小鼠胚胎干细胞诱导的血管细胞联合种植在PLGA上初步构建出血管组织
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摘要
目的:在我们已有小鼠胚胎干细胞诱导且经过RT-PCR和免疫组化鉴定血管平滑肌细胞(VSMc)和内皮细胞(EC)种子细胞的基础上,现在探索用诱导的内皮细胞和血管平滑肌细胞,联合种植在经明胶浸润处理的聚丙交酯/乙交酯(PLGA)静电纺丝材料上,进行体外构建小管径组织工程血管。
方法:①先将PLGA用1%明胶处理,并测量明胶浸润处理前后PLGA的接触角。②在明胶处理与否的PLGA片上种植血管平滑肌细胞,60分钟后即用甲苯胺蓝染色法观察材料对细胞的亲和力;用MTT、扫描电镜材料观察经培养1天,3天后细胞在材料上的生长情况。③在片状PLGA材料一侧隔日种植血管平滑肌细胞1次, 3天共2次,再于另外一侧种植内皮细胞1次,并进行联合培养2天后,作H&E和免疫组化检查。④将联合种植了血管平滑肌细胞和内皮细胞的PLGA裹成直经约1mm管状包埋于4周龄的裸鼠皮下,分别于3天,1,3周取出进行HE染色和免疫组化检查。⑤同时用放射免疫法测定单纯种植1次血管平滑肌细胞的A组,单纯种植内皮细胞的B组,联合种植了平滑肌和内皮细胞的C组这三组培养液中内皮素含量。
结果:①处理前的接触角是88.5°,处理后61.5°。②甲苯胺蓝染色见血管平滑肌细胞在材料上密集、均匀分布;扫描电镜观察到1小时细胞开始延展,1天开始长入孔隙中,3天呈多层分布,无序状生长;MTT检测支架上细胞在3天内的增殖情况等同于培养板。③H&E和免疫组化见材料两侧分别分布着血管平滑肌细胞和内皮。④包埋3周后材料结构仍存在,未被降解吸收。在材料周围包裹结缔组织,管壁外侧有多层血管平滑肌细胞,内侧有内皮细胞。⑤内皮素含量在A组为0.82±0.37 pg/ml,B组5.59±1.23 pg/ml,C组4.51±0.59 pg/ml。
结论:①用1%明胶浸润包被处理PLGA后能改善材料的细胞亲和力;②在PLGA上联合种植血管细胞的方法初步构建了具有血管平滑肌细胞和内皮细胞的组织工程血管片;③包埋裸鼠皮下后形成了具有三层细胞结构的组织工程血管。
Objective:Since we have the vessel seed cells that Cultured Esc as embryoid body, then use the inductive factors to induce mouse embryonic stem cells to differentiate into smooth muscle cells and endothelial cells, RT-PCR and immunohistochemistry were used to detect. The experiment use the cells that induced mouse embryonic stem cells to differentiate into smooth muscle cells and endothelial cells implanted on poly(lactide-co-glycolide)(PLGA)electrospinning,treated by gelatin infiltration, to construct a small-diameter vascular in vitro.
Methods:①Detected the contact angle of the scaffold treated with/without 1%gelatin.②VSMc was seeding onto PLGA treated by gelatin, Culture scaffold 60 min ,the compatibility detected at initial stage by Toluidine Bluestaining (TB),proliferation were observed for the 1d and 3d scaffolds by SEM and MTT.③Implanted smooth muscle cells on one side of the PLGA other day seeding again ,then seeding endothelial cells on other side after 3 days. Culture the PLGA with the sheet of smooth muscle cells and endothelial cells on the surface 2 days,examined with the H&E and. Immunohistochemistry.④4-weeks-old Nude mice were implanted subcutaneously with vascular scaffolds and sacrificed 3 days,1 and 3weeks after implantation. The scaffolds were anayslzd using H&E staining and immunohistochemistry.⑤Endothelin were examined by means of radioimmunology in the culture solution, group A simple seeding vascular smooth muscle cell,groupB merely seeding endothelial cell, and group C seeding combined with vascular smooth muscle cell and endothelial cell, each group cultured 2 days after sending cells.
