巯基烷基化壳聚糖介导共表达质粒pIRES-hVEGF_（121）cDNA/hBMP4体外转染的研究
|
摘要
目的:以新型巯基烷基化壳聚糖(TACS)为载体介导重组共表达质粒pIRES-hVEGFl2lcDNA/hBMP4体外转染大鼠骨髓间充质干细胞(MSCs),探讨TACS用作骨组织工程中基因载体的可行性,为进一步研究骨缺损的联合基因治疗提供实验基础。
方法:全骨髓培养法分离、培养大鼠MSCs ;真核共表达质粒pIRES-hVEGFl2lcDNA/hBMP4经酶切鉴定后,复凝聚法制备TACS-基因纳米粒,用透射电镜对其形态和粒径进行观察和表征,凝胶阻滞分析其对基因的保护情况;将纳米粒转染第3代大鼠MSCs,并设壳聚糖组、脂质体组、及裸质粒组分别为实验对照、阳性对照及阴性对照,噻唑蓝(MTT)法比较不同基因载体细胞毒性的差异;分别于转染后3,4d提取MSCs的总RNA、总蛋白,逆转录-聚合酶链反应(RT-PCR)、Western blot检测目的基因的表达情况。
结果:全骨髓培养法可获得大量MSCs并具有活跃增殖能力;共表达质粒经酶切鉴定,与预期结果相符;TACS/pDNA复合纳米粒形态不很均一,平均粒径约264nm;凝胶阻滞分析证明TACS能有效地包裹和保护共表达质粒不受DNaseⅠ酶的消化;对转染后各组MSCs相关指标检测显示,TACS组细胞存活率(73.18±6.56)%,明显高于脂质体组(45.92±4.93)%(P<0.01);除阴性对照组外, RT-PCR和Western blot均检测到转染后MSCs中hVEGF121及hBMP4的表达,对Western blot所得条带的灰度值相对半定量分析表明,TACS组目的蛋白表达量低于脂质体组(P<0.05),但明显高于壳聚糖组(P<0.01)。
结论:共表达质粒pIRES-hVEGFl2lcDNA/hBMP4在TACS介导下成功转染大鼠MSCs并获得表达。TACS细胞毒性小,且较未改性壳聚糖转染效率有明显提高。
Objective Recombinant coexpression plasmid of pIRES-hVEGFl2lcDNA/hBMP4 was transfected into rat bone marrow mesenchymal stem cells(MSCs) mediated by thiolated N-alkylated chitosan(TACS).To investigate the feasibility of using TACS as a gene vector in bone tissue engineering and build an experimental base on conbination gene therapy of bone defects.
Methods MSCs were isolated and cultivated from the bone marrow of rat. Recombinant coexpression plasmid of pIRES-hVEGFl2lcDNA/hBMP4 was confirmed by restriction enzymolysis,then TACS-pDNA nanoparticles were prepared by complex coacervation.The morphology and particle size of nanoparticles were observed by transmission electronic microscopy and nanoparticle size analyzer. The protection of the nanoparticles for pDNA was observed by gel electrophoresis.TACS bearing pIRES-hVEGFl2lcDNA/hBMP4 was transfected into the third generation of MSCs.Meanwhile,Chitosan group, liposome group and naked pDNA group was respectively experimental control,positive control and negative control.Different effects of the nanoparticles on cell viability were illustrated by MTT assay.After 3 days and 4 days of transfection, the total RNA and total protein were extracted from MSCs respectively.The target gene expressions were detected by RT-PCR and Western blot .
Results Lots of MSCs were separated from bone marrow of rat and the cells showed active proliferative ability.We got expected results on identification of pIRES-hVEGFl2lcDNA/hBMP4 by restriction enzymolysis.The shape of nanoparticles was not very homogeneous and the average particle size was about 264nm determined by nanoparticle size analyzer. TACS could protect encapsulated pDNA effectively from nuclease degradation as shown by electrophoretic mobility analysis.The related indexes of MSCs were inspected after transfection,and we found that cell viability of TACS group(73.18±6.56)% was significantly higher than liposome group(45.92±4.93)%(P<0.01).HVEGF121 and hBMP4 were expressed well in transfected MSCs detected by RT-PCR and Western blot except the negative control.We also conducted relative semi-quantitative analysis on the grey value of Western blot strips and got the result that the target protein expressions of TACS group were lower than liposome group(P<0.05) but significantly higher than chitosan group(P<0.01).
Conclusion The coexpression plasmid was transfected into MSCs successfully by TACS. TACS has low cytotoxicity and its transfection efficiency is obviously higher than non-modified chitosan.
