同时分离和培养兔外周血平滑肌祖细胞和内皮祖细胞

南京大学学报(自然科学版) ›› 2010, Vol. 46 ›› Issue (1): 92–99.

同时分离和培养兔外周血平滑肌祖细胞和内皮祖细胞

杨　斌 , 陈　贇 , 周六化 , 孙则禹 , 戴玉田

出版日期:2015-03-27 发布日期:2015-03-27
作者简介: ( 南京大学医学院附属鼓楼医院泌尿外科 , 南京 ,210008)
基金资助:
南京市卫生局医学科技发展重点资助项目 (ZKX07008)

Simultaneous isolation and culture of smooth muscle progenitor cell and endothelial progenitor cell from rabbit peripheral blood
)

Yang Bin , Chen Yun , Zhou L iu 2 Hua , S un Ze 2 Yu , Dai Yu 2 Tian

Online:2015-03-27 Published:2015-03-27
About author: (Department of Urology , Affiliated Drum Tower Hospital of Nanjing University Medical School , Nanjing , 210008 , China

摘要/Abstract

摘要： 同时分离和培养兔外周血平滑肌祖细胞 (SPC) 和内皮祖细胞 (EPC) , 为组织工程膀胱的构建和血管化提供种子细胞 . 分离新西兰兔外周血中的单个核细胞 , 分别进行 SPC 和 EPC 的分离和培养 ; 同
时 , 培养兔膀胱平滑肌细胞 (BSMC) 作为对照 . 细胞爬片 , 观察细胞摄取 Dil - AcLDL 和结合 FITC -UEA-1 的能力 ; 间接免疫荧光染色观察平滑肌肌动蛋白 (SMA) 、结蛋白 (Desmin) 、 KDR 、 eNOS 和 vWF 的表达
情况 ; 进行细胞增殖实验 , 观察 PDGF -BB ,VEGF 对 SPC 和 EPC 的增殖作用 . 结果发现 , 培养 1 周后出现 SPC 克隆和 EPC 克隆 ,SPC 呈梭形 , 长短不一 ;EPC 形态均一 , 呈典型的鹅卵石形 ; 培养的 BSMC 形
态均一 , 为长梭形 , 呈峰谷样形态 . 利用克隆环分离 SPC 和 EPC 克隆 , 继续培养可得到高纯度的 SPC 和EPC. SPC 不摄取 Dil - AcLDL , 不结合 FITC - UEA - 1 ;SPC 表达 SMA 、 Desmin 和 KDR , 不表达 eNOS 和
vWF. EPC 同时摄取 Dil - AcLDL 和结合 FITC -UEA- 1 ; EPC 表达 eNOS 、 vWF 和 KDR , 不表达 SMA 和Desmin.BSMC 仅表达 SMA 和 Desmin. PDGF - BB 仅能促进 SPC 增殖 ,VEGF 仅能促进 EPC 增殖 , 均具
有剂量依赖效应 . 本研究建立了同时分离和培养外周血 SPC 和 EPC 的技术 , 所培养的细胞具有较高的纯度 , 可以作为种子细胞进行组织工程膀胱及其血管化的研究和应用

Abstract: The objective of this study was to simultaneously isolate and culture smooth muscle progenitor cell (SPC) and endothelial progenitor cell (EPC) from rabbit peripheral blood for use as cell sources in tissue engineering
of bladder and vascularization. In this study , SPC and EPC were simultaneously isolated and cultured from the peripheral blood of New Zealand Rabbit , and rabbit bladder smooth muscle (BSMC) were cultured and used as
smooth muscle cell positive control. The cells were cultured on coverslips and applied for uptake of Dil -AcLDL and binding of FITC -UEA - 1. Indirect immunofluorescent staining was performed to characterize the cultured cells using
monoclonal antibodies against smooth muscle actin (SMA) , Desmin , KDR , eNOS and vWF. Cell proliferation assay was also used to investigate the mitogenic effect of PDGF 2 BB and VEGF on both the SPC and EPC. Both the SPC
and EPC clones emerged after one week of culture. The SPC displayed fusiform shape with different size. The EPC showed the cobblestone morphology typical of endothelial cells. The BSMC cultured from rabbit exhibited the
elongated shape and hill and valleys pattern before confluence. The cluster of SPC and EPC were isolated and ubcultured using cloning cylinders , after which the highly purified SPC and EPC had been obtained. The SPC
display no ability of uptake of Dil -AcLDL and binding of FITC -UEA - 1 , while the EPC took - up Dil- AcLDL and showed FITC-UEA -1 binding affinity. In indirect immunofluorescent staining , the SPC demonstrated positive
staining for SMA , Desmin and KDR , while negative staining for eNOS and vWF. The EPC showed positive staining for KDR , eNOS and vWF , while negative staining for SMA and Desmin. The BSMC stained only positive for SMA
and Desmin. PDGF-BB had dose-dependent mitogenic effect on SPC , and VEGF had dose -dependent mitogenic effect on EPC. In conclusion , the highly purified SPC and EPC had been simultaneously isolated and cultured from rabbit
peripheral blood , and can be used as cell sources for tissue engineering of bladder and vascularization.

