胶原凝胶及壳聚糖基因载体的制备
摘要
本文为载基因的壳聚糖胶原凝胶基因治疗提供了前期准备工作。
选用酶消化法,以牛腱为原料提取胶原,保证提取的胶原具有三股螺旋结构。并对提取的胶原进行的相应的表征工作。测定了胶原提取液的粘度和固体含量。研究了胃蛋白酶消化法中酶与材料的最佳配比为E：B=1：10。经红外、紫外光谱分析确定提取物为胶原。同时用提取的胶原制备胶原凝胶,测定胶原凝胶中固体含量。在胶原凝胶中培养细胞,用光学显微镜进行观察细胞生长状态,证明胶原凝胶可以作为细胞生长的立体三维生长支架。
另外,对天然的壳聚糖进行了一系列生物学表征工作,凝胶阻滞电泳实验证明壳聚糖具有一定的DNA结合能力。粒径电位分析表明壳聚糖与DNA形成了粒径在100-300nm,表面电位稳定的纳米复合物颗粒。MTT实验测试了壳聚糖的没有细胞毒性。证明了壳聚糖可以作为基因载体。
This paper provides prerequisite preparation for gene therapy of Collagen gel containing chitosan gene.
The method of enzyme digestion was used to extract collagen from bovine tendon so that triple helix of collagen molecules can be maintained. And the extracted collagen was characterized. The extracted collagen viscosity and solid content were measured. In pepsin digest method, the best ratio for enzymes and material is that E to B equals 1 to 10. The extracted protein was confirmed to be collagen by FTIR and UV. At the same time, collagen gel was prepared by the extracted collagen, and collagen gel solid content was measured. The cell was seeded in collagen gel. The cell growth state was observed by optical microscope and the results show that the collagen gel can be used as 3D cell scaffold.
In addition, a series of biology characterizations of natural chitosan were made. The results of gel retardation experiment indicated the chitosan had strong affinity to DNA. CS-DNA could form composite nano-particles with diameters from 100 to 300 nm and the surface potential is stable. The cytotoxicity was measured by MTT experiment that the results showed chitosan had no cytotoxicity and proved that chitosan can be used as gene carrier.
选用酶消化法,以牛腱为原料提取胶原,保证提取的胶原具有三股螺旋结构。并对提取的胶原进行的相应的表征工作。测定了胶原提取液的粘度和固体含量。研究了胃蛋白酶消化法中酶与材料的最佳配比为E：B=1：10。经红外、紫外光谱分析确定提取物为胶原。同时用提取的胶原制备胶原凝胶,测定胶原凝胶中固体含量。在胶原凝胶中培养细胞,用光学显微镜进行观察细胞生长状态,证明胶原凝胶可以作为细胞生长的立体三维生长支架。
另外,对天然的壳聚糖进行了一系列生物学表征工作,凝胶阻滞电泳实验证明壳聚糖具有一定的DNA结合能力。粒径电位分析表明壳聚糖与DNA形成了粒径在100-300nm,表面电位稳定的纳米复合物颗粒。MTT实验测试了壳聚糖的没有细胞毒性。证明了壳聚糖可以作为基因载体。
This paper provides prerequisite preparation for gene therapy of Collagen gel containing chitosan gene.
The method of enzyme digestion was used to extract collagen from bovine tendon so that triple helix of collagen molecules can be maintained. And the extracted collagen was characterized. The extracted collagen viscosity and solid content were measured. In pepsin digest method, the best ratio for enzymes and material is that E to B equals 1 to 10. The extracted protein was confirmed to be collagen by FTIR and UV. At the same time, collagen gel was prepared by the extracted collagen, and collagen gel solid content was measured. The cell was seeded in collagen gel. The cell growth state was observed by optical microscope and the results show that the collagen gel can be used as 3D cell scaffold.
In addition, a series of biology characterizations of natural chitosan were made. The results of gel retardation experiment indicated the chitosan had strong affinity to DNA. CS-DNA could form composite nano-particles with diameters from 100 to 300 nm and the surface potential is stable. The cytotoxicity was measured by MTT experiment that the results showed chitosan had no cytotoxicity and proved that chitosan can be used as gene carrier.
引文
[1]景沛.胶原蛋白质(Ⅰ-ⅩⅧ)[J].生命化学,1995,15(6)：30-31
[2]李国英.胶原的类型及其结构特征[J].中国皮革,2002,31(17)：20-21
[3]王一方.思想史视野中的DNA双螺旋发现[M].华夏出版社,2003,24(7)：17-18
[4]K. Gelse, E. Poschl, T. Aigner. Collagens-structure, function, and biosynthesis[J]. Advanced Drug Delivery Re views,2003,55(12):1531-1546
[5]W. Miltyk, J. A. Palka. Potential role of pyrroline 5-carboxylate in regulation of collagen biosynthesis in cultured human skin fibroblasts[J]. Comparative Biochemistry and Physiology Part A,2000,125(2):265-271
[6]M. E. Nimni, R. D. Harkness. Molecular Structure and Functions of Collagen[J]. Collagen Chemistry,1988:1-77
[7]D. Chan, S. R. Lamande, D. J. McQuillan, et al. In vitro expression analysis of collagen biosynthesis and ass essmbly[J]. Journal of Biochemical and Biophysical Methods,1997,36(1):11-29
[8]施辉阳.酶法提取生猪皮胶原的研究[D].北京化工大学.2004
[9]吴志谷,盛志勇,孙国柱.几种胶原型创伤敷料制作的实验研究[J].生物医学工程学杂志,1999,16(2)：147-150
[10]Davison PF. Collagen extracted from animal collagen-containing tissue[P]. US4983721-A,1991-05-20
[11]J. H. Muyongaa, C. G. B. Cole, K. G. Duodu. Characterisation of acid soluble collagen from skins of young and adult Nile perch(Lates niloticus)[J]. Food Chemistry,2004,85(1):81-89
[12]Maki Nishimoto, Shoshi Mizuta, Reiji Yoshinaka. Characterization of molecular species of collagen in muscles of Japanese amberjack, Seriola quinqueradiata[J]. Food Chemistry,2004,84(1):127-132
[13]Nagai, T. Suzuki, N. Partial characterization of collagen from purple sea urchin(Anthocidaris crassispina)test [J]. Inter-national Journal of Food Science and Technology,2000,35(5):497-501
[14]鲍士宝,王璋,许时婴.鲴鱼鱼皮胶原的提取新工艺研究[J].食品科学,2009,30(10)：101-105
[15]张艳,李国英,缪煜清,陈建荣.草鱼皮胶原的提取及性能的初步研究[J].水产科学,2006,25(3)：136-139
[16]赵帅,李国英.猪皮胶原的研究进展[J].皮革化工,2006,23(2)：19-23
[17]韩德权,金明姬,吴桐,曹相辉.猪皮胶原粉的制备工艺及功能特性研究[J].食品科学,2009,30(10)：60-64
[18]高杨,董秀萍,肖桂华等.海参体壁酸溶性胶原提取及氨基酸组成分析[J].食品与发酵工业,2008,34(11)：171-174
[19]Mario HUM, Joe LAM, et al. Jumbo squid(Dosidicus gigas)mantle collagen:Extrcation, characterization, and potential application in the preparation of chitosan-collagen biofilms[J]. Bioresource Technology,2010,101(11):4212-4219
[20]Roreger, Michael.Collagen preparation for the controlled release of active substances[P]. USA:US 6761908, 2004-07-13
[21]李贺,郑庚修,王秋芬,董海军,刘白玲.生物医学材料胶原蛋白的研究进展(Ⅰ)—胶原的分离纯化及交联[J].中国皮革,2005,34(11)：24-27
[22]刘白玲.生物酶法洁净化生产明胶研究[C].2000年亚洲国际皮革科学与技术研讨会论文集.2000：62-70
[23]张文熊.李欣,魏娜,马明霞.酶法提取胶原的研究[J].中国皮革,2006,35(23)：15-17
[24]朱梅湘,皮胶原的提取及胶原生物膜材料的研究[D].四川大学.2003
[25]周文常,但卫华,廖隆理等.猪皮胶原的提取及其结构表征[J].中国皮革,2004,33(13)：36-38
[26]Deyl, Z., Mikik, I., Eckhardt, A. Preparative procedures and purity assessment of collagen proteins[J]. Journal of Chro-matography B-Analytical Technologies in the Biomedical and Life Sciences,2003,790(1-2):245-275
[27]李建武,余瑞元,袁明秀等.生物化学实验原理和方法[M].北京大学出版社.1994.16-82
[28]Florin A, Shuichi M, Karim E, et al. Effects of hyaluronan on engineered articular cartilage extracelluar matrix gene expression in 3-dimensional collage scaffolds[J]. Journal of Biomedical Materials Research,2001,55(1):13-19