方法:全骨髓培养法分离、培养大鼠MSCs ;真核共表达质粒pIRES-hVEGFl2lcDNA/hBMP4经酶切鉴定后,复凝聚法制备TACS-基因纳米粒,用透射电镜对其形态和粒径进行观察和表征,凝胶阻滞分析其对基因的保护情况;将纳米粒转染第3代大鼠MSCs,并设壳聚糖组、脂质体组、及裸质粒组分别为实验对照、阳性对照及阴性对照,噻唑蓝(MTT)法比较不同基因载体细胞毒性的差异;分别于转染后3,4d提取MSCs的总RNA、总蛋白,逆转录-聚合酶链反应(RT-PCR)、Western blot检测目的基因的表达情况。
结果:全骨髓培养法可获得大量MSCs并具有活跃增殖能力;共表达质粒经酶切鉴定,与预期结果相符;TACS/pDNA复合纳米粒形态不很均一,平均粒径约264nm;凝胶阻滞分析证明TACS能有效地包裹和保护共表达质粒不受DNaseⅠ酶的消化;对转染后各组MSCs相关指标检测显示,TACS组细胞存活率(73.18±6.56)%,明显高于脂质体组(45.92±4.93)%(P<0.01);除阴性对照组外, RT-PCR和Western blot均检测到转染后MSCs中hVEGF121及hBMP4的表达,对Western blot所得条带的灰度值相对半定量分析表明,TACS组目的蛋白表达量低于脂质体组(P<0.05),但明显高于壳聚糖组(P<0.01)。
结论:共表达质粒pIRES-hVEGFl2lcDNA/hBMP4在TACS介导下成功转染大鼠MSCs并获得表达。TACS细胞毒性小,且较未改性壳聚糖转染效率有明显提高。
Objective Recombinant coexpression plasmid of pIRES-hVEGFl2lcDNA/hBMP4 was transfected into rat bone marrow mesenchymal stem cells(MSCs) mediated by thiolated N-alkylated chitosan(TACS).To investigate the feasibility of using TACS as a gene vector in bone tissue engineering and build an experimental base on conbination gene therapy of bone defects.
Methods MSCs were isolated and cultivated from the bone marrow of rat. Recombinant coexpression plasmid of pIRES-hVEGFl2lcDNA/hBMP4 was confirmed by restriction enzymolysis,then TACS-pDNA nanoparticles were prepared by complex coacervation.The morphology and particle size of nanoparticles were observed by transmission electronic microscopy and nanoparticle size analyzer. The protection of the nanoparticles for pDNA was observed by gel electrophoresis.TACS bearing pIRES-hVEGFl2lcDNA/hBMP4 was transfected into the third generation of MSCs.Meanwhile,Chitosan group, liposome group and naked pDNA group was respectively experimental control,positive control and negative control.Different effects of the nanoparticles on cell viability were illustrated by MTT assay.After 3 days and 4 days of transfection, the total RNA and total protein were extracted from MSCs respectively.The target gene expressions were detected by RT-PCR and Western blot .
Results Lots of MSCs were separated from bone marrow of rat and the cells showed active proliferative ability.We got expected results on identification of pIRES-hVEGFl2lcDNA/hBMP4 by restriction enzymolysis.The shape of nanoparticles was not very homogeneous and the average particle size was about 264nm determined by nanoparticle size analyzer. TACS could protect encapsulated pDNA effectively from nuclease degradation as shown by electrophoretic mobility analysis.The related indexes of MSCs were inspected after transfection,and we found that cell viability of TACS group(73.18±6.56)% was significantly higher than liposome group(45.92±4.93)%(P<0.01).HVEGF121 and hBMP4 were expressed well in transfected MSCs detected by RT-PCR and Western blot except the negative control.We also conducted relative semi-quantitative analysis on the grey value of Western blot strips and got the result that the target protein expressions of TACS group were lower than liposome group(P<0.05) but significantly higher than chitosan group(P<0.01).
Conclusion The coexpression plasmid was transfected into MSCs successfully by TACS. TACS has low cytotoxicity and its transfection efficiency is obviously higher than non-modified chitosan.
引文
1. Yoon ST,Boden SD. Osteoinductive molecules in orthopaedics:basic science and preclinical studies[J]. Clin Orthop Relat Res,2002,(395):33- 43.
2. Winn SR, Hu YH, Sfeir C, et al.Gene therapy approaches for modulating bone regeneration [J]. Adv Dru Del Rev,2000,42(1-2):121-138.
3. Patil SD,Rhodes DG,Burgess DJ.DNA-based therapeutics and DNA delivery systems:a comprehensive review[J].AAPS J,2005,7(1):E61-77.
4. Borchard G. Chitosans for gene delivery [J].Adv Drug Deliver Rev, 2001, 52(2): 145-150.
5. Mumper RJ, Wang J, Klakamp SL, et al. Protective interactive noncondensing (PINC) polymers for enhanced plasmid distribution and expression[J]. J Control Release, 1998, 52(1-2):191-203.
6. Erwin A, Ramos JV, Relucio CT,et al. Gene expression in tilapia following oral delivery of chitosan-encapsulated plasmid DNA incorporated into fish feeds [J]. Marine Biotech,2005,7(3):89-94.
7. Lee MH, Nah JW, Kwon YM, et al. Water-soluble and low molecular weight chitosan-based plasmid DNA delivery [J]. Pharma Res,2001,18(4):427-431.
8. Lee KY, Kwon IC, Kim YH, et al. Preparation of chitosan self-aggregatesas a gene delivery system [J]. J Control Release,1998,51(2-3):213-220.
9. Kim YH, Gihm SH, Park C R, et al. Structural characteristics of sizecontroll- ed selfaggregates of deoxycholic acid-modified chitosanand their application as a DNA delivery carrier [J]. Bioconjug Chem,2001,12(6):932-938.
10. Mao HQ, Roy K, Troung-Le VL, et al. Chitosan-DNA nanoparticlesas gene carriers:synthesis, characterization and transfection efficiency[J].J Control Release, 2001,70(3):399-421.
11.白欣,慕卿,李树宝等.新型基因载体巯基烷基化壳聚糖的制备及体外性质的初步研究[J].安徽医科大学学报,2009,44(2):162-167.
12. Wozney JM,Rosen V,Celeste AJ,et al.Novel regulators of bone formation:molecular clones and activities[J].Science,1988,242(4885):1528-1534.
13.李长有,原银栋,宋今丹.血管内皮生长因子在骨折愈合中的自分泌作用[J].中华实验外科杂志,2005,22(11):1385-1386.
14.韩雷,汪春兰,赵宇,等.真核双表达载体pIRES-hVEGF121cDNA/hBMP-4的构建与鉴定[J].安徽医科大学学报,2007,42(2):133-136.
15. KNIPPENBERG M,HELDER MN,DOULABI BZ,et al.Adipose tissue-derived mesenchymal stem cells acquire bone cell-like responsiveness to fluid shear stress on osteogenic stim-ulation[J].Tissue Engin,2005,11(11-12):1780-1788.
16.王荣祺,张军,王常勇,等.壳聚糖-hVEGF165基因微粒的制备及体外转染的实验研究[J].中国生物医学工程学报,2006,25(1):14-18.
17. Anderson W F. Human gene therapy[J].Science, 1992, 256(5058):808-813.
18. Fox JL.Gene therapy safety issues come to fore[J].Nat Biotechnol,1999,17(12): 1153.
19. Kohn DB, Sadelain M, Glorioso JC, et al. Occurrence of leukaemia following gene therapy of X-linked SCID [J]. Nat Rev Cancer, 2003, 3(7): 477-488.
20.Alesci S,Ramsey WJ,Bornstein SR,et al.Adenoviral vectors can impair adrenocortical steroidogenesis:clinical implications for natural infections and gene therapy[J].Proc Natl Acad Sci USA,2002,99(11):7484.
21. Sadelain M. Insertional oncogenesis in gene therapy: how much of a risk? [J] Gene Ther, 2004, 11(7): 569-573.
22. Colin W,Pouton,Leonard WS.Key issues in non-viral gene delivery[J].Adv Drug Delve Rev,1998,34(1):3-19.
23. Cristiano,Richard J.Targeted,non-viral gene delivery for cancer gene therapy[J].Front Biosci,1998,3:D1161-D1170.
24. Mansouri S,Cuie Y,Winnik F,et al.Characterization of folate-chitosan-DNA nanoparticles for gene therapy [J].Biomaterials,2006,27(9):2060-2065.
25. Suphasiriroj W, Yotnuengnit P, Surarit R, et al. The fundamental parameters of chitosan in polymer scaffolds affecting osteoblasts (MC3T3-E1) [J].J Mater Sci Mater Med ,2009 ,20(1):309-320.
26. Zheng F,Shi XW,Yang GF,et a1.Chitosan nanoparticle as gene therapy vector via gastrointestinal mucosa administration:Results of an in vitro and in vivo study[J].Life Sci,2007,80(4):388-396.
27. Kim TH,lhm JE,Choi YJ,et a1.Efficient gene delivery by urocanic acid-modified chitosan[J].J Control Release,2003,93(3):389-402.
28. Kawamura K, Oishi J, Kang JH, et al. Intracellular signal-responsive gene carrier for cell-specific gene expression [J]. Biomacromolecules, 2005, 6(2): 908-913.
29. Friedenstein AJ, Gorskaja JF, Kulagina NN. Fibroblast precursors in normal and irradiated mouse hematopoietic organs [J]. Exp Hematol, 1976, 4(5): 267-274.
30. Lee RH, Kim B, Choi I, et al. Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue [J].Cell P- hysiol Biochem, 2004, 14(4-6): 311-324.
31. Gang EJ, Jeong JA, Hong SH, et al. Skeletal myogenic differentiation of mesenchymal stem cells isolated from human umbilicalcord blood [J]. Stem Cells,2004, 22(4): 617-624.
32. Zhang Y, Li CD, Jiang XX, et al. Comparison of mesenchymal stem cells from human placenta and bone marrow [J]. Chin Med J (Engl), 2004, 117(6): 882-887.
33. In’t Anker PS, Scherjon SA, Kleijburg-van der Keur C, et al. Isolation of mesenchymal stem cells of fetal or maternal origin from human placenta [J].Stem Cells, 2004, 22(7):1338-1345.
34. Barry FP, Murphy JM. Mesenchymal stem cells: clinical applications and biological characterization [J]. Int J Biochem Cell Biol,2004, 36(4): 568-584.
35.路继业,王岩,蔡胥,等.骨髓基质干细胞成骨的研究进展[J].中华创伤骨科杂志, 2004, 6(5): 578-580.
36. Lisignoli G, Remiddi G, Cattini L, et al. An elevated number of differentiated osteoblast colonies can be obtained from rat bone marrow stromal cells using a gradient isolation procedure [J]. Connect Tissue Res,2001, 42(1): 49-58.
37.周志林,汪春兰,赵宇,等.重组共表达质粒pIRES-hVEGF121cDNA/hBMP-4在大鼠骨髓间充质干细胞中的共表达[J].安徽医科大学学报,2007,42(4):371-374.
38. Izadpanah R,Joswig T, Tsien F, et al. Characterization of multi potent mesenchymal stem cells from the bone marrow of rhesus macaques [J].Stem Cells Dev, 2005, 14(4): 440-451.
39. Sekiya I, Larson BL, Smith JR, et al. Expansion of human adult stem cells from bone marrow stroma: conditions that maximize the yields of early progenitors and evaluate their quality [J]. Stem Cells, 2002, 20(6):530-541.
40. Pittenger MF,Mackay AM,Jaiswal SC,et al.Multilineage potential of adult human mesenchymal stem cells[J].Science,1999,284(5411):143-147.
