异体骨髓间充质干细胞促进扩张皮肤新生的研究
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摘要
目的：探讨异体骨髓间充质干细胞(Bone marrow mesenchymal stem cells, BM-MSCs)在扩张皮肤中发挥的作用,为临床更好的使用皮肤扩张术提供新的方法。
方法：无菌条件下解剖乳猪双侧股骨,冲出骨髓后淋巴细胞分离液分离,接种于培养基中。取培养第三至四代的BM-MSCs,分别加入FITC标记的鼠抗猪CD90、CD34、CD45、CD29抗体,PBS重悬后上流式细胞仪检测。16头小型家猪随机分为4组,每组4头。A组：埋植扩张器后,局部皮下注射细胞悬液；B组：埋植扩张器后,耳缘静脉注射细胞悬液；C组：仅埋植扩张器；D组：空白对照组。在猪的脊柱两侧各设计3个皮瓣,皮瓣中心上文出4×4 cm2大小面积。距脊柱2 cm作纵行切口,按扩张器基底部大小在深筋膜浅层分离出4×6 cm2的皮下腔隙,置入80ml矩形单向扩张器。各组每隔三天注水一次,每次注水10ml。扩张后第7,14,28天分别测量各组标记面积,取出扩张器后即时测量标记面积。取各组扩张第28天的皮肤标本,检测4组皮肤血管内皮细胞、增殖细胞核抗原、表皮干细胞的表达量,分别用抗体CD31、PCNA(proliferating cell nuclear antigen)、K19(epidermal stem keratinocytes)进行免疫荧光染色标记。取各组扩张第14天的皮肤组织,实时定量PCR分析目的基因血管内皮细胞生长因子(vascular endothelial cell growth factor, VEGF)、表皮生长因子(epidermal growth factor, EGF)、碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)及基质细胞衍生因子-1 (Stromal cell-derived factor-1, SDF-1)相对表达量。
结果：体外培养的BM-MSCs呈多角形或梭形,贴壁细胞呈旋涡状、网状、辐射状排列生长。流式细胞检测结果表明BM-MSCs不表达CD34和CD45,但表达CD29和CD90。随着扩张时间延长,各组标记面积增加,A、B、C组扩张明显,与D组相比有统计学意义(P<0.01)。取出扩张器后测量标记面积,A、B、C组均有一定程度回缩,回缩后的剩余面积A、B组仍大于C组,A组与C组比较有统计学意义(P<0.05)。随着扩张时间延长,表皮层明显增厚,细胞层次增多,以A组扩张第28天最明显,扩张后真皮层变薄,胶原纤维排列松散,纤维间隙增加。A组扩张皮肤第7、14、28天均可看到明显的DiI荧光标记细胞分布,主要分布在真皮下层。随着时间延长,B组皮肤中DiI细胞分布增多,主要在真皮层。CD31检测：A.B.C.D四组血管断面密度(条/视野)分别为9.1±2.31、7.85±1.93、6.5±1.64、4.2±1.54。PCNA检测：A.B.C.D四组PCNA阳性细胞(个/视野)分别为163.25±35.4、108.62±8.93、52±8.07、13.63±3.96。K19检测：A.B.C.D四组K19阳性细胞(个/视野)分别为42±5.24、31.75±4.33、20.88±3.27、13.75±2.38,各组之间比较均具有统计学意义(P<0.01)。实时定量PCR检查结果示VEGF、SDF-1、EGF、bFGF的表达差异倍数,A组比B组分别为为5.4、1.07、1.21、6.03倍。
结论：
1、局部及全身移植BM-MSCs于扩张器模型可以促进扩张皮肤的生物性生长。
2、局部移植组效果好于全身移植组,是一种比较好的移植方案。
Objective:To investigate the feasibility of transplantation of mesenchymal stem cells into expanion skin.
Methods:The femurs of the porket were harvested in asterile fashion and the marrow was flushed with phosphate buffered saline (PBS).The mononuclear cell layer of the density medium's upper layer was collected to begin the culture. Passage 3 BM-MSCs were resuspended in PBS. Cell aliquots were incubated with fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies specific for CD29, CD90, CD34 and CD45.Cells cultured were analyzed by fluorescence-activated cell sorting.Sixteen pigs divided into four groups randomly with four each. In Group A. each flap received suspended stem cells around the wound. In Group B, stem cells in medium were injected into the ear vein. In Group C, each wound received medium and Group D was the blank control group.After hair removal from the dorsal surface and anesthesia, six flaps were marked on either side of the spine midline by injection of ink in dermal and the area was 4cm×4cm.The 80 ml silicone expanders were implanted under the flap as described previously. The animals were injected the normal saline (N. S.) (10 ml every three days until day 28).The marked area was measured at day 7,14,28 after the expansion. After the expanders were removed, the area was measured immediately. Skin tissue specimens taken after 28 days of expansion were tested the content of vascular endothelial cells, proliferating cell nuclear antigen, and the epidermal stem keratinocytes. They were identified with an antibody against CD31、PCNA and K19 respectively, compare the positive cells between four groups.Skin tissue specimens taken after 14 days of expansion were tested the target gene expression of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and stromal cell-derived factor-1 (SDF-1) by real-time PCR.
Result:Mesenchymal stem cells appeared morphologically to be ahomogenous population of fibroblast shaped cells. The results showed that the BM-MSCs did not express CD34 nor CD45. However, almost all the cells expressed CD29 and CD90.With the expansion prolonged, the marked area in each group increased. Compared to Group D, the marked areas of Groups A, B and C increased significantly (P<0.01). Measurements of the marked area after removing the expander showed that Groups A, B, and C all had different degrees of retraction, and the remained areas of Group A and Group B after retraction were still larger than that of Group C. Group A's was statistically larger than Group C's (P <0.05).BM-MSCs labeled with CM-DiI were observed throughout the subcutaneous fascial layer of flaps on day 7,14, and 28 after the implantation. There was obviously distribution in group A. With the extending of expansion, the cells labeled with CM-DiI increased in group B, which were mainly distributed in the dermis. None CM-DiI-Labeled BM-MSCs were detected in groups C and D. The vascular cross section densities (article/field) of groups A, B, C and D were 9.1±2.31,7.85±1.93,6.5±1.64,4.2±1.54 respectively. PCNA staining showed that positive cells in group A were 5-6 layers under the base of Epidermis, while there were 2-3 layers for group B and there were only scattered single cells for group C. K19 staining showed that under the base of epidermis there were full thickness positive cells for group A and part of the stratified region, while the continuous cells distribution were seen in group B, and there were only scattered cells in group C. Real-time quantitative PCR test results showed the multiples of difference in target gene expression of VEGF, SDF, EGF and bFGF were 5.4,1.07,4.03 and 6.03 times respectively for group A, as compared with group B.
Conclusion:
1、Transplant of BM-MSCs into local or systemic of expansion model can promote the growth of biological skin.
2、local transplantation achieves better results than systemic transplantation.
方法：无菌条件下解剖乳猪双侧股骨,冲出骨髓后淋巴细胞分离液分离,接种于培养基中。取培养第三至四代的BM-MSCs,分别加入FITC标记的鼠抗猪CD90、CD34、CD45、CD29抗体,PBS重悬后上流式细胞仪检测。16头小型家猪随机分为4组,每组4头。A组：埋植扩张器后,局部皮下注射细胞悬液；B组：埋植扩张器后,耳缘静脉注射细胞悬液；C组：仅埋植扩张器；D组：空白对照组。在猪的脊柱两侧各设计3个皮瓣,皮瓣中心上文出4×4 cm2大小面积。距脊柱2 cm作纵行切口,按扩张器基底部大小在深筋膜浅层分离出4×6 cm2的皮下腔隙,置入80ml矩形单向扩张器。各组每隔三天注水一次,每次注水10ml。扩张后第7,14,28天分别测量各组标记面积,取出扩张器后即时测量标记面积。取各组扩张第28天的皮肤标本,检测4组皮肤血管内皮细胞、增殖细胞核抗原、表皮干细胞的表达量,分别用抗体CD31、PCNA(proliferating cell nuclear antigen)、K19(epidermal stem keratinocytes)进行免疫荧光染色标记。取各组扩张第14天的皮肤组织,实时定量PCR分析目的基因血管内皮细胞生长因子(vascular endothelial cell growth factor, VEGF)、表皮生长因子(epidermal growth factor, EGF)、碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)及基质细胞衍生因子-1 (Stromal cell-derived factor-1, SDF-1)相对表达量。
结果：体外培养的BM-MSCs呈多角形或梭形,贴壁细胞呈旋涡状、网状、辐射状排列生长。流式细胞检测结果表明BM-MSCs不表达CD34和CD45,但表达CD29和CD90。随着扩张时间延长,各组标记面积增加,A、B、C组扩张明显,与D组相比有统计学意义(P<0.01)。取出扩张器后测量标记面积,A、B、C组均有一定程度回缩,回缩后的剩余面积A、B组仍大于C组,A组与C组比较有统计学意义(P<0.05)。随着扩张时间延长,表皮层明显增厚,细胞层次增多,以A组扩张第28天最明显,扩张后真皮层变薄,胶原纤维排列松散,纤维间隙增加。A组扩张皮肤第7、14、28天均可看到明显的DiI荧光标记细胞分布,主要分布在真皮下层。随着时间延长,B组皮肤中DiI细胞分布增多,主要在真皮层。CD31检测：A.B.C.D四组血管断面密度(条/视野)分别为9.1±2.31、7.85±1.93、6.5±1.64、4.2±1.54。PCNA检测：A.B.C.D四组PCNA阳性细胞(个/视野)分别为163.25±35.4、108.62±8.93、52±8.07、13.63±3.96。K19检测：A.B.C.D四组K19阳性细胞(个/视野)分别为42±5.24、31.75±4.33、20.88±3.27、13.75±2.38,各组之间比较均具有统计学意义(P<0.01)。实时定量PCR检查结果示VEGF、SDF-1、EGF、bFGF的表达差异倍数,A组比B组分别为为5.4、1.07、1.21、6.03倍。
结论：
1、局部及全身移植BM-MSCs于扩张器模型可以促进扩张皮肤的生物性生长。
2、局部移植组效果好于全身移植组,是一种比较好的移植方案。
Objective:To investigate the feasibility of transplantation of mesenchymal stem cells into expanion skin.