[29]胡学庆,韩春茂,岑航辉等.胶原-壳聚糖人工真皮的研制[A].第二届全国组织工程学术大会论文汇编[C],2002.39-241
[30]马列,高长友,毛峰伟等.胶原/壳聚糖真皮支架的制备与性能研究[A].第三界全国组织工程学术会议论文汇编[C],2002.220-222
[31]Tan W. Krishnaraj R, Desai TA. Evalution of nanostructured composite-chi-tosan matrices for tissue engineering[J]. Tissue Eng,2001.7(2):203-210
[32]杨洪义,马东瑞,张其清等.胶原-粘多糖复合组织工程支架的制备及性能研究[A].第二届全国组织工程学术会议论文汇编[C],2000.271-272
[33]吴志谷,盛志勇,孙同栓等.几种胶原型创伤敷料制作的实验研究[J].生物医学工程学杂志,1999,16(2)：147-150
[34]Pieper JS, Van der Kran PM, Hafmans T, et al. Crosslinked type Ⅱ collagen matrices:preparation, characterization, and potential for cartilage engineering[J]. Biomaterials,2002,23(15):3183-3192
[35]Ithoh S, Kikuchi M, Koyama Y, Takakuda K, Tanaka J. Development of an artificial vertebral boby using a novel biomaterial, hydroxyapatite/collge composite[J]. Biomaterials,2002,23(19):3919-3926
[36]马佳,赵铱民,王晓红等.胶原凝胶复合快速成型材料异位成软骨的研究[J].实用口腔医学杂志,2009,25(1)：9-12
[37]李忠,杨柳,何创龙,陈光兴等.胶原凝胶复合骨胶原基质(BCM)负载关节软骨细胞的实验研究[J].中国骨与关节损伤杂志,2005,20(10)：685-687
[38]杨小红,李斯明,戴丽冰等.胶原凝胶复合物治疗褥疮的临床研究[J].创伤外科杂志,2000,2(4)：219-221
[39]Graham F L, Prevec L.Methods for construction of adenovirus vectors[J]. Molecular Biotechnology,1995,3(3): 207-220
[40]张继文,杨桂连,王春凤.逆转录病毒载体的应用现状及前景展望[J].生物技术,2010,20(4)：89-92
[41]吴乃虎.基因工程原理[M].北京：北京科学出版社.2001.338-342
[42]Romano G. Mmichel P, Pacilio C, Giordano A. Latest developments in gene transfer technology:Achievemen ts, pe-rspectives, and controversies over therapeutic applications[J]. Stem Cells,2000,18(1):19-39
[43]Romano G, Pacilio C, Giordano A. Gene transfer technology in therapy:Current applications and future goals[J]. Stem Cells,1999,17(4):191-202
[44]Lundstrom K. Latest development in viral vectors for gene therapy[J]. Trends in Biotechnology,2003,21(3):117-122
[45]Neumann J, Stitz J, Konig R, et al. Retroviral vectors for vaccine development:induction of HIV-1-specific humor-al and cellular immune responses in rhesus macaques using a novel MLV(HIV-])pseud-otype vector[J]. Jour nal of Biotechnology,2006,124(3):615-625
[46]范凌云,谢庆军.腺病毒载体的研究进展[J].中国生物学制品杂志.2008,21(2)：153-157
[47]Engelhardt JF,Ye X,Doranz B.et al.Ablation of E2A in recombinant adenoviruses improves transgene persistence and decreases inflammatory response in mouse liver[J]. Proc Nat 1 Acad Sci USA,1994,91(13):6196-6200
[48]王玉泉,马立宾,于秋莲.动物腺病毒载体的研究进展[J].内蒙古民族大学学报.2009,24(2)：180-183
[49]姚二梅,冯泽华,侯云德.腺病毒伴随病毒载体的研究进展[J].中华实验和临床病毒学杂志,1997,11(1)：97-100
[50]Laughlin C A, Trat schin J D, Coon H, et al. Cloning of in fectious adeno-associated virus genomes in bacterial plasmids[J]. Gene,1983,23(1):65-73
[51]Samulski R J, Berns K I, Tan M, et al. Cloning of Adenoass ociated Virus into pBR322-Rescue of intact virus from the recombinant plasmid in human cells[J], Proceedings of the National Academy of Sciences of the United States of America-Biological Sciences,1982,79(6):2077-2081
[52]Srivastava A, Lusby E W, Berns K I. Nucleotide sequence and organization of the adeno-associated virus-2 genome[J]. Journal of Virology,1983,45(2):555-564