41. BARRY FP. Mesenchynal stem cell therapy in joint disease[J].Novartis Found Symp,2003,249:86-96.
42. Makino S,Fukuda K,Miyoshi S,et al.Cardiomyocytes can be generated from marrow stromal cells in vitro[J].J Clin Invest,1999,103(5):697-705.
43.Cesare C,Irene AG,Salesh K,et al.Identification of mesenchymal stem/progenit- or cells in human first trimester fetal blood,liver and bone marrow[J].Blood,2001,98(8):2396-2402.
44.周志林.重组共表达质粒pIRES-hVEGFl2lcDNA/hBMP4在大鼠骨髓间充质干细胞中的表达及对其分化的影响[D].合肥:安徽医科大学,2008.
45. Urist MR. Bone:formation by autoindution [J].Science,1965,150(698):893-899.
46. Reddi AH,Cunningham NS.Initiation and promotion of bone differentiation by bone morphogenetic proteins[J].J Bone Miner Res,1993,8(Suppl2):S499-S502.
47. Helm GA,Alden TD,Sheehan JP,et al.Bone morphogenetic proteins and bone morphogenetic protein gene therapy in neurological surgery:A review[J].Neurosur- gery,2000,46(5):1213-1222.
48. Sekiya I, Larson BL, Vuoristo JT, et al. Comparison of effect of BMP-2,-4,and-6 on in vitro cartilage formation of human adult stem cells from bone marrow stroma [J]. Cell Tissue Res,2005,320(2):269-276.
49.Ferrara N,Henzel WJ.Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells[J].Biochem Biophys Res Commun,1989,161(2):851-858.
50. Ennett AB,Kaigler D,Mooney DJ.Temporally regulated delivery of VEGF in vitro and in vivo[J].J Biomed Mater Res A,2006,79(1):176-184.
51. Lubiatowski P,Goldman CK,Gurunluoglu R,et al.Enhancement of epigastric skin flap survival by adenovirus-mediated VEGF gene therapy[J].Plast Reconstr Surg,2002,109(6):1986-1993.
52. Yang LW,Zhang JX,Zeng L,et al. vascular endothelial growth factor gene therapy with intramuscular injections of plasmid DNA enhances the survival of random pattern flaps in a rat model [J].Br J Plast Surg,2005,58(3):339-347.
53. Galiano RD,Tepper OM,Pelo CR,et al.Topical vascular endothelial growth factor accelerates diabetic wound healing through increased angiogenesis and mobilizing and recruiting bone marrow-derived cells[J].Am J Pathol,2004,164(6):1935-1947.
54. Deckers MM,van Bezooijen RL,van der Horst G,et al.Bone morphogenetic proteins stimulate angiogenesis through osteoblast-derived vascular endothelial growth factor A[J].Endocrinology,2002,143(4):1545-1553.
55. Bouletreau PJ,Warren SM,Spector JA,et al.Hypoxia and VEGF up-regulate BMP-2 mRNA and protein expression in microvascular endothelial cells:implications forfracture healing [J].Plast Reconstr Surg,2002,109(7):2384-2397.
56. Kang R,Chivizzani SC,Muzzonigro TS,et al.Orthopaedic applications of gene therapy.From concept to clinic [J]. Clin Orthop Relat Res,2000,(375):324-337.
57. Fransceschi RT. Biological approaches to bone regeneration by gene therapy [J]. J Dent Res,2005,84(12):1093-1103.
1. Service RF. Tissue engineers build new bone. Science 2000;289(5484):1498-1500
2. Shi C, Zhu Y, Ran X, et al. Therapeutic potential of chitosan and its derivatives in regenerative medicine. J Surg Res 2006;133(2):185-192
3. Khor E,Lim LY.Implantable applications of chitin and chitosan. Biomaterials 2003;24(13):2339-2349
4. Prabaharan M, Mano JF. Chitosan-based particles as controlled drug deliverysystems. Drug Deliv 2005;12(1):41-57
5. Suphasiriroj W, Yotnuengnit P, Surarit R, et al. The fundamental parameters of chitosan in polymer scaffolds affecting osteoblasts (MC3T3-E1). J Mater Sci Mater Med 2009 ;20(1):309-320
6. Jeong B, Kim SW, Bae YH. Thermosensitive sol-gel reversible hydrogels. Adv Drug Deliv Rev 2002;54(1):37-51
7. Jayakumar R, Prabaharan M, Reis RL, et al.Graft copolymerized chitosan–present status and applications. Carbohydr Polym 2005;62(2):142-158
8. Costa-Pinto AR, Correlo VM, Sol PC, et al. Osteogenic differentiation of human bone marrow mesenchymal stem cells seeded on melt based chitosan scaffolds for bone tissue engineering applications. Biomacromolecules 2009;10(8):2067-2073
9. Vandevord PJ,Matthew HW,Desiwa SP,et al.Evaluation of the biocompatibility of a chitosan scaffold in mice.J Biomed Mater Res 2002;59(3):585-590
10.Tomihata K, Ikada Y. In vitro and in vivo degradation of films of chitin and its deacetylated derivatives. Biomaterials 1997;18(7):567-575
11. Kim IY, Seo SJ, Moon HS, et al. Chitosan and its derivatives for tissue engineering applications.Biotechnol Adv 2008;26(1):1-21