Results:①Untreated group contact angle were 88.5°and treated group contact angle were 61.5°.②The VSMc distributed even dense on the treated PLGA ,SME observed cells extended 1h ,1d cell have grown into the pore ,and much layer cell distribution disorder; MTT find that cells proliferation on the treated PLGA is as well as the culture plate in the primary 3days.③H&E and immunohistochemistry prospected the VSMc and Ec distribution the scaffold each side.④The PLGA still conserved after implanted the Nude mice 3W, did not been completely absorbation or degradation ,the graft closed by the connect tissue, examined much layer VSMc at the lateral and one layer Ec on the tubule inside.⑤The levels of endothelin in the culture solution were 0.82±0.37 pg/mml, 5.59±1.23 pg/mml, 4.51±0.59 pg/mml respectively in the group A ,B and C.
Conclusion:①The scaffold treated by gelatin improved the vascular smooth muscle cells adsorbability .②The tissue engineering vessel film have two-layer cells of vascular smooth muscle cells and endothelial cells by union seeding the VSMc and EC.③It harvest the engineering vessel with three- lay cells by implanted the Nude mice.
方法:①先将PLGA用1%明胶处理,并测量明胶浸润处理前后PLGA的接触角。②在明胶处理与否的PLGA片上种植血管平滑肌细胞,60分钟后即用甲苯胺蓝染色法观察材料对细胞的亲和力;用MTT、扫描电镜材料观察经培养1天,3天后细胞在材料上的生长情况。③在片状PLGA材料一侧隔日种植血管平滑肌细胞1次, 3天共2次,再于另外一侧种植内皮细胞1次,并进行联合培养2天后,作H&E和免疫组化检查。④将联合种植了血管平滑肌细胞和内皮细胞的PLGA裹成直经约1mm管状包埋于4周龄的裸鼠皮下,分别于3天,1,3周取出进行HE染色和免疫组化检查。⑤同时用放射免疫法测定单纯种植1次血管平滑肌细胞的A组,单纯种植内皮细胞的B组,联合种植了平滑肌和内皮细胞的C组这三组培养液中内皮素含量。
结果:①处理前的接触角是88.5°,处理后61.5°。②甲苯胺蓝染色见血管平滑肌细胞在材料上密集、均匀分布;扫描电镜观察到1小时细胞开始延展,1天开始长入孔隙中,3天呈多层分布,无序状生长;MTT检测支架上细胞在3天内的增殖情况等同于培养板。③H&E和免疫组化见材料两侧分别分布着血管平滑肌细胞和内皮。④包埋3周后材料结构仍存在,未被降解吸收。在材料周围包裹结缔组织,管壁外侧有多层血管平滑肌细胞,内侧有内皮细胞。⑤内皮素含量在A组为0.82±0.37 pg/ml,B组5.59±1.23 pg/ml,C组4.51±0.59 pg/ml。
结论:①用1%明胶浸润包被处理PLGA后能改善材料的细胞亲和力;②在PLGA上联合种植血管细胞的方法初步构建了具有血管平滑肌细胞和内皮细胞的组织工程血管片;③包埋裸鼠皮下后形成了具有三层细胞结构的组织工程血管。
Objective:Since we have the vessel seed cells that Cultured Esc as embryoid body, then use the inductive factors to induce mouse embryonic stem cells to differentiate into smooth muscle cells and endothelial cells, RT-PCR and immunohistochemistry were used to detect. The experiment use the cells that induced mouse embryonic stem cells to differentiate into smooth muscle cells and endothelial cells implanted on poly(lactide-co-glycolide)(PLGA)electrospinning,treated by gelatin infiltration, to construct a small-diameter vascular in vitro.
Methods:①Detected the contact angle of the scaffold treated with/without 1%gelatin.②VSMc was seeding onto PLGA treated by gelatin, Culture scaffold 60 min ,the compatibility detected at initial stage by Toluidine Bluestaining (TB),proliferation were observed for the 1d and 3d scaffolds by SEM and MTT.③Implanted smooth muscle cells on one side of the PLGA other day seeding again ,then seeding endothelial cells on other side after 3 days. Culture the PLGA with the sheet of smooth muscle cells and endothelial cells on the surface 2 days,examined with the H&E and. Immunohistochemistry.④4-weeks-old Nude mice were implanted subcutaneously with vascular scaffolds and sacrificed 3 days,1 and 3weeks after implantation. The scaffolds were anayslzd using H&E staining and immunohistochemistry.⑤Endothelin were examined by means of radioimmunology in the culture solution, group A simple seeding vascular smooth muscle cell,groupB merely seeding endothelial cell, and group C seeding combined with vascular smooth muscle cell and endothelial cell, each group cultured 2 days after sending cells.