[53]Hermonat P L, Muzyczka N. Use of Adeno-associated Virus as a mammalian DNA cloning vector-T ransduction of neomycin resistance into mammalian tissue culture cells[J]. Proceedings of the National Academy of Sciences of the United States of America-Biological Sciences,1984,81(20):6466-6470
[54]Tratschin J D, Miller I L, Smith MG, et al. Adeno-associated virus vector for high-frequency integration, expression and rescue of genes in mammalian cells[J]. Molecular and Cellular Biology,1985,5(11):3251-3260
[55]Pitard B, Aguerre O, Airiau M, et al. Virus-sized self-assembling lamellar complexes between plasmid DNA and cationic micelles promote gene transfer[J]. Proceedings of the National Academy of Sciences of the United States of America-Biological Sciences,1997,94(26):14412-14417
[56]Templeton NS, Lasic DD, Frederik PM, et al. Improved DNA:Liposome complexes for increased systemic delivery and gene expression[J]. Narure Biotechnology,1997,15(7):647-652
[57]Radler JO, Koltover I, Salditt T, Safinya CR. Structure of DNA-cationic liposome complexes:DNA intercalation in multilamellar membranes in distinct interhelical packing regimes[J]. Science,1997,275(5301):810-814
[58]Schwartz B, Ivanov MA, Pitard B, et al. Synthetic DNA-compacting peptides derived from human sequence enhance cationic lipid-mediated gene transfer in vitro and in vivo[J]. Gene Therapy,1999,6(2):282-292
[59]Zhang G F, Vargo D, Budker V, et al. Expression of naked plasmid DNA injected into the afferent and efferent vessels of rodent and dog livers[J]. Human Gene Therapy,1997.8(15):1763-1772
[60]Choate K A. Khavari P A, Direct cutaneous gene delivery in a human genetic skin disease[J]. Human Gene Therapy. 1997,8(14):1659-1665
[61]Liu F, Song Y K, Liu D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA[J]. Gene Therapy,1999,6(7):1258-1266
[62]Trollet C, Benstead J, Bales O, et al.Gene therapy strategy for occulopharyngeal muscular dystrophy (OPMD)[J]. Human Gene Therapy,2008,19(10):1080-1081
[63]Miyazaki S, Miyazaki.J I. In vivo DNA electrotransfer into muscle[J]. Development Growth & Differentiatio n,2008,50(6):479-483
[64]付爱玲,裸DNA治疗研究[J].生命的化学,2009,29(3)：327-329
[65]赵亮,苏畅,马洪林.阳离子聚合物基因载体的理论研究[J].中国组织工程研究与临床康复,2009,13(42)：8329-8332
[66]杨洪,魏婉,田月等.壳聚糖及其衍生物作为基因载体的研究进展[J].医药导报,2008,27(10)：1229-1231
[67]安江洪,敖绪军,钱莘等.壳聚糖纳米颗粒作为基因载体的初步研究[J].医学研究生学报,2008,21(8)：790-794
[68]魏涛,唐粉芳,高兆兰,王卫平,金宗镰.壳聚糖降血脂、降血糖及增强免疫作用的研究[J].食品工程,2001,2(4)：48-52
[69]马宁,汪琴,孙胜玲,王爱勤.甲壳素和壳聚糖化学改性研究进展[J].化学进展,2006,16(4)：643-653
[70]姬振行,安静,王德松,罗青枝.壳聚糖微球的制备与应用[J].材料导报,2010,2(7)：128-132
[71]丁明,苏丹.低聚壳聚糖制备及产物的膜分离技术研究[J].合肥学院学报,2009,19(3)：63-67
[72]AI-Helw AA, AI-Angary AA. Mahous GM, et al. Preparation and evaluation of Sustained release cross-linked chitosan microspheres containing phenobarbitone[J]. Joernal of Microencapsulation,1998,15(3):373-382
[73]Ohya Y, Shiratani M, Kobayashi H, et al. Release behavior of 5-fluorouraeil from chitosan-gel nanospheres immobilizings 5-fluorouracil coated with Polysaceharides and their cell specific cytotoxieity[J]. Journal of Macromolecular Science-Pure and Applied Chemistry,1994, A31(5):629-642