12.Zhang Y,Zhang M.Synthesis and characterization of macroprons chitosan/calcium phosphate composite scaffolds for tissue engineering.J Biomed Mater Res 2001;55(3):304-312
13. Di Martino A, Sittinger M, Risbud MV . Chitosan: a versatile biopolymer for orthopaedic tissue-engineering. Biomaterials 2005;26(30):5983-5990
14.季娟娟,丁仲鹃,杨雪莲,等.壳聚糖膜对成骨细胞的毒性研究.实用口腔医学杂志,2008,24(5):630-634
15.金黎明,魏长征,田文杰,等.壳寡糖及其衍生物对体外培养成骨细胞增殖的作用特点.中国组织工程研究与临床康复,2008,12(19):3637-3640
16. Liuyun J, Yubao L, Chengdong X. Preparation and biological properties of a novelcomposite scaffold of nano-hydroxyapatite/chitosan/carboxymethyl cellulose for bone tissue engineering. J Biomed Sci 2009;16:65
17. Li LH, Kommareddy KP, Pilz C, et al. In vitro bioactivity of bioresorbable porous polymeric scaffolds incorporating hydroxyapatite microspheres. Acta Biomater 2009 [Epub ahead of print]
18.孔丽君,敖强,王爱军,等.仿生多层纳米羟基磷灰石/壳聚糖复合支架对兔骨缺损的修复.中国组织工程研究与修复康复,2007,11(5):815-818
19. Lee SH, Shin H. Matrices and scaffolds for delivery of bioactive molecules in bone and cartilage tissue engineering. Adv Drug Deliv Rev 2007;59(4-5):339–359
20. Yanagisawa M, Takizawa T, Ochiai W, et al. Fate alteration of neuroepithelial cells from neurogenesis to astrocytogenesis by bone morphogenetic proteins. Neurosci Res 2001;41(4):391-396
21.Sinha VR,Singla AK, Wadhawan S,et al.Chitosan microspheres as a potential carrier for drugs. Int J Pharm 2004; 274 (1–2): 1–33
22.Varshosaz J, The promise of chitosan microspheres in drug delivery systems.Expert Opin Drug Deliv 2007;4(3):263-273
23. De la Riva B, Nowak C, Sánchez E, et al. VEGF-controlled release within a bone defect from alginate/chitosan/PLA-H scaffolds. Eur J Pharm Biopharm 2009;73(1):50-58
24. Yilgor P, Tuzlakoglu K, Reis RL, et al. Incorporation of a sequential BMP-2/BMP-7 delivery system into chitosan-based scaffolds for bone tissue engineering. Biomaterials 2009;30(21):3551-3559
25. Roy I, Gupta MN. Smart polymeric materials: emerging biochemical applications. Chem Biol 2003;10(12):1161-1171
26. Couto DS, Hong Z, Mano JF. Development of bioactive and biodegradable chitosan-based injectable systems containing bioactive glass nanoparticles. Acta Biomater 2009; 5(1):115-123
27.Ahmadi R, de Bruijn JD. Biocompatibility and gelation of chitosan-glycerolphosphate hydrogels. J Biomed Mater Res A 2008;86(3):824-832
28.Madhumathi K, Shalumon KT, Rani VV, et al. Wet chemical synthesis of chitosan hydrogel-hydroxyapatite composite membranes for tissue engineering applications. Int J Biol Macromol 2009; 45(1):12-15
29. Cruz DM, Ivirico JL, Gomes MM, et al. Chitosan microparticles as injectable scaffolds for tissue engineering. J Tissue Eng Regen Med 2008 ;2(6):378-380
30. Fransceschi RT.Biological approaches to bone regeneration by gene therapy. J Dent Res 2005;84(12):1093-1103
31.严孟宁,戴魁戎,汤亭亭.假体周围骨溶解性骨缺损的转骨形态发生蛋白-2基因治疗.中华实验外科杂志,2008,25(6):702-704
32.Sato T,Ishii T, Okahata Y.In vitro gene delivery mediated by chitosan.effect of pH, serum, andmolecular mass of chitosan on the transfection efficiency. Biomaterials2001;22(15):2075-2080
33. Nie H, Ho ML, Wang CK, et al. BMP-2 plasmid loaded PLGA/HAp composite scaffolds for treatment of bone defects in nude mice. Biomaterials 2009;30(5):892-901
34.邢艳莉,汪春兰,赵宇,等.巯基烷基化壳聚糖介导pIRES-hVEGFl2lcDNA/hBMP-4真核双表达质粒修复兔桡骨缺损的研究.组织工程与重建外科杂志,2009,5(3):138-142.
2. Winn SR, Hu YH, Sfeir C, et al.Gene therapy approaches for modulating bone regeneration [J]. Adv Dru Del Rev,2000,42(1-2):121-138.
3. Patil SD,Rhodes DG,Burgess DJ.DNA-based therapeutics and DNA delivery systems:a comprehensive review[J].AAPS J,2005,7(1):E61-77.
4. Borchard G. Chitosans for gene delivery [J].Adv Drug Deliver Rev, 2001, 52(2): 145-150.
5. Mumper RJ, Wang J, Klakamp SL, et al. Protective interactive noncondensing (PINC) polymers for enhanced plasmid distribution and expression[J]. J Control Release, 1998, 52(1-2):191-203.
6. Erwin A, Ramos JV, Relucio CT,et al. Gene expression in tilapia following oral delivery of chitosan-encapsulated plasmid DNA incorporated into fish feeds [J]. Marine Biotech,2005,7(3):89-94.
7. Lee MH, Nah JW, Kwon YM, et al. Water-soluble and low molecular weight chitosan-based plasmid DNA delivery [J]. Pharma Res,2001,18(4):427-431.