　

杨　斌 , 陈　贇 , 周六化 , 孙则禹 , 戴玉田. 同时分离和培养兔外周血平滑肌祖细胞和内皮祖细胞

[J]. 南京大学学报(自然科学版), 2010, 46(1): 92–99.

Yang Bin , Chen Yun , Zhou L iu 2 Hua , S un Ze 2 Yu , Dai Yu 2 Tian
. Simultaneous isolation and culture of smooth muscle progenitor cell and endothelial progenitor cell from rabbit peripheral blood
)[J]. Journal of Nanjing University(Natural Sciences), 2010, 46(1): 92–99.

参考文献

[ 1 ] 　 Griffith L G, Naughton G. Tissue engineer -ing — Current challenges and expanding opportu -nities. Science , 2002 ,295(5557) :1009 ～ 1014.
[ 2 ] 　 Atala A , Bauer S B , Soker S , et al . Tissue - en-gineered autologous bladders for patients nee-ding cystoplasty. Lancet , 2006 , 367 ( 9518) : 1241 ～ 1246.
[ 3 ] 　 Baker S C , Southgate J. Towards control of smooth muscle cell differentiation in synthetic 3D scaffolds. Biomaterials , 2008 , 29 ( 23 ) : 3357 ～ 3366.
[ 4 ] 　 Frimberger D , Lin H K, Kropp B P. The use of tissue engineering and stem cells in bladder regeneration. Regenerative Medicine , 2006 , 1(4) :425 ～ 435.
[ 5 ] 　 Kanematsu A , Yamamoto S , Ogawa O. Chan -ging concepts of bladder regeneration. Interna -tional Journal of Urology , 2007 , 14 ( 8 ) : 673 ～ 678.
[ 6 ] 　 Lin H K, Cowan R , Moore P , et al . Charac -terization of neuropathic bladder smooth muscle cells in culture. The Journal of Urology , 2004 , 171(3) :1348 ～ 1352.
[ 7 ] 　 Ma N T , Gao P J. Research advancement of smooth muscle progenitor cell. Progress in Physiological Sciences , 2008 ,39 (1) : 87 ～ 90. ( 马宁涛 , 高平进 . 平滑肌祖细胞研究进展 . 生理科学进展 , 39(1) :87 ～ 90) .
[ 8 ] 　 Simper D , Stalboerger P G, Panetta C J , et al . Smooth muscle progenitor cells in human blood. Circulation , 2002 ,106(10) :1199 ～ 1204.
[ 9 ] 　 Yamashita J , Itoh H , Hirashima M , et al . Flk1 - positive cells derived from embryonic stem cells serve as vascular progenitors. Nature , 2000 ,408(6808) :92 ～ 96.
[10] 　 Levenberg S , Rouwkema J , Macdonald M , et al . Engineering vascularized skeletal muscle tis -sue. Nature Biotechnology , 2005 , 23 ( 7 ) : 879 ～ 884.
[11] 　 Ott H C , Matthiesen T S , Goh S K, et al . Per -fusion - decellularized matrix: Using nature ’ splatform to engineer a bioartificial heart. Nature Medicine , 2008 ,14(2) :213 ～ 221.
[12] 　 Khakoo A Y, Finkel T. Endothelial progenitor cells. Annual Review of Medicine , 2005 , 56 : 79 ～ 101.
[13] 　 Wu X, Rabkin -Aikawa E , Guleserian K J , et al . Tissue - engineered microvessels on three- di- mensional biodegradable scaffolds using human
endothelial progenitor cells. The American Journal of Physiology- Heart and Circulatory Physiology , 2004 ,287(2) :480 ～ 487.
[14] 　 Shepherd B R , Enis D R , Wang F , et al . Vas -cularization and engraftment of a human skin substitute using circulating progenitor cell - de -rived endothelial cells. Journal of the Federation
of American Societies for Experimental Biology , 2006 ,20(10) :1739 ～ 1741.
[15] 　 Xie S T , Chen B , Tao K, et al . Construction and transplantation of tissue engineering skin with EPCs in athymic mice. Chinese Journal of Experimental Surgery , 2006 , 23 (12) : 1531 ～
1532. ( 谢松涛 , 陈　璧 , 陶　克等 . 含内皮祖细胞的组织工程皮肤体外构建及裸鼠移植研究 . 中华实验外科杂志 , 2006 , 23 (12) : 1531 ～ 1532) .
[16] 　 Kalka C , Masuda H , Takahashi T , et al . Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovasculariza -tion. The Proceedings of the National Academy of Sciences USA , 2000 ,97(7) :3422 ～ 3427.
[17] 　 Ma L L , Huang H M , Jiang Z M , et al . Differ -entiation of endothelial progenitor cells into smooth muscle cells. Acta Universitatis Medici -nalis Secondae Shanghai , 2006 , 26 (8) : 865 ～
868. ( 马良龙 , 黄惠民 , 蒋祖明等 . 内皮祖细胞定向分化平滑肌细胞的实验研究 . 上海交通大学学报 ( 医学版 ) , 2006 ,27(8) :865 ～ 868) .
[18] 　 Broxmeyer H E , Srour E , Orschell C , et al .Cord blood stem and progenitor cells. Methods Enzymol , 2006 ,419 :439 ～ 473.

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed