聚己内酯囊管化骨髓间充质干细胞活性研究
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摘要
目的分离培养大鼠骨髓间充质干细胞为研究对象,建立囊管化细胞的回收方法和囊管化细胞活性测定方法,研究聚己内酯半透膜囊管化对细胞活性的影响,探讨半透膜影响细胞活性的机制,为聚己内酯半透膜免疫隔离移植技术的进一步研究和应用提供依据。
方法分离、纯化和培养SD大鼠骨髓间充质干细胞,第三代细胞用于实验研究。细胞悬液置聚己内酯半透膜管内,半透膜管两端70℃热夹闭封装,细胞贴壁后0.25%胰酶消化回收细胞,测定不同消化时间对细胞活性和回收率的影响。以添加10%胎牛血清的完全培养液平皿法细胞培养为对照组,设置3个实验组进行聚己内酯囊管化骨髓间充质干细胞培养活性研究。实验组1:细胞以完全培养液悬浮后封装,完全培养液培养,小分子营养物质能够通过渗透有效补充;实验组2:细胞以完全培养液悬浮后封装,不含血清的基础培养液培养,血清小分子活性物质不能有效补充;实验组3:细胞以基础培养液悬浮后封装,完全培养液培养,小分子营养物质能够通过渗透补充,但细胞缺乏血清大分子的营养。囊管内细胞胰酶消化回收,以细胞计数、MTT法、CCK-8法测定细胞的活性,并进行生长曲线分析。以完全培养液平皿法细胞培养为对照组,设2个实验组进行聚己内酯囊管化骨髓间充质干细胞移植细胞活性研究。两个实验组分别以完全培养液和基础培养液悬浮细胞后封装移植,囊管内细胞回收后以细胞计数、MTT法、CCK-8法测定细胞的活性,共8天,进行生长曲线分析。
结果分离培养得到典型的大鼠骨髓间充质干细胞,细胞活性良好。70℃热封装及采用0.25%胰酶消化3分钟对细胞无明显损伤,活细胞超过95%,细胞回收率不低于97%的。完全培养液悬浮细胞封装,以完全培养液培养的骨髓间充质干细胞成活良好,细胞增殖率达到150%,低于对照组的210%。完全培养液悬浮封装,基础培养液培养,骨髓间充质干细胞活性迅速下降,一周之内全部死亡;基础培养液悬浮封装,以完全培养液培养,骨髓间充质干细胞活性下降,2-3天后逐渐上升,细胞增殖率低于10%,3个实验组与对照组均具有显著性差异。异体移植实验中,完全培养液悬浮和封装后移植的骨髓间充质干细胞增殖率约为140%;较对照组200%为低,基础培养液悬浮细胞封装后细胞活性下降,其后逐渐上升,但增殖率低于10%,2个实验组与对照组均具有显著性差异。
结论建立了囊管化细胞的回收方法和以细胞回收为基础的囊管化细胞活性测定方法,发现囊管化封装对细胞培养与细胞移植均有一定的的抑制作用。血清中小分子活性物质对细胞具有重要的意义,是细胞存活和生长的关键。血清中大分子对细胞活性具有一定的作用,缺乏血清大分子细胞的增殖明显减弱,囊内血清大分子的耗竭可能是半透膜包裹细胞难以长期存活的原因之一。由于低分子物质的通透性是细胞活性的关键,半透膜免疫隔离技术能够保证细胞一定时间内的活性,可以作为解决异体移植排斥反应的一种手段。
Objective To separate and culture rat bone mesenchymal stem cells as study object, establish retrieve method for encapsulated cells and develop cell bioactivity determination methods,investigate the impact of polycaprolactone(PCL) semipermeable membrane tube encapsulation on bioactivity of cells and explore its mechanism,it is expected to offer better help for related research and application.
Method Bone mesenchymal stem cells isolated,purified and cultured from SD rat,third generation cells prepared for experiments.Cell suspension implanted into PCL semipermeable membrane tube and encapsulated at 70 centigrade,cell activity and retrieving rate were measured in varies time of 0.25%trypsin digestion.Impact of PCL capsulation on the mesenchymal stem cells activity studied in vitro studied,one plate culture with complete culture medium for control group and three encapsulated culture for experimental groups installed.Experimental group 1:complete culture medium for cell suspended before package and for culture,in which small molecule nutrients can be maintained through infiltration;experimental group 2:complete culture medium for cell suspended before package and serum free basic culture medium for culture,in which small molecule nutrients can not be supplemented through infiltration;experimental group 3: basic culture medium for cell suspended before package and complete culture medium for culture,in which small molecule nutrients can be supplemented through infiltration without serum macromolecules nutrition included.Retrieving capsulated cells for cell counting, MTT assay,CCK-8 assay daily for 8days to determine cell activity,and analysis growth curve.Impact of PCL capsulation on the mesenchymal stem cell activity studied in vivo, one plate culture with complete culture medium for control group with and two encapsulated transplantation for experimental groups(complete culture medium or basic culture medium for cell suspended) installed,after package and transplantation,retrieving capsulated cells for cell count,MTT assay,CCK-8 assay Daily for 8days to determine cell activity,and analysis growth curve.
Result Typical rat bone marrow-derived mesenchymal stem cells isolated and cultured, 70℃packaging and three minutes 0.25%trypsin digestion retrieve no less than 97%cells in which more than 95%of living cells without obvious injury.Mesenchymal stem cells in experimental group 1 survived well,cell proliferation rate reach to 150%,activity of mesenchymal stem cells in experimental group 2 decreased rapidly and all died within one week,bioactivity of mesenchymal stem cells in experimental group 3 gradually increased after a decline,but cell proliferation rate less than 10%,all three experimental groups have significant differences compare with control group in which proliferation rate is 210%.For in vivo experiment,mesenchymal stem cells in experimental group 1 survived well,cell proliferation rate reach to 140%;bioactivity of mesenchymal stem cells in experimental group 2 gradually increase after a decline,but cell proliferation rate less than 10%,.two experimental groups have significant differences compare with control group in which proliferation rate is 200%
Convludion Retrieve method for encapsulated cells established and cell bioactivity determination methods developed based on cell retrieve,encapsulation in some degree decreased cell activity in vitro and in vivo.We confirmed small molecular substances in serum is of great significance,and is the key to cell survival and growth,macromolecules in serum play a role in proliferation of the cells,lack of serum hinder cell proliferation, intracapsular macromolecules depletion may be one of the reasons for the decline of cells for long-term transplantation.Low molecular weight substances permeability provide a certain time of cell activity,semipermeable membrane immunoisolation technology can be potential method for allograft rejection
方法分离、纯化和培养SD大鼠骨髓间充质干细胞,第三代细胞用于实验研究。细胞悬液置聚己内酯半透膜管内,半透膜管两端70℃热夹闭封装,细胞贴壁后0.25%胰酶消化回收细胞,测定不同消化时间对细胞活性和回收率的影响。以添加10%胎牛血清的完全培养液平皿法细胞培养为对照组,设置3个实验组进行聚己内酯囊管化骨髓间充质干细胞培养活性研究。实验组1:细胞以完全培养液悬浮后封装,完全培养液培养,小分子营养物质能够通过渗透有效补充;实验组2:细胞以完全培养液悬浮后封装,不含血清的基础培养液培养,血清小分子活性物质不能有效补充;实验组3:细胞以基础培养液悬浮后封装,完全培养液培养,小分子营养物质能够通过渗透补充,但细胞缺乏血清大分子的营养。囊管内细胞胰酶消化回收,以细胞计数、MTT法、CCK-8法测定细胞的活性,并进行生长曲线分析。以完全培养液平皿法细胞培养为对照组,设2个实验组进行聚己内酯囊管化骨髓间充质干细胞移植细胞活性研究。两个实验组分别以完全培养液和基础培养液悬浮细胞后封装移植,囊管内细胞回收后以细胞计数、MTT法、CCK-8法测定细胞的活性,共8天,进行生长曲线分析。
结果分离培养得到典型的大鼠骨髓间充质干细胞,细胞活性良好。70℃热封装及采用0.25%胰酶消化3分钟对细胞无明显损伤,活细胞超过95%,细胞回收率不低于97%的。完全培养液悬浮细胞封装,以完全培养液培养的骨髓间充质干细胞成活良好,细胞增殖率达到150%,低于对照组的210%。完全培养液悬浮封装,基础培养液培养,骨髓间充质干细胞活性迅速下降,一周之内全部死亡;基础培养液悬浮封装,以完全培养液培养,骨髓间充质干细胞活性下降,2-3天后逐渐上升,细胞增殖率低于10%,3个实验组与对照组均具有显著性差异。异体移植实验中,完全培养液悬浮和封装后移植的骨髓间充质干细胞增殖率约为140%;较对照组200%为低,基础培养液悬浮细胞封装后细胞活性下降,其后逐渐上升,但增殖率低于10%,2个实验组与对照组均具有显著性差异。
