘要
背景：角质化细胞生长因子(Keratinocyte Growth Factor, KGF)是一种备受关注的细胞生长因子，其对肺泡上皮细胞(Alveolar Epithelial Cells，AECs)的多途径、多水平的保护作用已经广为认可，既往研究表明KGF对急性肺损伤／急性呼吸窘迫综合征(Acute Lung Injury／Acute Respiratory Distress Syndrome，ALI／ARDS)，以及肺纤维化(Pulmonary Fibrosis，PF)均有预防作用，能够明显缓解病情改善预后。但是，就其应用而言，主要存在如下问题：(1)KGF蛋白昂贵；(2)KGF目前仅限于预防。因此，构建KGF、角质化细胞生长因子受体(Keratinocyte Growth Factor，KGFR)的重组腺病毒载体对于探索肺部疾病的治疗；探讨KGF，及其他细胞生长因子在肺损伤免疫进程中的作用都将是一个有力的工具。
目的：构建表达小鼠KGF、KGFR基因的重组腺病毒。
方法：(1)根据小鼠KGF、KGFR基因序列，设计正向、反向引物；同时，提取小鼠肺组织RNA，进行逆转录聚合酶链反应(reverse transcription-polymerase chain raction，RT-PCR)反应，构建cDNA文库。构建聚合酶链式反应(polymerase chain reaction，PCR)反应体系，扩增目的基因片段。(2)选择pShuttle-CMV(卡那霉素抗性)作为载体质粒，根据引物设计所选择的核酸内切酶，对载体质粒和目的基因片段进行双酶切。将线性化的两条DNA连接后，进行扩增，提取穿梭质粒pShuttle-CMV-KGF(pKGF)、pShuttle-CMV-KGF(pKGFR)。(3)核酸内切酶PmeⅠ线性化pKGF、pKGFR，通过转化含病毒骨架质粒pAdeasy-1(氨苄西林抗性)的大肠杆菌BJ5183以连接病毒骨架DNA，提取病毒质粒pAdeasy-1-pShuttle-CMV-KGF(pAd-KGF)，pAdeasy-1-pShuttle-CMV-KGFR(pAd-KGFR)，PacⅠ酶切鉴定后，大量扩增质粒，转染HEK293细胞。提取重组腺病毒Adeasy-1-pShuttle-CMV-KGF(Ad-KGF)，Adeasy-1-pShuttle-CMV-KGFR(Ad-KGFR)，大量扩增，氯化铯梯度离心纯化，测量病毒滴度。
结果：(1)DNA测序结果表明pKGF所载基因仅于第48bp处G突变为A，考虑氨基酸翻译简并性，不影响KGF表达；pKGFR则完全符合，提示成功构建穿梭质粒。(2)线性化穿梭质粒转化BJ5183细菌后，细菌生长于含Kan的LB平板，同时PacⅠ酶切显示目的条带，提示成功构建含KGF、KGFR基因的病毒质粒。(3)显微镜下观察HEK293细胞出现病理形态变化，表明病毒合成并进行复制。(4)测得Ad-KGF、Ad-KGFR病毒滴度，分别为3.0×10~(11)PFU／ml、2.25×10~(11)PFU／ml。
结论：本实验成功构建小鼠KGF、KGFR基因重组腺病毒载体Ad-KGF、Ad-KGFR，测得病毒滴度，表明可以作为进一步的体外细胞、体内动物实验的实验工具。本实验是在DNA水平上初步证实病毒的成功构建，进一步的体内、体外实验中，将在蛋白水平上明确病毒构建成功，明确目的基因的稳定表达。
Background: As a classic growth factor, KGF has multiple protective effects on AECs to obviate future harm, which has been proven in the past experiments, from the acute course of Acute Lung Injury/ Acute Respiratory Distress Syndrome ALI/ARDS to the chronic course of PF.However, there are still many problems for its cost in protein therapy, for its only contribution of prevention but not protection which may have something with the decrease of KGFR on epithelial cells after the injuries to the lung, and its effects of a kind of cytokines associated with immune network which has been ignored. Therefore, successful construction of recombinant adenovirus vector of KGF and KGFR may be a better way to cure the pulmonary diseases such as PF, and to discover the roles of KGF and even other growth factor in the immune course of lung injuries.
Objective: To construct the recombinant adenovirus vectors expressing mouse KGF or KGFR.
Methods: (1)According to the gene order of mouse KGF and KGFR provided by the gene Bank in National Center for Biotechnology Information, we had designed the sense and the antisense primer, with which we amplified the KGF/KGFR gene by PCR, with cDNA acquired from mouse lung RNA using RT-PCR. (2)pShuttle-CMV with the drug resistance of kanamycin to be the carrying plasmid, we inserted the KGF or KGFR gene into it, to construct the shuttle vector pKGF and pKGFR. (3) We then linearized pKGF and pKGFR, transformed them, respectively, into the E.Coli BJ5183 which contain virus backbones, followed by the step transfection of HEK293
cells after identification of endonuclease Pac I and amplification. Finally, we harvested the virus after amplification, caesium chloride density gradient centrifugation purification and measurement of virus titers.
Results: (1)The results of DNA sequencing suggested the success of construction of shuttle vector pKGF and pKGFR. (2)The bacteria could grow in the culture with kanamycin, as well as the agarose gel electrophoresis showed the proper strap, which had shown the success of construction of virus plasmid expressing KGF or KGFR. (3)HFK293 cells showed the pathologic morphologic changes, under microscope, which suggested the synthesis and duplication of recombinant adenovirus. (4)Finally, we had measured the virus titers, which had achieved the requirement of the future experiments in vivo and in vitro.