8. Lee KY, Kwon IC, Kim YH, et al. Preparation of chitosan self-aggregatesas a gene delivery system [J]. J Control Release,1998,51(2-3):213-220.
9. Kim YH, Gihm SH, Park C R, et al. Structural characteristics of sizecontroll- ed selfaggregates of deoxycholic acid-modified chitosanand their application as a DNA delivery carrier [J]. Bioconjug Chem,2001,12(6):932-938.
10. Mao HQ, Roy K, Troung-Le VL, et al. Chitosan-DNA nanoparticlesas gene carriers:synthesis, characterization and transfection efficiency[J].J Control Release, 2001,70(3):399-421.
11.白欣,慕卿,李树宝等.新型基因载体巯基烷基化壳聚糖的制备及体外性质的初步研究[J].安徽医科大学学报,2009,44(2):162-167.
12. Wozney JM,Rosen V,Celeste AJ,et al.Novel regulators of bone formation:molecular clones and activities[J].Science,1988,242(4885):1528-1534.
13.李长有,原银栋,宋今丹.血管内皮生长因子在骨折愈合中的自分泌作用[J].中华实验外科杂志,2005,22(11):1385-1386.
14.韩雷,汪春兰,赵宇,等.真核双表达载体pIRES-hVEGF121cDNA/hBMP-4的构建与鉴定[J].安徽医科大学学报,2007,42(2):133-136.
15. KNIPPENBERG M,HELDER MN,DOULABI BZ,et al.Adipose tissue-derived mesenchymal stem cells acquire bone cell-like responsiveness to fluid shear stress on osteogenic stim-ulation[J].Tissue Engin,2005,11(11-12):1780-1788.
16.王荣祺,张军,王常勇,等.壳聚糖-hVEGF165基因微粒的制备及体外转染的实验研究[J].中国生物医学工程学报,2006,25(1):14-18.
17. Anderson W F. Human gene therapy[J].Science, 1992, 256(5058):808-813.
18. Fox JL.Gene therapy safety issues come to fore[J].Nat Biotechnol,1999,17(12): 1153.
19. Kohn DB, Sadelain M, Glorioso JC, et al. Occurrence of leukaemia following gene therapy of X-linked SCID [J]. Nat Rev Cancer, 2003, 3(7): 477-488.
20.Alesci S,Ramsey WJ,Bornstein SR,et al.Adenoviral vectors can impair adrenocortical steroidogenesis:clinical implications for natural infections and gene therapy[J].Proc Natl Acad Sci USA,2002,99(11):7484.
21. Sadelain M. Insertional oncogenesis in gene therapy: how much of a risk? [J] Gene Ther, 2004, 11(7): 569-573.
22. Colin W,Pouton,Leonard WS.Key issues in non-viral gene delivery[J].Adv Drug Delve Rev,1998,34(1):3-19.
23. Cristiano,Richard J.Targeted,non-viral gene delivery for cancer gene therapy[J].Front Biosci,1998,3:D1161-D1170.
24. Mansouri S,Cuie Y,Winnik F,et al.Characterization of folate-chitosan-DNA nanoparticles for gene therapy [J].Biomaterials,2006,27(9):2060-2065.
25. Suphasiriroj W, Yotnuengnit P, Surarit R, et al. The fundamental parameters of chitosan in polymer scaffolds affecting osteoblasts (MC3T3-E1) [J].J Mater Sci Mater Med ,2009 ,20(1):309-320.
26. Zheng F,Shi XW,Yang GF,et a1.Chitosan nanoparticle as gene therapy vector via gastrointestinal mucosa administration:Results of an in vitro and in vivo study[J].Life Sci,2007,80(4):388-396.
27. Kim TH,lhm JE,Choi YJ,et a1.Efficient gene delivery by urocanic acid-modified chitosan[J].J Control Release,2003,93(3):389-402.
28. Kawamura K, Oishi J, Kang JH, et al. Intracellular signal-responsive gene carrier for cell-specific gene expression [J]. Biomacromolecules, 2005, 6(2): 908-913.
29. Friedenstein AJ, Gorskaja JF, Kulagina NN. Fibroblast precursors in normal and irradiated mouse hematopoietic organs [J]. Exp Hematol, 1976, 4(5): 267-274.
30. Lee RH, Kim B, Choi I, et al. Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue [J].Cell P- hysiol Biochem, 2004, 14(4-6): 311-324.
31. Gang EJ, Jeong JA, Hong SH, et al. Skeletal myogenic differentiation of mesenchymal stem cells isolated from human umbilicalcord blood [J]. Stem Cells,2004, 22(4): 617-624.
32. Zhang Y, Li CD, Jiang XX, et al. Comparison of mesenchymal stem cells from human placenta and bone marrow [J]. Chin Med J (Engl), 2004, 117(6): 882-887.
33. In’t Anker PS, Scherjon SA, Kleijburg-van der Keur C, et al. Isolation of mesenchymal stem cells of fetal or maternal origin from human placenta [J].Stem Cells, 2004, 22(7):1338-1345.
34. Barry FP, Murphy JM. Mesenchymal stem cells: clinical applications and biological characterization [J]. Int J Biochem Cell Biol,2004, 36(4): 568-584.
35.路继业,王岩,蔡胥,等.骨髓基质干细胞成骨的研究进展[J].中华创伤骨科杂志, 2004, 6(5): 578-580.