结论建立了囊管化细胞的回收方法和以细胞回收为基础的囊管化细胞活性测定方法,发现囊管化封装对细胞培养与细胞移植均有一定的的抑制作用。血清中小分子活性物质对细胞具有重要的意义,是细胞存活和生长的关键。血清中大分子对细胞活性具有一定的作用,缺乏血清大分子细胞的增殖明显减弱,囊内血清大分子的耗竭可能是半透膜包裹细胞难以长期存活的原因之一。由于低分子物质的通透性是细胞活性的关键,半透膜免疫隔离技术能够保证细胞一定时间内的活性,可以作为解决异体移植排斥反应的一种手段。
Objective To separate and culture rat bone mesenchymal stem cells as study object, establish retrieve method for encapsulated cells and develop cell bioactivity determination methods,investigate the impact of polycaprolactone(PCL) semipermeable membrane tube encapsulation on bioactivity of cells and explore its mechanism,it is expected to offer better help for related research and application.
Method Bone mesenchymal stem cells isolated,purified and cultured from SD rat,third generation cells prepared for experiments.Cell suspension implanted into PCL semipermeable membrane tube and encapsulated at 70 centigrade,cell activity and retrieving rate were measured in varies time of 0.25%trypsin digestion.Impact of PCL capsulation on the mesenchymal stem cells activity studied in vitro studied,one plate culture with complete culture medium for control group and three encapsulated culture for experimental groups installed.Experimental group 1:complete culture medium for cell suspended before package and for culture,in which small molecule nutrients can be maintained through infiltration;experimental group 2:complete culture medium for cell suspended before package and serum free basic culture medium for culture,in which small molecule nutrients can not be supplemented through infiltration;experimental group 3: basic culture medium for cell suspended before package and complete culture medium for culture,in which small molecule nutrients can be supplemented through infiltration without serum macromolecules nutrition included.Retrieving capsulated cells for cell counting, MTT assay,CCK-8 assay daily for 8days to determine cell activity,and analysis growth curve.Impact of PCL capsulation on the mesenchymal stem cell activity studied in vivo, one plate culture with complete culture medium for control group with and two encapsulated transplantation for experimental groups(complete culture medium or basic culture medium for cell suspended) installed,after package and transplantation,retrieving capsulated cells for cell count,MTT assay,CCK-8 assay Daily for 8days to determine cell activity,and analysis growth curve.
Result Typical rat bone marrow-derived mesenchymal stem cells isolated and cultured, 70℃packaging and three minutes 0.25%trypsin digestion retrieve no less than 97%cells in which more than 95%of living cells without obvious injury.Mesenchymal stem cells in experimental group 1 survived well,cell proliferation rate reach to 150%,activity of mesenchymal stem cells in experimental group 2 decreased rapidly and all died within one week,bioactivity of mesenchymal stem cells in experimental group 3 gradually increased after a decline,but cell proliferation rate less than 10%,all three experimental groups have significant differences compare with control group in which proliferation rate is 210%.For in vivo experiment,mesenchymal stem cells in experimental group 1 survived well,cell proliferation rate reach to 140%;bioactivity of mesenchymal stem cells in experimental group 2 gradually increase after a decline,but cell proliferation rate less than 10%,.two experimental groups have significant differences compare with control group in which proliferation rate is 200%
Convludion Retrieve method for encapsulated cells established and cell bioactivity determination methods developed based on cell retrieve,encapsulation in some degree decreased cell activity in vitro and in vivo.We confirmed small molecular substances in serum is of great significance,and is the key to cell survival and growth,macromolecules in serum play a role in proliferation of the cells,lack of serum hinder cell proliferation, intracapsular macromolecules depletion may be one of the reasons for the decline of cells for long-term transplantation.Low molecular weight substances permeability provide a certain time of cell activity,semipermeable membrane immunoisolation technology can be potential method for allograft rejection
引文
1 Chang TM,Johnson LJ,Ransome OJ.Semipermeable aqueous microcapsules.Ⅳ.Nonthrombogenic microcapsules with heparin-complexed membranes.Can J Physiol Pharmacol,1967,45:705-15.