Conclusion: We have successfully constructed the recombinant adenovirus vector of KGF or KGFR, which had supplied a tool to understand more about the role of KGF in pulmonary disease, especially as a cytokine in immune reaction, and even might be a new, a lower cost way to cure PF.
目的：构建表达小鼠KGF、KGFR基因的重组腺病毒。
方法：(1)根据小鼠KGF、KGFR基因序列，设计正向、反向引物；同时，提取小鼠肺组织RNA，进行逆转录聚合酶链反应(reverse transcription-polymerase chain raction，RT-PCR)反应，构建cDNA文库。构建聚合酶链式反应(polymerase chain reaction，PCR)反应体系，扩增目的基因片段。(2)选择pShuttle-CMV(卡那霉素抗性)作为载体质粒，根据引物设计所选择的核酸内切酶，对载体质粒和目的基因片段进行双酶切。将线性化的两条DNA连接后，进行扩增，提取穿梭质粒pShuttle-CMV-KGF(pKGF)、pShuttle-CMV-KGF(pKGFR)。(3)核酸内切酶PmeⅠ线性化pKGF、pKGFR，通过转化含病毒骨架质粒pAdeasy-1(氨苄西林抗性)的大肠杆菌BJ5183以连接病毒骨架DNA，提取病毒质粒pAdeasy-1-pShuttle-CMV-KGF(pAd-KGF)，pAdeasy-1-pShuttle-CMV-KGFR(pAd-KGFR)，PacⅠ酶切鉴定后，大量扩增质粒，转染HEK293细胞。提取重组腺病毒Adeasy-1-pShuttle-CMV-KGF(Ad-KGF)，Adeasy-1-pShuttle-CMV-KGFR(Ad-KGFR)，大量扩增，氯化铯梯度离心纯化，测量病毒滴度。
结果：(1)DNA测序结果表明pKGF所载基因仅于第48bp处G突变为A，考虑氨基酸翻译简并性，不影响KGF表达；pKGFR则完全符合，提示成功构建穿梭质粒。(2)线性化穿梭质粒转化BJ5183细菌后，细菌生长于含Kan的LB平板，同时PacⅠ酶切显示目的条带，提示成功构建含KGF、KGFR基因的病毒质粒。(3)显微镜下观察HEK293细胞出现病理形态变化，表明病毒合成并进行复制。(4)测得Ad-KGF、Ad-KGFR病毒滴度，分别为3.0×10~(11)PFU／ml、2.25×10~(11)PFU／ml。
结论：本实验成功构建小鼠KGF、KGFR基因重组腺病毒载体Ad-KGF、Ad-KGFR，测得病毒滴度，表明可以作为进一步的体外细胞、体内动物实验的实验工具。本实验是在DNA水平上初步证实病毒的成功构建，进一步的体内、体外实验中，将在蛋白水平上明确病毒构建成功，明确目的基因的稳定表达。
Background: As a classic growth factor, KGF has multiple protective effects on AECs to obviate future harm, which has been proven in the past experiments, from the acute course of Acute Lung Injury/ Acute Respiratory Distress Syndrome ALI/ARDS to the chronic course of PF.However, there are still many problems for its cost in protein therapy, for its only contribution of prevention but not protection which may have something with the decrease of KGFR on epithelial cells after the injuries to the lung, and its effects of a kind of cytokines associated with immune network which has been ignored. Therefore, successful construction of recombinant adenovirus vector of KGF and KGFR may be a better way to cure the pulmonary diseases such as PF, and to discover the roles of KGF and even other growth factor in the immune course of lung injuries.
Objective: To construct the recombinant adenovirus vectors expressing mouse KGF or KGFR.
Methods: (1)According to the gene order of mouse KGF and KGFR provided by the gene Bank in National Center for Biotechnology Information, we had designed the sense and the antisense primer, with which we amplified the KGF/KGFR gene by PCR, with cDNA acquired from mouse lung RNA using RT-PCR. (2)pShuttle-CMV with the drug resistance of kanamycin to be the carrying plasmid, we inserted the KGF or KGFR gene into it, to construct the shuttle vector pKGF and pKGFR. (3) We then linearized pKGF and pKGFR, transformed them, respectively, into the E.Coli BJ5183 which contain virus backbones, followed by the step transfection of HEK293
cells after identification of endonuclease Pac I and amplification. Finally, we harvested the virus after amplification, caesium chloride density gradient centrifugation purification and measurement of virus titers.
Results: (1)The results of DNA sequencing suggested the success of construction of shuttle vector pKGF and pKGFR. (2)The bacteria could grow in the culture with kanamycin, as well as the agarose gel electrophoresis showed the proper strap, which had shown the success of construction of virus plasmid expressing KGF or KGFR. (3)HFK293 cells showed the pathologic morphologic changes, under microscope, which suggested the synthesis and duplication of recombinant adenovirus. (4)Finally, we had measured the virus titers, which had achieved the requirement of the future experiments in vivo and in vitro.
Conclusion: We have successfully constructed the recombinant adenovirus vector of KGF or KGFR, which had supplied a tool to understand more about the role of KGF in pulmonary disease, especially as a cytokine in immune reaction, and even might be a new, a lower cost way to cure PF.
引文
1.Rubin JS, Osada H, Finch DW, et al: Purfication and characterization of a newly identified growth factor specific for epithelial cell. [J] Proc Natl Acad Sci USA, 1989, 86:802-806.