36. Lisignoli G, Remiddi G, Cattini L, et al. An elevated number of differentiated osteoblast colonies can be obtained from rat bone marrow stromal cells using a gradient isolation procedure [J]. Connect Tissue Res,2001, 42(1): 49-58.
37.周志林,汪春兰,赵宇,等.重组共表达质粒pIRES-hVEGF121cDNA/hBMP-4在大鼠骨髓间充质干细胞中的共表达[J].安徽医科大学学报,2007,42(4):371-374.
38. Izadpanah R,Joswig T, Tsien F, et al. Characterization of multi potent mesenchymal stem cells from the bone marrow of rhesus macaques [J].Stem Cells Dev, 2005, 14(4): 440-451.
39. Sekiya I, Larson BL, Smith JR, et al. Expansion of human adult stem cells from bone marrow stroma: conditions that maximize the yields of early progenitors and evaluate their quality [J]. Stem Cells, 2002, 20(6):530-541.
40. Pittenger MF,Mackay AM,Jaiswal SC,et al.Multilineage potential of adult human mesenchymal stem cells[J].Science,1999,284(5411):143-147.
41. BARRY FP. Mesenchynal stem cell therapy in joint disease[J].Novartis Found Symp,2003,249:86-96.
42. Makino S,Fukuda K,Miyoshi S,et al.Cardiomyocytes can be generated from marrow stromal cells in vitro[J].J Clin Invest,1999,103(5):697-705.
43.Cesare C,Irene AG,Salesh K,et al.Identification of mesenchymal stem/progenit- or cells in human first trimester fetal blood,liver and bone marrow[J].Blood,2001,98(8):2396-2402.
44.周志林.重组共表达质粒pIRES-hVEGFl2lcDNA/hBMP4在大鼠骨髓间充质干细胞中的表达及对其分化的影响[D].合肥:安徽医科大学,2008.
45. Urist MR. Bone:formation by autoindution [J].Science,1965,150(698):893-899.
46. Reddi AH,Cunningham NS.Initiation and promotion of bone differentiation by bone morphogenetic proteins[J].J Bone Miner Res,1993,8(Suppl2):S499-S502.
47. Helm GA,Alden TD,Sheehan JP,et al.Bone morphogenetic proteins and bone morphogenetic protein gene therapy in neurological surgery:A review[J].Neurosur- gery,2000,46(5):1213-1222.
48. Sekiya I, Larson BL, Vuoristo JT, et al. Comparison of effect of BMP-2,-4,and-6 on in vitro cartilage formation of human adult stem cells from bone marrow stroma [J]. Cell Tissue Res,2005,320(2):269-276.
49.Ferrara N,Henzel WJ.Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells[J].Biochem Biophys Res Commun,1989,161(2):851-858.
50. Ennett AB,Kaigler D,Mooney DJ.Temporally regulated delivery of VEGF in vitro and in vivo[J].J Biomed Mater Res A,2006,79(1):176-184.
51. Lubiatowski P,Goldman CK,Gurunluoglu R,et al.Enhancement of epigastric skin flap survival by adenovirus-mediated VEGF gene therapy[J].Plast Reconstr Surg,2002,109(6):1986-1993.
52. Yang LW,Zhang JX,Zeng L,et al. vascular endothelial growth factor gene therapy with intramuscular injections of plasmid DNA enhances the survival of random pattern flaps in a rat model [J].Br J Plast Surg,2005,58(3):339-347.
53. Galiano RD,Tepper OM,Pelo CR,et al.Topical vascular endothelial growth factor accelerates diabetic wound healing through increased angiogenesis and mobilizing and recruiting bone marrow-derived cells[J].Am J Pathol,2004,164(6):1935-1947.
54. Deckers MM,van Bezooijen RL,van der Horst G,et al.Bone morphogenetic proteins stimulate angiogenesis through osteoblast-derived vascular endothelial growth factor A[J].Endocrinology,2002,143(4):1545-1553.
55. Bouletreau PJ,Warren SM,Spector JA,et al.Hypoxia and VEGF up-regulate BMP-2 mRNA and protein expression in microvascular endothelial cells:implications forfracture healing [J].Plast Reconstr Surg,2002,109(7):2384-2397.
56. Kang R,Chivizzani SC,Muzzonigro TS,et al.Orthopaedic applications of gene therapy.From concept to clinic [J]. Clin Orthop Relat Res,2000,(375):324-337.