2 Chang TM,MacIntosh FC,Mason SG.Semipermeable aqueous microcapsules.Ⅰ.Preparation and properties.Can J Physiol Pharmacol,1966,44:115-28.
3 Chang TM.Semipermeable aqueous microcapsules(“artificial cells”):with emphasis on experiments in an extracorporeal shunt system.Trans Am Soc Artif Intern Organs,1966,12:13-9.
4 丁惠锋,汤亭亭.微胶囊技术及其在骨科领域的应用.中国修复重建外科杂志,2005,19:1029-1032.
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6 Zondervan GJ,Hoppen HJ,Pennings AJ,et al.Design of polyurethane membrane for Strategy for the encapsulation of islet of Langerhan.Biomaterials,1992,13:136.
7 Ward RS,white KA,woleott CA,et al.Development of a hybrid artificial pancreas with a dense polyurethane membrane.ASAIO,1993,2:261.
8 Delaunay C,Darquy S,Honiger J,et al.Glucose-insulin kinetics of a bioartificial pancreas made of an AN69 hydrogel hollow fiber containing porcine islets and implanted in diabetic mice.Artif Organs,1998,22:291-9.
9 迟勇,罗毅.用聚砜中空纤维隔离的垂体与下丘脑垂体联合移植.中华器官移植杂志,1994,15:25-27.
10 de Groot JH,Zijlstra FM,Kuipers HW,et al.Meniscal tissue regeneration in porous 50/50 copoly(L-lactide/epsilon-caprolactone) implants.Biomaterials,1997,18:613-22.
11 Sun H,Mei L,Song C,et al.The in vivo degradation,absorption and excretion of PCL-based implant.Biomaterials,2006,27:1735-40.
12 de Groot M,Schuurs TA,van Schilfgaarde R.Causes of limited survival of microencapsulated pancreatic islet grafts.J Surg Res,2004,121:141-50.
13 de Vos P,de Haan BJ,de Haan A,et al.Factors influencing functional survival of microencapsulated islet grafts.Cell Transplant,2004,13:515-24.
14 King A,Andersson A,Sandler S.Cytokine-induced functional suppression of microencapsulated rat pancreatic islets in vitro.Transplantation,2000,70:380-3.
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1 Chang TM,Johnson LJ,Ransome OJ.Semipermeable aqueous microcapsules.Ⅳ.Nonthrombogenic microcapsules with heparin-complexed membranes.Can J Physiol Pharmacol,1967,45:705-15.
2 Chang TM,MacIntosh FC,Mason SG.Semipermeable aqueous microcapsules.Ⅰ.Preparation and properties.Can J Physiol Pharmacol,1966,44:115-28.
3 Chang TM.Semipermeable aqueous microcapsules(“artificial cells”):with emphasis on experiments in an extracorporeal shunt system.Trans Am Soc Artif Intern Organs,1966.12:13-9.
4 Zondervan GJ,Hoppen HJ,Pennings AJ,et al.Design of polyurethane membrane for Strategy for the encapsulation of islet of Langerhan.Biomaterials,1992,13:136.
5 Ward RS,white KA,woleott CA,et al.Development of a hybrid artificial pancreas with a dense polyurethane membrane.ASAIO,1993,2:261.
6 Delaunay C,Darquy S,Honiger J,et al.Glucose-insulin kinetics of a bioartificial pancreas made of an AN69 hydrogel hollow fiber containing porcine islets and implanted in diabetic mice.Artif Organs,1998,22:291-9.