[74]Hejazi R, Amiji M.Stomaeh-specific anti-H-pylori therapy Part 111:Effeet of ehitosan mierospheres crosslinkin g on the gastric residence and locale tracycline concentrations in fasted gerbils[J]. International Journal of Pharma ceutics,2004,272(1-2):99-108
[75]张祖菲,周建平,霍美蓉.壳聚糖微球给药系统[J].药学进展,2006,30(6)：261-266
[76]韩永萍,林强.低聚壳聚糖制备及其生理活性进展[J].化学工业与工程,2007,24(3)：272-276
[77]段杉,彭喜春,彭志英.低聚壳聚糖的制备及应用[J].中国食品添加剂,2002,5：66-71
[78]罗平,何波兵.蔺显俊等.水溶性低分子壳聚糖的制备[J].化学研究与应用,2000,12(6)：677-679
[79]Chen RH, Hwa HD. Effect of molecular weight of chitosan with the same degree of deacetylation on the th ermal, mechanical, and permeability properties of the prepared membrane[J]. Carbohydrate Polymers,1996(26): 353-358
[80]陈春刚,韩芬霞.不同因素影响壳聚糖降解速度的研究[J].安徽农业科学,2006,34(60)：1052-1056
[81]李治,刘晓非,杨冬芝,管云林等.壳聚糖降解研究进展[J].化工进展,2000,6：20-23
[82]KIM TH, IHM J E, CHOI Y J, et al. Efficient gene delivery by urocanic acid-modified chitosan[J]. Controlled Release, 2003,93(3):389-402
[83]Mumper RJ, Wang JM, et al. Novel polymeric condensing carriers for gene delivery[J]. Controlled Rel. Bioact.Mater, 1995,22(2):178-179
[84]李夷平,姚静,周建平等.尿刊酸偶联壳聚糖基因载体的合成和表征[J].药学学报,2008,43(12)：1233-1238
[85]杨洪,魏婉,田月,向明.壳聚糖及其衍生物作为基因载体的研究进展[J].医药导报,2008,27(10)：1229-1231
[86]Wei XH, Liang WQ, Pan YJ. Preparation and characterization of PEGylated chitosan/DNA self-assemble co mplex and the research on transfection on Hela cell in vitro[J]. Chemical Journal of Chinese Universities-Chinese, 2003,24(11):1993-1996
[87]林炜,穆畅道,朱梅湘.利用皮边角料提取食品级胶原蛋白/多肽(Ⅰ)—氨基酸组成分析和分子量测定[J].中国皮革,2003,32(17)：5-8
[2]李国英.胶原的类型及其结构特征[J].中国皮革,2002,31(17)：20-21
[3]王一方.思想史视野中的DNA双螺旋发现[M].华夏出版社,2003,24(7)：17-18
[4]K. Gelse, E. Poschl, T. Aigner. Collagens-structure, function, and biosynthesis[J]. Advanced Drug Delivery Re views,2003,55(12):1531-1546
[5]W. Miltyk, J. A. Palka. Potential role of pyrroline 5-carboxylate in regulation of collagen biosynthesis in cultured human skin fibroblasts[J]. Comparative Biochemistry and Physiology Part A,2000,125(2):265-271
[6]M. E. Nimni, R. D. Harkness. Molecular Structure and Functions of Collagen[J]. Collagen Chemistry,1988:1-77
[7]D. Chan, S. R. Lamande, D. J. McQuillan, et al. In vitro expression analysis of collagen biosynthesis and ass essmbly[J]. Journal of Biochemical and Biophysical Methods,1997,36(1):11-29
[8]施辉阳.酶法提取生猪皮胶原的研究[D].北京化工大学.2004
[9]吴志谷,盛志勇,孙国柱.几种胶原型创伤敷料制作的实验研究[J].生物医学工程学杂志,1999,16(2)：147-150
[10]Davison PF. Collagen extracted from animal collagen-containing tissue[P]. US4983721-A,1991-05-20
[11]J. H. Muyongaa, C. G. B. Cole, K. G. Duodu. Characterisation of acid soluble collagen from skins of young and adult Nile perch(Lates niloticus)[J]. Food Chemistry,2004,85(1):81-89
[12]Maki Nishimoto, Shoshi Mizuta, Reiji Yoshinaka. Characterization of molecular species of collagen in muscles of Japanese amberjack, Seriola quinqueradiata[J]. Food Chemistry,2004,84(1):127-132
[13]Nagai, T. Suzuki, N. Partial characterization of collagen from purple sea urchin(Anthocidaris crassispina)test [J]. Inter-national Journal of Food Science and Technology,2000,35(5):497-501