Results:①Untreated group contact angle were 88.5°and treated group contact angle were 61.5°.②The VSMc distributed even dense on the treated PLGA ,SME observed cells extended 1h ,1d cell have grown into the pore ,and much layer cell distribution disorder; MTT find that cells proliferation on the treated PLGA is as well as the culture plate in the primary 3days.③H&E and immunohistochemistry prospected the VSMc and Ec distribution the scaffold each side.④The PLGA still conserved after implanted the Nude mice 3W, did not been completely absorbation or degradation ,the graft closed by the connect tissue, examined much layer VSMc at the lateral and one layer Ec on the tubule inside.⑤The levels of endothelin in the culture solution were 0.82±0.37 pg/mml, 5.59±1.23 pg/mml, 4.51±0.59 pg/mml respectively in the group A ,B and C.
Conclusion:①The scaffold treated by gelatin improved the vascular smooth muscle cells adsorbability .②The tissue engineering vessel film have two-layer cells of vascular smooth muscle cells and endothelial cells by union seeding the VSMc and EC.③It harvest the engineering vessel with three- lay cells by implanted the Nude mice.
引文
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[2]. 曹谊林. 组织工程学理论与实践.[M]第一版. 上海: 上海科学技术出版社
[3]. Luscher TF, Barton M. Biology of the endothelium.[J] Clin Cardiol 1997;20:II-3-10.
[4].Mikos AG, Lyman MD, Freed LE, et al.Wetting of poly(L-lactic acid) and poly (DL-lactic-co-glycolic acid) foams for tissue culture. Biomaterials 1994;15:55-58.
[5]. 何创龙,黄争鸣, 张彦中等. 静电纺丝法制备组织工程纳/微米纤维支架[J].自然科学进展,2005,15(10):1175-1182.
[6]. Fridrikh SV, Yu JH, Brenner MP,et al. Controlling the fiber diameter during electrospinning.[J] Phys Rev Lett 2003;90:144502.
[7]. Ma Z,Kotaki M,Inai R,et al. Potential of nanofiber matrix as tissueengineering scaffolds[J]. Tissue Engineering,2005,11.(1-2).101-109.
[8]. Taylor GI. Disintegration of water drops in an electric field. [J]Proceedings of Royal Society London(A),1964,280:383.
[9].LI.D,Xia.YN. Electrospinning of nanofibers: reinventing the wheel?[J]Advanced Materials, 2004,16,1151-1170
[10]. Courtney T, Sacks MS, Stankus J, et al. Design and analysis of tissue engineering scaffolds that mimic soft tissue mechanical anisotropy. [J]Biomaterials 2006;27:3631-3638.
[11]. Boland ED, Matthews JA, Pawlowski KJ, et al. Electrospinning collagen and elastin: preliminary vascular tissue engineering. [J]Front Biosci 2004;9:1422-1432.
[12]. Stitzel J, Liu J, Lee SJ, et al. Controlled fabrication of a biological vascular substitute. [J]Biomaterials 2006;27:1088-1094.
[13]. Freed LE, Vunjak-Novakovic G, Biron RJ, et al. Biodegradable polymer scaffolds for tissue engineering.[J] Biotechnology (N Y) 1994;12:689-693.
[14]. 熊吉信,刘兆轩,刘小春等.体外定向诱导小鼠胚胎干细胞向内皮分化的研究.[J]中国重建外科杂志,2007,21(9):994-998
[15]. Herring M, Gardner A, Glover J. Seeding endothelium onto canine arterial prostheses. The effects of graft design. [J]Arch Surg 1979;114:679-682.
[16]. Schnell AM, Hoerstrup SP, Zund G, et al. Optimal cell source for cardiovascular tissue engineering: venous vs. aortic human myofibroblasts. [J]Thorac Cardiovasc Surg 2001; 49: 221-225.
[17]. Pasic M, Muller-Glauser W, Odermatt B, et al. Seeding with omental cells prevents late neointimal hyperplasia in small-diameter Dacron grafts. [J]Circulation 1995;92:2605-2616.
[18]. Wen SJ, Zhao LM, Li P, et al. [Blood vessel tissue engineering: seeding vascular smooth muscle cells and endothelial cells sequentially on biodegradable scaffold in vitro [J]Zhonghua Yi Xue Za Zhi 2005;85:816-818.
[19]. Zhang X, Baughman CB, Kaplan DL. In vitro evaluation of electrospun silk fibroin scaffolds for vascular cell growth.[J] Biomaterials 2008;29:2217-2227.
[20]. Shin H, Jo S, Mikos AG. Biomimetic materials for tissue engineering.[J] Biomaterials 2003;24:4353-4364.
[21]. Chun KW, Yoo HS, Yoon JJ, et al. Biodegradable PLGA microcarriers for injectable delivery of chondrocytes: effect of surface modification on cell attachment and function. [J]Biotechnol Prog 2004;20:1797-1801.
[22]. Miller DC, Thapa A, Haberstroh KM, et al. Endothelial and vascular smooth muscle cell function on poly(lactic-co-glycolic acid) with nano-structured surface features.[J] Biomaterials 2004;25:53-61.
[23]. Li M, Guo Y, Wei Y, et al. Electrospinning polyaniline-contained gelatin nanofibers for tissue engineering applications. [J]Biomaterials 2006;27:2705-2715.
[24]. Buijtenhuijs P, Buttafoco L, Poot AA, et al. Tissue engineering of blood vessels: characterization of smooth-muscle cells for culturing on collagen-and-elastin-based scaffolds. [J]Biotechnol Appl Biochem 2004;39:141-149.
[25]. Rho KS, Jeong L, Lee G, et al. Electrospinning of collagen nanofibers: effects on the behavior of normal human keratinocytes and early-stage wound healing. [J]Biomaterials 2006; 27:1452-1461.
[26]. Matthews JA, Wnek GE, Simpson DG, et al. Electrospinning of collagen nanofibers. [J]Biomacromolecules 2002;3:232-238.
[27]. Buttafoco L, Kolkman NG, Poot AA, et al. Electrospinning collagen and elastin for tissue engineering small diameter blood vessels.[ J] Control Release 2005;101:322-324.
[28]. Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. [J]Nature 1981;292:154-156.
[29]. Wang R, Clark R, Bautch VL. Embryonic stem cell-derived cystic embryoid bodies form vascular channels: an in vitro model of blood vessel development. [J]Development 1992;114: 303-316.
[30]. Doetschman T, Shull M, Kier A, et al. Embryonic stem cell model systems for vascular morphogenesis and cardiac disorders. [J]Hypertension 1993;22:618-629.
[31]. Vittet D, Prandini MH, Berthier R, et al. Embryonic stem cells differentiate in vitro to endothelial cells through successive maturation steps.[J] Blood 1996;88:3424-3431.
[32]. Balconi G, Spagnuolo R, Dejana E. Development of endothelial cell lines from embryonic stem cells: A tool for studying genetically manipulated endothelial cells in vitro. [J]Arterioscler Thromb Vasc Biol 2000;20:1443-1451.
[33]. Herring M, Gardner A, Glover J. A single-staged technique for seeding vascular grafts with autogenous endothelium.[J] Surgery 1978;84:498-504.
[34]. Matsuda T. Recent progress of vascular graft engineering in Japan. [J]Artif Organs 2004;28:64-71.
[35]. Brandt J, Nilsson A, Kanje M, et al. Acutely-dissociated Schwann cells used in tendon autografts for bridging nerve defects in rats: a new principle for tissue engineering in nerve reconstruction.[J] Scand J Plast Reconstr Surg Hand Surg 2005;39:321-325.
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[2]. 曹谊林. 组织工程学理论与实践.[M]第一版. 上海: 上海科学技术出版社
[3]. Luscher TF, Barton M. Biology of the endothelium.[J] Clin Cardiol 1997;20:II-3-10.
[4].Mikos AG, Lyman MD, Freed LE, et al.Wetting of poly(L-lactic acid) and poly (DL-lactic-co-glycolic acid) foams for tissue culture. Biomaterials 1994;15:55-58.
[5]. 何创龙,黄争鸣, 张彦中等. 静电纺丝法制备组织工程纳/微米纤维支架[J].自然科学进展,2005,15(10):1175-1182.
[6]. Fridrikh SV, Yu JH, Brenner MP,et al. Controlling the fiber diameter during electrospinning.[J] Phys Rev Lett 2003;90:144502.
[7]. Ma Z,Kotaki M,Inai R,et al. Potential of nanofiber matrix as tissueengineering scaffolds[J]. Tissue Engineering,2005,11.(1-2).101-109.
[8]. Taylor GI. Disintegration of water drops in an electric field. [J]Proceedings of Royal Society London(A),1964,280:383.
[9].LI.D,Xia.YN. Electrospinning of nanofibers: reinventing the wheel?[J]Advanced Materials, 2004,16,1151-1170
[10]. Courtney T, Sacks MS, Stankus J, et al. Design and analysis of tissue engineering scaffolds that mimic soft tissue mechanical anisotropy. [J]Biomaterials 2006;27:3631-3638.