2. YanoT, Deterding RR, Simonet WS, Shannon JM, and Mason RJ. Keratinocyte growth factor reduces lung damage due to acid instillation in rats. Am J Respir Cell Mol Biol 15: 433-442, 1996.
3. Nemzek JA, Ebong SJ, Kim J, Bolgos GL, and Remick DG. Keratinocyte growth factor pretreatment is associated with decreased MIP-2_concentrations and reduced neutrophil recruitment in acid aspiration lung injury. Shock 18: 501-506, 2002.
4. Guo J, Yi ES, Havill AM, Sarosi I, Whitcomb L, Yin S, Middleton SC, Piguet P, and Ulich TR. Intravenous keratinocyte growth factor protects against experimental pulmonary injury. Am J Physiol Lung Cell Mol Physiol 275: L800 - L805, 1998.
5. Sugahara K, Iyama KI, Kuroda MJ, and Sano K. Double intratracheal instillation of keratinocyte growth factor prevents bleomycin-induced lung fibrosis in rats. J Pathol 186: 90-98, 1998.
6. Barazzone C, Donati YR, Rochat AF, Vesin C, Kan CD, Pache JC, and Piguet PF. Keratinocyte growth factor protects alveolar epithelium and endothelium from oxygen-induced injury in mice. Am J Pathol 154: 1479-1487, 1999.
7. Viget N, Guery BP, Ader F, Alfandari S, Creuzy C, Roussel-Delvallez M, Foucher C, Mason CM, Beaucaire G, and Pittet JF. Keratinocyte growth factor protects against Pseudomonas aeruginosa-induced lung injury. Am J Physiol Lung Cell Mol Physiol 279: L1199 - L1209, 2000.
8.Yang Y. et al. Recombinant IL-12 prevent formation of blocking antibodies to recombinant adenovirus and allows repeated gene therapy to mouse lung. Nat .Med. 1996:9:890-893
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14. Nicola M D., Milanesi M., Magni M., et al. Recombinant adenoviral vector-lipofectamine complex for gene transduction into human T lymphocytes. Hum Gene Ther. 1999, 10: 1875-1884.
15．黎檀实，吴旭辉等．生长激素对大鼠肺泡Ⅱ型上皮细胞凋亡抑制作用的观察．中国危重病急救医学．2002，14(5)：290-293．
16．黎檀实，吴旭辉等．生长激素对酸吸入性肺损伤大鼠肺组织细胞凋亡的作用．中国危重病急救医学．2002，14(12)：728-731．
17．吴旭辉，黎檀实．生长激素对阿霉素中毒大鼠心肌细胞的抗凋亡作用．心肺血管病杂志．2003，22(4)：236-238．
18．黎檀实，尹明，吴旭辉等．生长激素对盐酸致大鼠肺泡Ⅱ型上皮细胞凋亡的保护．世界急危重病医学杂志．2004，1(2)：113-116．
19．李银平，黎檀实．胰岛素样生长因子-1对海水浸泡大鼠肺泡Ⅱ上皮细胞的抗凋亡作用．中华急诊医学杂志．2004，13(4)：248-250．
20．黎檀实，尹明等．生长激素在酸吸入性肺损伤大鼠肺组织中的修复作用．世界急危重病医学杂志．2005，2(2)：608-611．
21. Frank,-L: Protective effect of keratinocyte growth factor against lung abnormalities associated with hyperoxia in prematurely born rats. Biol-Neonate. 2003; 83(4): 263-72
22. Sugahara,-K; Tokumine,-J; Teruya,-K; et al: Alveolar epithelial cells: differentiation and lung injury. Respirology. 2006 Jan; 11 Suppl: S28-31
23. Chelly N, Henrion A, Pinteut C, et al: Role of keratinocyte growth factor in the control of surfactant synthesis by fetal lung mesenchyme. Endocrinology 2001; 142(5): 1814
24. Matthay,-M-A; Fukuda,-N; Frank,-J; et al: Alveolar epithelial barrier. Role in lung fluid balance in clinical lung injury. Clin-Chest-Med. 2000 Sep; 21(3): 477-90
25. Prince L. S., Philip H. K., Thomas O.M. et al. KGF alters gene expression in human airway epithelia: potential regulation of the inflammatory response. Physiol Genomics 2001, 6: 81～89
26. Stern B, Fierobe L, Paugam C, et al. Keratinocyte growth factor and hepatocyte growth factor in bronchoalveolar lavage fluid in acute respiratory distress syndrome patients. CritCareMed 2000; 28(7): 2326
27. Ulrich,-K; Stern,-M; Goddard,-M-E; et al: Keratinocyte growth factor therapy in murine oleic acid-induced acute lung injury, Am-J-Physiol-Lung-Cell-Mol-Physiol. 2005 Jun; 288(6): L1179-92.
28．刘斌剑、王建民、陈林等：角质细胞生长因子受体腺病毒表达载体在脂多糖所致肺泡上皮细胞损伤修复中的应用．中华急诊医学杂志，2005．12，14(12)：1004-1006．
29. Panoskaltsis-Mortari A, Ingbar DH, Jung P, et al: KGF pretreatment decreases B7 and granzyme B expression and hastens repair in lungs of mice after allogeneic BMT. Am J Physiol Lung Cell Mol Physiol. 2000 May; 278(5): L988-99.
30. Haddad IY, Milla C, Yang S, et al: Surfactant protein A is a required mediator of keratinocyte growth factor after experimental marrow transplantation. Am J Physiol Lung Cell Mol Physiol. 2003 Sep; 285(3): L602-10. Epub 2003 May 9.