[14]鲍士宝,王璋,许时婴.鲴鱼鱼皮胶原的提取新工艺研究[J].食品科学,2009,30(10)：101-105
[15]张艳,李国英,缪煜清,陈建荣.草鱼皮胶原的提取及性能的初步研究[J].水产科学,2006,25(3)：136-139
[16]赵帅,李国英.猪皮胶原的研究进展[J].皮革化工,2006,23(2)：19-23
[17]韩德权,金明姬,吴桐,曹相辉.猪皮胶原粉的制备工艺及功能特性研究[J].食品科学,2009,30(10)：60-64
[18]高杨,董秀萍,肖桂华等.海参体壁酸溶性胶原提取及氨基酸组成分析[J].食品与发酵工业,2008,34(11)：171-174
[19]Mario HUM, Joe LAM, et al. Jumbo squid(Dosidicus gigas)mantle collagen:Extrcation, characterization, and potential application in the preparation of chitosan-collagen biofilms[J]. Bioresource Technology,2010,101(11):4212-4219
[20]Roreger, Michael.Collagen preparation for the controlled release of active substances[P]. USA:US 6761908, 2004-07-13
[21]李贺,郑庚修,王秋芬,董海军,刘白玲.生物医学材料胶原蛋白的研究进展(Ⅰ)—胶原的分离纯化及交联[J].中国皮革,2005,34(11)：24-27
[22]刘白玲.生物酶法洁净化生产明胶研究[C].2000年亚洲国际皮革科学与技术研讨会论文集.2000：62-70
[23]张文熊.李欣,魏娜,马明霞.酶法提取胶原的研究[J].中国皮革,2006,35(23)：15-17
[24]朱梅湘,皮胶原的提取及胶原生物膜材料的研究[D].四川大学.2003
[25]周文常,但卫华,廖隆理等.猪皮胶原的提取及其结构表征[J].中国皮革,2004,33(13)：36-38
[26]Deyl, Z., Mikik, I., Eckhardt, A. Preparative procedures and purity assessment of collagen proteins[J]. Journal of Chro-matography B-Analytical Technologies in the Biomedical and Life Sciences,2003,790(1-2):245-275
[27]李建武,余瑞元,袁明秀等.生物化学实验原理和方法[M].北京大学出版社.1994.16-82
[28]Florin A, Shuichi M, Karim E, et al. Effects of hyaluronan on engineered articular cartilage extracelluar matrix gene expression in 3-dimensional collage scaffolds[J]. Journal of Biomedical Materials Research,2001,55(1):13-19
[29]胡学庆,韩春茂,岑航辉等.胶原-壳聚糖人工真皮的研制[A].第二届全国组织工程学术大会论文汇编[C],2002.39-241
[30]马列,高长友,毛峰伟等.胶原/壳聚糖真皮支架的制备与性能研究[A].第三界全国组织工程学术会议论文汇编[C],2002.220-222
[31]Tan W. Krishnaraj R, Desai TA. Evalution of nanostructured composite-chi-tosan matrices for tissue engineering[J]. Tissue Eng,2001.7(2):203-210
[32]杨洪义,马东瑞,张其清等.胶原-粘多糖复合组织工程支架的制备及性能研究[A].第二届全国组织工程学术会议论文汇编[C],2000.271-272
[33]吴志谷,盛志勇,孙同栓等.几种胶原型创伤敷料制作的实验研究[J].生物医学工程学杂志,1999,16(2)：147-150
[34]Pieper JS, Van der Kran PM, Hafmans T, et al. Crosslinked type Ⅱ collagen matrices:preparation, characterization, and potential for cartilage engineering[J]. Biomaterials,2002,23(15):3183-3192
[35]Ithoh S, Kikuchi M, Koyama Y, Takakuda K, Tanaka J. Development of an artificial vertebral boby using a novel biomaterial, hydroxyapatite/collge composite[J]. Biomaterials,2002,23(19):3919-3926
[36]马佳,赵铱民,王晓红等.胶原凝胶复合快速成型材料异位成软骨的研究[J].实用口腔医学杂志,2009,25(1)：9-12
[37]李忠,杨柳,何创龙,陈光兴等.胶原凝胶复合骨胶原基质(BCM)负载关节软骨细胞的实验研究[J].中国骨与关节损伤杂志,2005,20(10)：685-687
[38]杨小红,李斯明,戴丽冰等.胶原凝胶复合物治疗褥疮的临床研究[J].创伤外科杂志,2000,2(4)：219-221
[39]Graham F L, Prevec L.Methods for construction of adenovirus vectors[J]. Molecular Biotechnology,1995,3(3): 207-220
[40]张继文,杨桂连,王春凤.逆转录病毒载体的应用现状及前景展望[J].生物技术,2010,20(4)：89-92
[41]吴乃虎.基因工程原理[M].北京：北京科学出版社.2001.338-342
[42]Romano G. Mmichel P, Pacilio C, Giordano A. Latest developments in gene transfer technology:Achievemen ts, pe-rspectives, and controversies over therapeutic applications[J]. Stem Cells,2000,18(1):19-39
[43]Romano G, Pacilio C, Giordano A. Gene transfer technology in therapy:Current applications and future goals[J]. Stem Cells,1999,17(4):191-202