[11]. Boland ED, Matthews JA, Pawlowski KJ, et al. Electrospinning collagen and elastin: preliminary vascular tissue engineering. [J]Front Biosci 2004;9:1422-1432.
[12]. Stitzel J, Liu J, Lee SJ, et al. Controlled fabrication of a biological vascular substitute. [J]Biomaterials 2006;27:1088-1094.
[13]. Freed LE, Vunjak-Novakovic G, Biron RJ, et al. Biodegradable polymer scaffolds for tissue engineering.[J] Biotechnology (N Y) 1994;12:689-693.
[14]. 熊吉信,刘兆轩,刘小春等.体外定向诱导小鼠胚胎干细胞向内皮分化的研究.[J]中国重建外科杂志,2007,21(9):994-998
[15]. Herring M, Gardner A, Glover J. Seeding endothelium onto canine arterial prostheses. The effects of graft design. [J]Arch Surg 1979;114:679-682.
[16]. Schnell AM, Hoerstrup SP, Zund G, et al. Optimal cell source for cardiovascular tissue engineering: venous vs. aortic human myofibroblasts. [J]Thorac Cardiovasc Surg 2001; 49: 221-225.
[17]. Pasic M, Muller-Glauser W, Odermatt B, et al. Seeding with omental cells prevents late neointimal hyperplasia in small-diameter Dacron grafts. [J]Circulation 1995;92:2605-2616.
[18]. Wen SJ, Zhao LM, Li P, et al. [Blood vessel tissue engineering: seeding vascular smooth muscle cells and endothelial cells sequentially on biodegradable scaffold in vitro [J]Zhonghua Yi Xue Za Zhi 2005;85:816-818.
[19]. Zhang X, Baughman CB, Kaplan DL. In vitro evaluation of electrospun silk fibroin scaffolds for vascular cell growth.[J] Biomaterials 2008;29:2217-2227.
[20]. Shin H, Jo S, Mikos AG. Biomimetic materials for tissue engineering.[J] Biomaterials 2003;24:4353-4364.
[21]. Chun KW, Yoo HS, Yoon JJ, et al. Biodegradable PLGA microcarriers for injectable delivery of chondrocytes: effect of surface modification on cell attachment and function. [J]Biotechnol Prog 2004;20:1797-1801.
[22]. Miller DC, Thapa A, Haberstroh KM, et al. Endothelial and vascular smooth muscle cell function on poly(lactic-co-glycolic acid) with nano-structured surface features.[J] Biomaterials 2004;25:53-61.
[23]. Li M, Guo Y, Wei Y, et al. Electrospinning polyaniline-contained gelatin nanofibers for tissue engineering applications. [J]Biomaterials 2006;27:2705-2715.
[24]. Buijtenhuijs P, Buttafoco L, Poot AA, et al. Tissue engineering of blood vessels: characterization of smooth-muscle cells for culturing on collagen-and-elastin-based scaffolds. [J]Biotechnol Appl Biochem 2004;39:141-149.
[25]. Rho KS, Jeong L, Lee G, et al. Electrospinning of collagen nanofibers: effects on the behavior of normal human keratinocytes and early-stage wound healing. [J]Biomaterials 2006; 27:1452-1461.
[26]. Matthews JA, Wnek GE, Simpson DG, et al. Electrospinning of collagen nanofibers. [J]Biomacromolecules 2002;3:232-238.
[27]. Buttafoco L, Kolkman NG, Poot AA, et al. Electrospinning collagen and elastin for tissue engineering small diameter blood vessels.[ J] Control Release 2005;101:322-324.
[28]. Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. [J]Nature 1981;292:154-156.
[29]. Wang R, Clark R, Bautch VL. Embryonic stem cell-derived cystic embryoid bodies form vascular channels: an in vitro model of blood vessel development. [J]Development 1992;114: 303-316.
[30]. Doetschman T, Shull M, Kier A, et al. Embryonic stem cell model systems for vascular morphogenesis and cardiac disorders. [J]Hypertension 1993;22:618-629.
[31]. Vittet D, Prandini MH, Berthier R, et al. Embryonic stem cells differentiate in vitro to endothelial cells through successive maturation steps.[J] Blood 1996;88:3424-3431.
[32]. Balconi G, Spagnuolo R, Dejana E. Development of endothelial cell lines from embryonic stem cells: A tool for studying genetically manipulated endothelial cells in vitro. [J]Arterioscler Thromb Vasc Biol 2000;20:1443-1451.
[33]. Herring M, Gardner A, Glover J. A single-staged technique for seeding vascular grafts with autogenous endothelium.[J] Surgery 1978;84:498-504.
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