31. A. Bergeronl, P. Solerl, M. Kambouchner2, et al: Cytokine profiles in idiopathic pulmonary fibrosis suggest an important role for TGF-beta and IL-10. Eur Respir J. 2003 Jul; 22(1): 69-76.
32. Kahoru Nishina, Feijie Zhang, Larry D. et al: Expression of CINC-2beta is related to the state of differentiation of alveolar epithelial cells. Am J Respir Cell Mol Biol. 2005 Nov; 33(5): 505-12. Epub 2005 Jul 29.
33. Prince LS, Karp PH, Moninger TO, et al: KGF alters gene expression in human airway epithelia: potential regulation of the inflammatory response. Physiol Genomics. 2001 Jul 17; 6(2): 81-9.
34. Chen,-L; Brizel,-D-M; Rabbani,-Z-N; et al: The protective effect of recombinant human keratinocyte growth factor on radiation-induced pulmonary toxicity in rats. Int-J-Radiat-Oncol-Biol-Phys. 2004 Dec 1; 60(5): 1520-9
35. Marchand-Adam,-S; Plantier,-L; Bernuau,-D; et al: Keratinocyte growth factor expression by fibroblasts in pulmonary fibrosis: poor response to interleukin-1beta. Am-J-Respir-Cell-Mol-Biol. 2005 May; 32(5): 470-7
36. Upadhyay,-D; Panduri,-V; Kamp,-D-W: Fibroblast growth factor-10 prevents asbestos-induced alveolar epithelial cell apoptosis by a mitogen-activated protein kinase-dependent mechanism. Am-J-Respir-Cell-Mol-Biol. 2005 Mar; 32(3): 232-8.
37. Pereira CT, Herndon DN, Rocker R, et al: Liposomal Gene Transfer of Keratinocyte Growth Factor Improves Wound Healing by Altering Growth Factor and Collagen Expression. J Surg Res. 2007 Feb 8; [Epub ahead of print]
38. Simonian PL, Roark CL, Diaz del Valle F, et al: Regulatory role of gammadelta T cells in the recruitment of CD4+ and CD8+ T cells to lung and subsequent pulmonary fibrosis. J Immunol. 2006 Oct 1; 177(7): 4436-43.
1. Kcane MP, et al: J Immunoloy. 1999; 163: 5686-5692.
2. Davies HR, Richeldi L. Idiopathic pulmonary fibrosis: current and future treatment options, Am-J-Respir-Med, 2002, 1(3): 211-224.
3. King JR, Ulrish C, Jean F. Idiopathic pulmonary fibrosis: diagnosis and treatment international consensus statement. Am J-Respir and Crit Care Med, 2000, 161: 651-660.
4．黎檀实，吴旭辉等．生长激素对大鼠肺泡Ⅱ型上皮细胞凋亡抑制作用的观察．中国危重病急救医学．2002，14(5)：290-293．
5．黎檀实，吴旭辉等．生长激素对酸吸入性肺损伤大鼠肺组织细胞凋亡的作用．中国危重病急救医学．2002，14(12)：728-731．
6．吴旭辉，黎檀实．生长激素对阿霉素中毒大鼠心肌细胞的抗凋亡作用．心肺血管病杂志．2003，22(4)：236-238．
7．黎檀实，尹明，吴旭辉等．生长激素对盐酸致大鼠肺泡Ⅱ型上皮细胞凋亡的保护．世界急危重病医学杂志．2004，1(2)：113-116．
8．李银平，黎檀实．胰岛素样生长因子-1对海水浸泡大鼠肺泡Ⅱ上皮细胞的抗凋亡作用．中华急诊医学杂志．2004，13(4)：248-250．
9．黎檀实，尹明等．生长激素在酸吸入性肺损伤大鼠肺组织中的修复作用．世界急危重病医学杂志．2005，2(2)：608-611．
10．黎檀实，吴旭辉等．重型颅脑损伤患者血浆肾上腺髓质素变化及临床意义．中国急救医学．2003，23(1)：30-31．
11. Rubin JS, Osada H, Finch DW, et al: Purfication and characterization of a newly identified growth factor specific for epithelial cell.[J] Proc Natl Acad Sci USA, 1989, 86: 802-806.
12. Yano T, Deterding RR, Simonet WS, Shannon JM, and Mason RJ. Keratinocyte growth factor reduces lung damage due to acid instillation in rats. Am J Respir Cell Mol Biol 15: 433-442, 1996.
13. Nemzek JA, Ebong SJ, Kim J, Bolgos GL, and Remick DG. Keratinocyte growth factor pretreatment is associated with decreased MIP-2_concentrations and reduced neutrophil recruitment in acid aspiration lung injury. Shock 18: 501-506, 2002.
14. Guo J, Yi ES, Havill AM, Sarosi I, Whitcomb L, Yin S, Middleton SC, Piguet P, and Ulich TR. Intravenous keratinocyte growth factor protects against experimental pulmonary injury. Am J Physiol Lung Cell Mol Physiol 275: L800-L805, 1998.
15. Sugahara K, Iyama KI, Kuroda MJ, and Sano K. Double intratracheal instillation of keratinocyte growth factor prevents bleomycin-induced lung fibrosis in rats. J Pathol 186: 90-98, 1998.
16. BarazzoneC, Donati YR, Rochat AF, VesinC, Kan CD, Pache JC, and Piguet PF. Keratinocyte growth factor protects alveolar epithelium and endothelium from oxygen-induced injury in mice. Am J Pathol 154: 1479-1487, 1999.