7 迟勇,罗毅.用聚砜中空纤维隔离的垂体与下丘脑垂体联合移植.中华器官移植杂志,1994,15:25-27.
8 de Groot M,Schuurs TA,van Schifgaarde R.Causes of limited survival of microencapsulated pancreatic islet grafts.J Surg Res,2004,121:141-50.
9 de Vos P,de Haan BJ,de Haan A,et al.Factors influerlcing functional survival of microencapsulated islet grafts.Cell Transplant,2004,13:515-24.
10 King A,Andersson A,Sandler S.Cytokine-induced functional suppression of microencapsulated rat pancreatic islets in vitro.Transplantation,2000,70:380-3.
11 Chen LG,Qu Y,Xiao J,et al.[Encapsulation of atrial natriuretic peptide(ANP) cDNA transfection cells.and ANP circadian expression].space Med Med Eng(Beijing),2003.16:179-83.
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1 Zondervan GJ,Hoppen HJ,Pennings AJ,et al.Design of polyurethane membrane for Strategy for the encapsulation of islet of Langerhan.Biomaterials,1992,13:136.
2 Ward RS,white KA,woleott CA,et al.Development of a hybrid artificial pancreas with a dense polyurethane membrane.ASAIO,1993,2:261.
3 Delaunay C,Darquy S,Honiger J,et al.Glucose-insulin kinetics of a bioartificial pancreas made of an AN69 hydrogel hollow fiber containing porcine islets and implanted in diabetic mice.Artif Organs,1998,22:291-9.
4 迟勇,罗毅.用聚砜中空纤维隔离的垂体与下丘脑垂体联合移植.中华器官移植杂志,1994,15:25-27.
5 de Groot JH,zijlstra FM,Kuipers HW,et al.Meniscal tissue regeneration in porous 50/50 copoly(L-lactide/epsilon-caprolactone)implants.Biomaterials,1997,18:613-22.
6 Sun H,Mei L,Song C,et.al.The in vivo degradation,absorption and excretion of PCL-based implant.Biomaterials,2006,27:1735-40.
7 陈立国[1],屈艺[1],肖静[1],等.心房肽cDNA转染细胞微囊化及心房肽近日节律性表达.航天医学与医学工程,2003,16:179-183.
1 de Groot JH,zijlstra FM,Kuipers HW,et al.Meniseal tissue regeneration in porous 50/50 copoly(L-lactide/epsilon-caprolactone)implants.Biomaterials,1997,18:613-22.
2 Sun H,Mei L,Song C,et.al.The in vivo degradation,absorption and excretion of PCL-based implant.Biomaterials,2006,27:1735-40.
3 Rohner D,Hutmacher DW,See P,et al.[Individually CAD-CAM technique designed,bioresorbable 3-dimensional polycaprolactone framework for experimental reconstruction of craniofacial defects in the pig].Mund Kiefer Gesichtschir,2002.6:162-7.
4 孙洪范,胡刚.聚己内酯微孔膜囊管化胰组织及其胰岛素释放的观察.中国生物医学工程学报,1995,14:289-291.
5 Chen LG,Qu Y,Xiao J,et al.[Encapsulation of atrial natriuretic peptide(ANP)cDNA transfection cells and ANP circadian expression].Space Med Med Eng(Beijing),2003.16:179-83.
6 陈立国,屈艺,等.微囊化的ANP cDNA转染细胞降低高血压大鼠血压.航天医学与医学工程,2003,16:77-78.
7 Chang TM,Johnson LJ,Ransome OJ.Semipermeable aqueous microcapsules.Ⅳ.Nonthrombogenic microcapsules with heparin-complexed membranes.Can J Physiol Pharmacol,1967,45:705-15.
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9 Chang TM.Semipermeable aqueous microcapsules(“artificial cells”):with emphasis on experiments in an extracorporeal shunt system.Trans Am Soc Artif Intern Organs,1966.12:13-9.
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7 Ward RS,white KA,woleott CA,et al.Development of a hybrid artificial pancreas with a dense polyurethane membrane.ASAIO,1993,2:261.
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