57. Fransceschi RT. Biological approaches to bone regeneration by gene therapy [J]. J Dent Res,2005,84(12):1093-1103.
1. Service RF. Tissue engineers build new bone. Science 2000;289(5484):1498-1500
2. Shi C, Zhu Y, Ran X, et al. Therapeutic potential of chitosan and its derivatives in regenerative medicine. J Surg Res 2006;133(2):185-192
3. Khor E,Lim LY.Implantable applications of chitin and chitosan. Biomaterials 2003;24(13):2339-2349
4. Prabaharan M, Mano JF. Chitosan-based particles as controlled drug deliverysystems. Drug Deliv 2005;12(1):41-57
5. Suphasiriroj W, Yotnuengnit P, Surarit R, et al. The fundamental parameters of chitosan in polymer scaffolds affecting osteoblasts (MC3T3-E1). J Mater Sci Mater Med 2009 ;20(1):309-320
6. Jeong B, Kim SW, Bae YH. Thermosensitive sol-gel reversible hydrogels. Adv Drug Deliv Rev 2002;54(1):37-51
7. Jayakumar R, Prabaharan M, Reis RL, et al.Graft copolymerized chitosan–present status and applications. Carbohydr Polym 2005;62(2):142-158
8. Costa-Pinto AR, Correlo VM, Sol PC, et al. Osteogenic differentiation of human bone marrow mesenchymal stem cells seeded on melt based chitosan scaffolds for bone tissue engineering applications. Biomacromolecules 2009;10(8):2067-2073
9. Vandevord PJ,Matthew HW,Desiwa SP,et al.Evaluation of the biocompatibility of a chitosan scaffold in mice.J Biomed Mater Res 2002;59(3):585-590
10.Tomihata K, Ikada Y. In vitro and in vivo degradation of films of chitin and its deacetylated derivatives. Biomaterials 1997;18(7):567-575
11. Kim IY, Seo SJ, Moon HS, et al. Chitosan and its derivatives for tissue engineering applications.Biotechnol Adv 2008;26(1):1-21
12.Zhang Y,Zhang M.Synthesis and characterization of macroprons chitosan/calcium phosphate composite scaffolds for tissue engineering.J Biomed Mater Res 2001;55(3):304-312
13. Di Martino A, Sittinger M, Risbud MV . Chitosan: a versatile biopolymer for orthopaedic tissue-engineering. Biomaterials 2005;26(30):5983-5990
14.季娟娟,丁仲鹃,杨雪莲,等.壳聚糖膜对成骨细胞的毒性研究.实用口腔医学杂志,2008,24(5):630-634
15.金黎明,魏长征,田文杰,等.壳寡糖及其衍生物对体外培养成骨细胞增殖的作用特点.中国组织工程研究与临床康复,2008,12(19):3637-3640
16. Liuyun J, Yubao L, Chengdong X. Preparation and biological properties of a novelcomposite scaffold of nano-hydroxyapatite/chitosan/carboxymethyl cellulose for bone tissue engineering. J Biomed Sci 2009;16:65
17. Li LH, Kommareddy KP, Pilz C, et al. In vitro bioactivity of bioresorbable porous polymeric scaffolds incorporating hydroxyapatite microspheres. Acta Biomater 2009 [Epub ahead of print]
18.孔丽君,敖强,王爱军,等.仿生多层纳米羟基磷灰石/壳聚糖复合支架对兔骨缺损的修复.中国组织工程研究与修复康复,2007,11(5):815-818
19. Lee SH, Shin H. Matrices and scaffolds for delivery of bioactive molecules in bone and cartilage tissue engineering. Adv Drug Deliv Rev 2007;59(4-5):339–359
20. Yanagisawa M, Takizawa T, Ochiai W, et al. Fate alteration of neuroepithelial cells from neurogenesis to astrocytogenesis by bone morphogenetic proteins. Neurosci Res 2001;41(4):391-396
21.Sinha VR,Singla AK, Wadhawan S,et al.Chitosan microspheres as a potential carrier for drugs. Int J Pharm 2004; 274 (1–2): 1–33
22.Varshosaz J, The promise of chitosan microspheres in drug delivery systems.Expert Opin Drug Deliv 2007;4(3):263-273
23. De la Riva B, Nowak C, Sánchez E, et al. VEGF-controlled release within a bone defect from alginate/chitosan/PLA-H scaffolds. Eur J Pharm Biopharm 2009;73(1):50-58
24. Yilgor P, Tuzlakoglu K, Reis RL, et al. Incorporation of a sequential BMP-2/BMP-7 delivery system into chitosan-based scaffolds for bone tissue engineering. Biomaterials 2009;30(21):3551-3559
25. Roy I, Gupta MN. Smart polymeric materials: emerging biochemical applications. Chem Biol 2003;10(12):1161-1171
26. Couto DS, Hong Z, Mano JF. Development of bioactive and biodegradable chitosan-based injectable systems containing bioactive glass nanoparticles. Acta Biomater 2009; 5(1):115-123
27.Ahmadi R, de Bruijn JD. Biocompatibility and gelation of chitosan-glycerolphosphate hydrogels. J Biomed Mater Res A 2008;86(3):824-832
28.Madhumathi K, Shalumon KT, Rani VV, et al. Wet chemical synthesis of chitosan hydrogel-hydroxyapatite composite membranes for tissue engineering applications. Int J Biol Macromol 2009; 45(1):12-15
29. Cruz DM, Ivirico JL, Gomes MM, et al. Chitosan microparticles as injectable scaffolds for tissue engineering. J Tissue Eng Regen Med 2008 ;2(6):378-380
30. Fransceschi RT.Biological approaches to bone regeneration by gene therapy. J Dent Res 2005;84(12):1093-1103
31.严孟宁,戴魁戎,汤亭亭.假体周围骨溶解性骨缺损的转骨形态发生蛋白-2基因治疗.中华实验外科杂志,2008,25(6):702-704
32.Sato T,Ishii T, Okahata Y.In vitro gene delivery mediated by chitosan.effect of pH, serum, andmolecular mass of chitosan on the transfection efficiency. Biomaterials2001;22(15):2075-2080
33. Nie H, Ho ML, Wang CK, et al. BMP-2 plasmid loaded PLGA/HAp composite scaffolds for treatment of bone defects in nude mice. Biomaterials 2009;30(5):892-901
34.邢艳莉,汪春兰,赵宇,等.巯基烷基化壳聚糖介导pIRES-hVEGFl2lcDNA/hBMP-4真核双表达质粒修复兔桡骨缺损的研究.组织工程与重建外科杂志,2009,5(3):138-142.