17.Viget N, Guery BP, Ader F, Alfandari S, Creuzy C, Roussel-Delvallez M, Foucher C, Mason CM, Beaucaire G, and Pittet JF. Keratinocyte growth factor protects against Pseudomonas aeruginosa-induced lung injury. Am J Physiol Lung Cell Mol Physiol 279: L1199-L1209, 2000.
18. Frank, -L: Protective effect of keratinocyte growth factor against lung abnormalities associated with hyperoxia in prematurely born rats. Biol-Neonate. 2003; 83(4): 263-72
19. Sugahara, -K; Tokumine,-J; Teruya,-K; et al: Alveolar epithelial cells: differentiation and lung injury. Respirology. 2006 Jan; 11 Suppl: S28-31
20.Chelly N, Henrion A, Pinteut C, et al: Role of keratinocyte growth factor in the control of surfactant synthesis by fetal lung mesenchyme. Endocrinology 2001;142 (5):1814
21.Matthay,-M-A; Fukuda,-N; Frank,-J; et al: Alveolar epithelial barrier. Role in lung fluid balance in clinical lung injury. Clin-Chest-Med. 2000 Sep; 21(3): 477-90
22. Prince L. S., Philip H. K., Thomas O.M. et al. KGF alters gene expression in human airway epithelia: potential regulation of the inflammatory response. Physiol Genomics 2001, 6: 81～89
23. Stern B, Fierobe L, Paugam C, et al. Keratinocyte growth factor and hepatocyte growth factor in bronchoalveolar lavage fluid in acute respiratory distress syndrome patients. CritCareMed 2000; 28(7): 2326
24. Panoskaltsis-Mortari A, Ingbar DH, Jung P, et al: KGF pretreatment decreases B7 and granzyme B expression and hastens repair in lungs of mice after allogeneic BMT. Am J Physiol Lung Cell Mol Physiol. 2000 May; 278(5): L988-99.
25. Haddad IY, Milla C, Yang S, et al: Surfactant protein A is a required mediator of keratinocyte growth factor after experimental marrow transplantation. Am J Physiol Lung Cell Mol Physiol. 2003 Sep; 285(3): L602-10. Epub 2003 May 9.
26．田桂珍、高占成、徐钰等．γ干扰素转基因表达对博来霉素致小鼠肺纤维化的作用．中华结核和呼吸杂志．2003，26(5)：290-293．
27. Ulrich,-K; Stern,-M; Goddard,-M-E; et al: Keratinocyte growth factor therapy in murine oleic acid-induced acute lung injury. Am-J-Physiol-Lung-Cell-Mol-Physiol. 2005 Jun; 288(6): L1179-92.
28．刘斌剑、王建民、陈林等：角质细胞生长因子受体腺病毒表达载体在脂多糖所致肺泡上皮细胞损伤修复中的应用．中华急诊医学杂志，2005．12，14(12)：1004-1006．
29. Vorburger SA, Hunt KK. Adenoviral gene therapy[J]. The Oncologist 2002, 7: 46-59.
30. Simonian PL, Roark CL, Oiaz del Valle F, et al: Regulatory role of gammadelta T cells in the recruitment of CD4+ and CD8+ T cells to lung and subsequent pulmonary fibrosis. J Immunol. 2006 Oct 1; 177(7): 4436-43.
31. Prince LS, Karp PH, Moninger TO, et al: KGF alters gene expression in human airway epithelia: potential regulation of the inflammatory response. Physiol Genomics. 2001 Jul 17; 6(2): 81-9.
32. Chen,-L; Brizel,-D-M; Rabbani,-Z-N; et al: The protective effect of recombinant human keratinocyte growth factor on radiation-induced pulmonary toxicity in rats. Int-J-Radiat-Oncol-Biol-Phys. 2004 Dec 1; 60(5): 1520-9
33. Marchand-Adam,-S; Plantier,-L; Bernuau,-D; et al: Keratinocyte growth factor expression by fibroblasts in pulmonary fibrosis: poor response to interleukin-1beta. Am-J-Respir-Cell-Mol-Biol. 2005 May; 32(5): 470-7
34. Upadhyay,-D; Panduri,-V; Kamp,-D-W: Fibroblast growth factor-10 prevents asbestos-induced alveolar epithelial cell apoptosis by a mitogen-activated protein kinase-dependent mechanism. Am-J-Respir-Cell-Mol-Biol. 2005 Mar; 32(3): 232-8
35. Pereira CT, Herndon DN, Rocker R, et al: Liposomal Gene Transfer of Keratinocyte Growth Factor Improves Wound Healing by Altering Growth Factor and Collagen Expression. J Surg Res. 2007 Feb 8; [Epub ahead of print]
36. A. Bergeronl, P. Solerl, M. Kambouchner2, et al: Cytokine profiles in idiopathic pulmonary fibrosis suggest an important role for TGF-beta and IL-10. Eur Respir J. 2003 Jul; 22(1): 69-76.
37. Kahoru Nishina, Feijie Zhang, Larry D. et al: Expression of CINC-2beta is related to the state of differentiation of alveolar epithelial cells. Am J Respir Cell Mol Biol. 2005 Nov; 33(5): 505-12. Epub 2005 Jul 29.
2. YanoT, Deterding RR, Simonet WS, Shannon JM, and Mason RJ. Keratinocyte growth factor reduces lung damage due to acid instillation in rats. Am J Respir Cell Mol Biol 15: 433-442, 1996.
3. Nemzek JA, Ebong SJ, Kim J, Bolgos GL, and Remick DG. Keratinocyte growth factor pretreatment is associated with decreased MIP-2_concentrations and reduced neutrophil recruitment in acid aspiration lung injury. Shock 18: 501-506, 2002.