[44]Lundstrom K. Latest development in viral vectors for gene therapy[J]. Trends in Biotechnology,2003,21(3):117-122
[45]Neumann J, Stitz J, Konig R, et al. Retroviral vectors for vaccine development:induction of HIV-1-specific humor-al and cellular immune responses in rhesus macaques using a novel MLV(HIV-])pseud-otype vector[J]. Jour nal of Biotechnology,2006,124(3):615-625
[46]范凌云,谢庆军.腺病毒载体的研究进展[J].中国生物学制品杂志.2008,21(2)：153-157
[47]Engelhardt JF,Ye X,Doranz B.et al.Ablation of E2A in recombinant adenoviruses improves transgene persistence and decreases inflammatory response in mouse liver[J]. Proc Nat 1 Acad Sci USA,1994,91(13):6196-6200
[48]王玉泉,马立宾,于秋莲.动物腺病毒载体的研究进展[J].内蒙古民族大学学报.2009,24(2)：180-183
[49]姚二梅,冯泽华,侯云德.腺病毒伴随病毒载体的研究进展[J].中华实验和临床病毒学杂志,1997,11(1)：97-100
[50]Laughlin C A, Trat schin J D, Coon H, et al. Cloning of in fectious adeno-associated virus genomes in bacterial plasmids[J]. Gene,1983,23(1):65-73
[51]Samulski R J, Berns K I, Tan M, et al. Cloning of Adenoass ociated Virus into pBR322-Rescue of intact virus from the recombinant plasmid in human cells[J], Proceedings of the National Academy of Sciences of the United States of America-Biological Sciences,1982,79(6):2077-2081
[52]Srivastava A, Lusby E W, Berns K I. Nucleotide sequence and organization of the adeno-associated virus-2 genome[J]. Journal of Virology,1983,45(2):555-564
[53]Hermonat P L, Muzyczka N. Use of Adeno-associated Virus as a mammalian DNA cloning vector-T ransduction of neomycin resistance into mammalian tissue culture cells[J]. Proceedings of the National Academy of Sciences of the United States of America-Biological Sciences,1984,81(20):6466-6470
[54]Tratschin J D, Miller I L, Smith MG, et al. Adeno-associated virus vector for high-frequency integration, expression and rescue of genes in mammalian cells[J]. Molecular and Cellular Biology,1985,5(11):3251-3260
[55]Pitard B, Aguerre O, Airiau M, et al. Virus-sized self-assembling lamellar complexes between plasmid DNA and cationic micelles promote gene transfer[J]. Proceedings of the National Academy of Sciences of the United States of America-Biological Sciences,1997,94(26):14412-14417
[56]Templeton NS, Lasic DD, Frederik PM, et al. Improved DNA:Liposome complexes for increased systemic delivery and gene expression[J]. Narure Biotechnology,1997,15(7):647-652
[57]Radler JO, Koltover I, Salditt T, Safinya CR. Structure of DNA-cationic liposome complexes:DNA intercalation in multilamellar membranes in distinct interhelical packing regimes[J]. Science,1997,275(5301):810-814
[58]Schwartz B, Ivanov MA, Pitard B, et al. Synthetic DNA-compacting peptides derived from human sequence enhance cationic lipid-mediated gene transfer in vitro and in vivo[J]. Gene Therapy,1999,6(2):282-292
[59]Zhang G F, Vargo D, Budker V, et al. Expression of naked plasmid DNA injected into the afferent and efferent vessels of rodent and dog livers[J]. Human Gene Therapy,1997.8(15):1763-1772