4. Guo J, Yi ES, Havill AM, Sarosi I, Whitcomb L, Yin S, Middleton SC, Piguet P, and Ulich TR. Intravenous keratinocyte growth factor protects against experimental pulmonary injury. Am J Physiol Lung Cell Mol Physiol 275: L800 - L805, 1998.
5. Sugahara K, Iyama KI, Kuroda MJ, and Sano K. Double intratracheal instillation of keratinocyte growth factor prevents bleomycin-induced lung fibrosis in rats. J Pathol 186: 90-98, 1998.
6. Barazzone C, Donati YR, Rochat AF, Vesin C, Kan CD, Pache JC, and Piguet PF. Keratinocyte growth factor protects alveolar epithelium and endothelium from oxygen-induced injury in mice. Am J Pathol 154: 1479-1487, 1999.
7. Viget N, Guery BP, Ader F, Alfandari S, Creuzy C, Roussel-Delvallez M, Foucher C, Mason CM, Beaucaire G, and Pittet JF. Keratinocyte growth factor protects against Pseudomonas aeruginosa-induced lung injury. Am J Physiol Lung Cell Mol Physiol 279: L1199 - L1209, 2000.
8.Yang Y. et al. Recombinant IL-12 prevent formation of blocking antibodies to recombinant adenovirus and allows repeated gene therapy to mouse lung. Nat .Med. 1996:9:890-893
9. Vorburger SA, Hunt KK. Adenoviral gene therapy[J]. The Oncologist 2002, 7: 46-59.
10. Stephan A. et al. Adenoviral gene therapy. The Oncologist 2002; 7: 46-59.
11. Paul E. et al. Adeno-associated virus vectors for gene therapy: more pros than cons? Mol. Med. Today 2000; 8: 433-440.
12. HeTC, ZhOH S, Costal, et al. A simfled systemfor generation re-eombinant adenovirus [J]. Pme NatI Acad Sci USA, 1998; 95: 2509-2514.
13. Latiermann C, Oxner wm, Xiao X. et al. The adeno associated viral vector as a strategy for intradiscal gene transfer in immune competent and preexposed rabbits[J]. Spine, 2005, 30(5): 497-504.
14. Nicola M D., Milanesi M., Magni M., et al. Recombinant adenoviral vector-lipofectamine complex for gene transduction into human T lymphocytes. Hum Gene Ther. 1999, 10: 1875-1884.
15．黎檀实，吴旭辉等．生长激素对大鼠肺泡Ⅱ型上皮细胞凋亡抑制作用的观察．中国危重病急救医学．2002，14(5)：290-293．
16．黎檀实，吴旭辉等．生长激素对酸吸入性肺损伤大鼠肺组织细胞凋亡的作用．中国危重病急救医学．2002，14(12)：728-731．
17．吴旭辉，黎檀实．生长激素对阿霉素中毒大鼠心肌细胞的抗凋亡作用．心肺血管病杂志．2003，22(4)：236-238．
18．黎檀实，尹明，吴旭辉等．生长激素对盐酸致大鼠肺泡Ⅱ型上皮细胞凋亡的保护．世界急危重病医学杂志．2004，1(2)：113-116．
19．李银平，黎檀实．胰岛素样生长因子-1对海水浸泡大鼠肺泡Ⅱ上皮细胞的抗凋亡作用．中华急诊医学杂志．2004，13(4)：248-250．
20．黎檀实，尹明等．生长激素在酸吸入性肺损伤大鼠肺组织中的修复作用．世界急危重病医学杂志．2005，2(2)：608-611．
21. Frank,-L: Protective effect of keratinocyte growth factor against lung abnormalities associated with hyperoxia in prematurely born rats. Biol-Neonate. 2003; 83(4): 263-72
22. Sugahara,-K; Tokumine,-J; Teruya,-K; et al: Alveolar epithelial cells: differentiation and lung injury. Respirology. 2006 Jan; 11 Suppl: S28-31
23. Chelly N, Henrion A, Pinteut C, et al: Role of keratinocyte growth factor in the control of surfactant synthesis by fetal lung mesenchyme. Endocrinology 2001; 142(5): 1814
24. Matthay,-M-A; Fukuda,-N; Frank,-J; et al: Alveolar epithelial barrier. Role in lung fluid balance in clinical lung injury. Clin-Chest-Med. 2000 Sep; 21(3): 477-90
25. Prince L. S., Philip H. K., Thomas O.M. et al. KGF alters gene expression in human airway epithelia: potential regulation of the inflammatory response. Physiol Genomics 2001, 6: 81～89
26. Stern B, Fierobe L, Paugam C, et al. Keratinocyte growth factor and hepatocyte growth factor in bronchoalveolar lavage fluid in acute respiratory distress syndrome patients. CritCareMed 2000; 28(7): 2326
27. Ulrich,-K; Stern,-M; Goddard,-M-E; et al: Keratinocyte growth factor therapy in murine oleic acid-induced acute lung injury, Am-J-Physiol-Lung-Cell-Mol-Physiol. 2005 Jun; 288(6): L1179-92.
28．刘斌剑、王建民、陈林等：角质细胞生长因子受体腺病毒表达载体在脂多糖所致肺泡上皮细胞损伤修复中的应用．中华急诊医学杂志，2005．12，14(12)：1004-1006．
29. Panoskaltsis-Mortari A, Ingbar DH, Jung P, et al: KGF pretreatment decreases B7 and granzyme B expression and hastens repair in lungs of mice after allogeneic BMT. Am J Physiol Lung Cell Mol Physiol. 2000 May; 278(5): L988-99.
30. Haddad IY, Milla C, Yang S, et al: Surfactant protein A is a required mediator of keratinocyte growth factor after experimental marrow transplantation. Am J Physiol Lung Cell Mol Physiol. 2003 Sep; 285(3): L602-10. Epub 2003 May 9.
31. A. Bergeronl, P. Solerl, M. Kambouchner2, et al: Cytokine profiles in idiopathic pulmonary fibrosis suggest an important role for TGF-beta and IL-10. Eur Respir J. 2003 Jul; 22(1): 69-76.
32. Kahoru Nishina, Feijie Zhang, Larry D. et al: Expression of CINC-2beta is related to the state of differentiation of alveolar epithelial cells. Am J Respir Cell Mol Biol. 2005 Nov; 33(5): 505-12. Epub 2005 Jul 29.
33. Prince LS, Karp PH, Moninger TO, et al: KGF alters gene expression in human airway epithelia: potential regulation of the inflammatory response. Physiol Genomics. 2001 Jul 17; 6(2): 81-9.
34. Chen,-L; Brizel,-D-M; Rabbani,-Z-N; et al: The protective effect of recombinant human keratinocyte growth factor on radiation-induced pulmonary toxicity in rats. Int-J-Radiat-Oncol-Biol-Phys. 2004 Dec 1; 60(5): 1520-9
35. Marchand-Adam,-S; Plantier,-L; Bernuau,-D; et al: Keratinocyte growth factor expression by fibroblasts in pulmonary fibrosis: poor response to interleukin-1beta. Am-J-Respir-Cell-Mol-Biol. 2005 May; 32(5): 470-7
36. Upadhyay,-D; Panduri,-V; Kamp,-D-W: Fibroblast growth factor-10 prevents asbestos-induced alveolar epithelial cell apoptosis by a mitogen-activated protein kinase-dependent mechanism. Am-J-Respir-Cell-Mol-Biol. 2005 Mar; 32(3): 232-8.
37. Pereira CT, Herndon DN, Rocker R, et al: Liposomal Gene Transfer of Keratinocyte Growth Factor Improves Wound Healing by Altering Growth Factor and Collagen Expression. J Surg Res. 2007 Feb 8; [Epub ahead of print]
38. Simonian PL, Roark CL, Diaz del Valle F, et al: Regulatory role of gammadelta T cells in the recruitment of CD4+ and CD8+ T cells to lung and subsequent pulmonary fibrosis. J Immunol. 2006 Oct 1; 177(7): 4436-43.
1. Kcane MP, et al: J Immunoloy. 1999; 163: 5686-5692.
2. Davies HR, Richeldi L. Idiopathic pulmonary fibrosis: current and future treatment options, Am-J-Respir-Med, 2002, 1(3): 211-224.
3. King JR, Ulrish C, Jean F. Idiopathic pulmonary fibrosis: diagnosis and treatment international consensus statement. Am J-Respir and Crit Care Med, 2000, 161: 651-660.
4．黎檀实，吴旭辉等．生长激素对大鼠肺泡Ⅱ型上皮细胞凋亡抑制作用的观察．中国危重病急救医学．2002，14(5)：290-293．
5．黎檀实，吴旭辉等．生长激素对酸吸入性肺损伤大鼠肺组织细胞凋亡的作用．中国危重病急救医学．2002，14(12)：728-731．
6．吴旭辉，黎檀实．生长激素对阿霉素中毒大鼠心肌细胞的抗凋亡作用．心肺血管病杂志．2003，22(4)：236-238．
7．黎檀实，尹明，吴旭辉等．生长激素对盐酸致大鼠肺泡Ⅱ型上皮细胞凋亡的保护．世界急危重病医学杂志．2004，1(2)：113-116．
8．李银平，黎檀实．胰岛素样生长因子-1对海水浸泡大鼠肺泡Ⅱ上皮细胞的抗凋亡作用．中华急诊医学杂志．2004，13(4)：248-250．
9．黎檀实，尹明等．生长激素在酸吸入性肺损伤大鼠肺组织中的修复作用．世界急危重病医学杂志．2005，2(2)：608-611．
10．黎檀实，吴旭辉等．重型颅脑损伤患者血浆肾上腺髓质素变化及临床意义．中国急救医学．2003，23(1)：30-31．
11. Rubin JS, Osada H, Finch DW, et al: Purfication and characterization of a newly identified growth factor specific for epithelial cell.[J] Proc Natl Acad Sci USA, 1989, 86: 802-806.
12. Yano T, Deterding RR, Simonet WS, Shannon JM, and Mason RJ. Keratinocyte growth factor reduces lung damage due to acid instillation in rats. Am J Respir Cell Mol Biol 15: 433-442, 1996.
13. Nemzek JA, Ebong SJ, Kim J, Bolgos GL, and Remick DG. Keratinocyte growth factor pretreatment is associated with decreased MIP-2_concentrations and reduced neutrophil recruitment in acid aspiration lung injury. Shock 18: 501-506, 2002.
14. Guo J, Yi ES, Havill AM, Sarosi I, Whitcomb L, Yin S, Middleton SC, Piguet P, and Ulich TR. Intravenous keratinocyte growth factor protects against experimental pulmonary injury. Am J Physiol Lung Cell Mol Physiol 275: L800-L805, 1998.
15. Sugahara K, Iyama KI, Kuroda MJ, and Sano K. Double intratracheal instillation of keratinocyte growth factor prevents bleomycin-induced lung fibrosis in rats. J Pathol 186: 90-98, 1998.