[60]Choate K A. Khavari P A, Direct cutaneous gene delivery in a human genetic skin disease[J]. Human Gene Therapy. 1997,8(14):1659-1665
[61]Liu F, Song Y K, Liu D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA[J]. Gene Therapy,1999,6(7):1258-1266
[62]Trollet C, Benstead J, Bales O, et al.Gene therapy strategy for occulopharyngeal muscular dystrophy (OPMD)[J]. Human Gene Therapy,2008,19(10):1080-1081
[63]Miyazaki S, Miyazaki.J I. In vivo DNA electrotransfer into muscle[J]. Development Growth & Differentiatio n,2008,50(6):479-483
[64]付爱玲,裸DNA治疗研究[J].生命的化学,2009,29(3)：327-329
[65]赵亮,苏畅,马洪林.阳离子聚合物基因载体的理论研究[J].中国组织工程研究与临床康复,2009,13(42)：8329-8332
[66]杨洪,魏婉,田月等.壳聚糖及其衍生物作为基因载体的研究进展[J].医药导报,2008,27(10)：1229-1231
[67]安江洪,敖绪军,钱莘等.壳聚糖纳米颗粒作为基因载体的初步研究[J].医学研究生学报,2008,21(8)：790-794
[68]魏涛,唐粉芳,高兆兰,王卫平,金宗镰.壳聚糖降血脂、降血糖及增强免疫作用的研究[J].食品工程,2001,2(4)：48-52
[69]马宁,汪琴,孙胜玲,王爱勤.甲壳素和壳聚糖化学改性研究进展[J].化学进展,2006,16(4)：643-653
[70]姬振行,安静,王德松,罗青枝.壳聚糖微球的制备与应用[J].材料导报,2010,2(7)：128-132
[71]丁明,苏丹.低聚壳聚糖制备及产物的膜分离技术研究[J].合肥学院学报,2009,19(3)：63-67
[72]AI-Helw AA, AI-Angary AA. Mahous GM, et al. Preparation and evaluation of Sustained release cross-linked chitosan microspheres containing phenobarbitone[J]. Joernal of Microencapsulation,1998,15(3):373-382
[73]Ohya Y, Shiratani M, Kobayashi H, et al. Release behavior of 5-fluorouraeil from chitosan-gel nanospheres immobilizings 5-fluorouracil coated with Polysaceharides and their cell specific cytotoxieity[J]. Journal of Macromolecular Science-Pure and Applied Chemistry,1994, A31(5):629-642
[74]Hejazi R, Amiji M.Stomaeh-specific anti-H-pylori therapy Part 111:Effeet of ehitosan mierospheres crosslinkin g on the gastric residence and locale tracycline concentrations in fasted gerbils[J]. International Journal of Pharma ceutics,2004,272(1-2):99-108
[75]张祖菲,周建平,霍美蓉.壳聚糖微球给药系统[J].药学进展,2006,30(6)：261-266
[76]韩永萍,林强.低聚壳聚糖制备及其生理活性进展[J].化学工业与工程,2007,24(3)：272-276
[77]段杉,彭喜春,彭志英.低聚壳聚糖的制备及应用[J].中国食品添加剂,2002,5：66-71
[78]罗平,何波兵.蔺显俊等.水溶性低分子壳聚糖的制备[J].化学研究与应用,2000,12(6)：677-679
[79]Chen RH, Hwa HD. Effect of molecular weight of chitosan with the same degree of deacetylation on the th ermal, mechanical, and permeability properties of the prepared membrane[J]. Carbohydrate Polymers,1996(26): 353-358
[80]陈春刚,韩芬霞.不同因素影响壳聚糖降解速度的研究[J].安徽农业科学,2006,34(60)：1052-1056
[81]李治,刘晓非,杨冬芝,管云林等.壳聚糖降解研究进展[J].化工进展,2000,6：20-23
[82]KIM TH, IHM J E, CHOI Y J, et al. Efficient gene delivery by urocanic acid-modified chitosan[J]. Controlled Release, 2003,93(3):389-402
[83]Mumper RJ, Wang JM, et al. Novel polymeric condensing carriers for gene delivery[J]. Controlled Rel. Bioact.Mater, 1995,22(2):178-179
[84]李夷平,姚静,周建平等.尿刊酸偶联壳聚糖基因载体的合成和表征[J].药学学报,2008,43(12)：1233-1238
[85]杨洪,魏婉,田月,向明.壳聚糖及其衍生物作为基因载体的研究进展[J].医药导报,2008,27(10)：1229-1231
[86]Wei XH, Liang WQ, Pan YJ. Preparation and characterization of PEGylated chitosan/DNA self-assemble co mplex and the research on transfection on Hela cell in vitro[J]. Chemical Journal of Chinese Universities-Chinese, 2003,24(11):1993-1996
[87]林炜,穆畅道,朱梅湘.利用皮边角料提取食品级胶原蛋白/多肽(Ⅰ)—氨基酸组成分析和分子量测定[J].中国皮革,2003,32(17)：5-8