16. BarazzoneC, Donati YR, Rochat AF, VesinC, Kan CD, Pache JC, and Piguet PF. Keratinocyte growth factor protects alveolar epithelium and endothelium from oxygen-induced injury in mice. Am J Pathol 154: 1479-1487, 1999.
17.Viget N, Guery BP, Ader F, Alfandari S, Creuzy C, Roussel-Delvallez M, Foucher C, Mason CM, Beaucaire G, and Pittet JF. Keratinocyte growth factor protects against Pseudomonas aeruginosa-induced lung injury. Am J Physiol Lung Cell Mol Physiol 279: L1199-L1209, 2000.
18. Frank, -L: Protective effect of keratinocyte growth factor against lung abnormalities associated with hyperoxia in prematurely born rats. Biol-Neonate. 2003; 83(4): 263-72
19. Sugahara, -K; Tokumine,-J; Teruya,-K; et al: Alveolar epithelial cells: differentiation and lung injury. Respirology. 2006 Jan; 11 Suppl: S28-31
20.Chelly N, Henrion A, Pinteut C, et al: Role of keratinocyte growth factor in the control of surfactant synthesis by fetal lung mesenchyme. Endocrinology 2001;142 (5):1814
21.Matthay,-M-A; Fukuda,-N; Frank,-J; et al: Alveolar epithelial barrier. Role in lung fluid balance in clinical lung injury. Clin-Chest-Med. 2000 Sep; 21(3): 477-90
22. Prince L. S., Philip H. K., Thomas O.M. et al. KGF alters gene expression in human airway epithelia: potential regulation of the inflammatory response. Physiol Genomics 2001, 6: 81～89
23. Stern B, Fierobe L, Paugam C, et al. Keratinocyte growth factor and hepatocyte growth factor in bronchoalveolar lavage fluid in acute respiratory distress syndrome patients. CritCareMed 2000; 28(7): 2326
24. Panoskaltsis-Mortari A, Ingbar DH, Jung P, et al: KGF pretreatment decreases B7 and granzyme B expression and hastens repair in lungs of mice after allogeneic BMT. Am J Physiol Lung Cell Mol Physiol. 2000 May; 278(5): L988-99.
25. Haddad IY, Milla C, Yang S, et al: Surfactant protein A is a required mediator of keratinocyte growth factor after experimental marrow transplantation. Am J Physiol Lung Cell Mol Physiol. 2003 Sep; 285(3): L602-10. Epub 2003 May 9.
26．田桂珍、高占成、徐钰等．γ干扰素转基因表达对博来霉素致小鼠肺纤维化的作用．中华结核和呼吸杂志．2003，26(5)：290-293．
27. Ulrich,-K; Stern,-M; Goddard,-M-E; et al: Keratinocyte growth factor therapy in murine oleic acid-induced acute lung injury. Am-J-Physiol-Lung-Cell-Mol-Physiol. 2005 Jun; 288(6): L1179-92.
28．刘斌剑、王建民、陈林等：角质细胞生长因子受体腺病毒表达载体在脂多糖所致肺泡上皮细胞损伤修复中的应用．中华急诊医学杂志，2005．12，14(12)：1004-1006．
29. Vorburger SA, Hunt KK. Adenoviral gene therapy[J]. The Oncologist 2002, 7: 46-59.
30. Simonian PL, Roark CL, Oiaz del Valle F, et al: Regulatory role of gammadelta T cells in the recruitment of CD4+ and CD8+ T cells to lung and subsequent pulmonary fibrosis. J Immunol. 2006 Oct 1; 177(7): 4436-43.
31. Prince LS, Karp PH, Moninger TO, et al: KGF alters gene expression in human airway epithelia: potential regulation of the inflammatory response. Physiol Genomics. 2001 Jul 17; 6(2): 81-9.
32. Chen,-L; Brizel,-D-M; Rabbani,-Z-N; et al: The protective effect of recombinant human keratinocyte growth factor on radiation-induced pulmonary toxicity in rats. Int-J-Radiat-Oncol-Biol-Phys. 2004 Dec 1; 60(5): 1520-9
33. Marchand-Adam,-S; Plantier,-L; Bernuau,-D; et al: Keratinocyte growth factor expression by fibroblasts in pulmonary fibrosis: poor response to interleukin-1beta. Am-J-Respir-Cell-Mol-Biol. 2005 May; 32(5): 470-7
34. Upadhyay,-D; Panduri,-V; Kamp,-D-W: Fibroblast growth factor-10 prevents asbestos-induced alveolar epithelial cell apoptosis by a mitogen-activated protein kinase-dependent mechanism. Am-J-Respir-Cell-Mol-Biol. 2005 Mar; 32(3): 232-8
35. Pereira CT, Herndon DN, Rocker R, et al: Liposomal Gene Transfer of Keratinocyte Growth Factor Improves Wound Healing by Altering Growth Factor and Collagen Expression. J Surg Res. 2007 Feb 8; [Epub ahead of print]
36. A. Bergeronl, P. Solerl, M. Kambouchner2, et al: Cytokine profiles in idiopathic pulmonary fibrosis suggest an important role for TGF-beta and IL-10. Eur Respir J. 2003 Jul; 22(1): 69-76.
37. Kahoru Nishina, Feijie Zhang, Larry D. et al: Expression of CINC-2beta is related to the state of differentiation of alveolar epithelial cells. Am J Respir Cell Mol Biol. 2005 Nov; 33(5): 505-12. Epub 2005 Jul 29.
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