Methods:The femurs of the porket were harvested in asterile fashion and the marrow was flushed with phosphate buffered saline (PBS).The mononuclear cell layer of the density medium's upper layer was collected to begin the culture. Passage 3 BM-MSCs were resuspended in PBS. Cell aliquots were incubated with fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies specific for CD29, CD90, CD34 and CD45.Cells cultured were analyzed by fluorescence-activated cell sorting.Sixteen pigs divided into four groups randomly with four each. In Group A. each flap received suspended stem cells around the wound. In Group B, stem cells in medium were injected into the ear vein. In Group C, each wound received medium and Group D was the blank control group.After hair removal from the dorsal surface and anesthesia, six flaps were marked on either side of the spine midline by injection of ink in dermal and the area was 4cm×4cm.The 80 ml silicone expanders were implanted under the flap as described previously. The animals were injected the normal saline (N. S.) (10 ml every three days until day 28).The marked area was measured at day 7,14,28 after the expansion. After the expanders were removed, the area was measured immediately. Skin tissue specimens taken after 28 days of expansion were tested the content of vascular endothelial cells, proliferating cell nuclear antigen, and the epidermal stem keratinocytes. They were identified with an antibody against CD31、PCNA and K19 respectively, compare the positive cells between four groups.Skin tissue specimens taken after 14 days of expansion were tested the target gene expression of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and stromal cell-derived factor-1 (SDF-1) by real-time PCR.
Result:Mesenchymal stem cells appeared morphologically to be ahomogenous population of fibroblast shaped cells. The results showed that the BM-MSCs did not express CD34 nor CD45. However, almost all the cells expressed CD29 and CD90.With the expansion prolonged, the marked area in each group increased. Compared to Group D, the marked areas of Groups A, B and C increased significantly (P<0.01). Measurements of the marked area after removing the expander showed that Groups A, B, and C all had different degrees of retraction, and the remained areas of Group A and Group B after retraction were still larger than that of Group C. Group A's was statistically larger than Group C's (P <0.05).BM-MSCs labeled with CM-DiI were observed throughout the subcutaneous fascial layer of flaps on day 7,14, and 28 after the implantation. There was obviously distribution in group A. With the extending of expansion, the cells labeled with CM-DiI increased in group B, which were mainly distributed in the dermis. None CM-DiI-Labeled BM-MSCs were detected in groups C and D. The vascular cross section densities (article/field) of groups A, B, C and D were 9.1±2.31,7.85±1.93,6.5±1.64,4.2±1.54 respectively. PCNA staining showed that positive cells in group A were 5-6 layers under the base of Epidermis, while there were 2-3 layers for group B and there were only scattered single cells for group C. K19 staining showed that under the base of epidermis there were full thickness positive cells for group A and part of the stratified region, while the continuous cells distribution were seen in group B, and there were only scattered cells in group C. Real-time quantitative PCR test results showed the multiples of difference in target gene expression of VEGF, SDF, EGF and bFGF were 5.4,1.07,4.03 and 6.03 times respectively for group A, as compared with group B.
Conclusion:
1、Transplant of BM-MSCs into local or systemic of expansion model can promote the growth of biological skin.
2、local transplantation achieves better results than systemic transplantation.
引文
[1]丁锐,谭敏,傅晓源,柳辉.皮肤扩张后修复过程的实验观察研究.国际医药卫生导报.2007,13(20)：14-16
[2]Herzog EL, Chai L, and Krause DS. Plasticity of marrow-derived stem cells. Blood. 2003,102:3483-3493
[3]Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science,1997,276:71-74
[4]Wu Y, Wang J, Scott PG, Tredget EE. Bone marrow-derived stem cells in wound healing:a review. Wound Repair Regen.2007,15 Suppl 1:S 18-26
[5]Fox JM, Chamberlain G, Ashton BA, Middleton J. Recent advances into the understanding of mesenchymal stem cell trafficking. Br J Haematol.2007,137(6):491-502
[6]Bauer SM, Bauer RJ, Liu ZJ, Chen H, Goldstein L, Velazquez OC. Vascular endothelial growth factor-C promotes vasculogenesis, angiogenesis, and collagen constriction in three-dimensional collagen gels. J Vasc Surg.2005,41(4):699-707
[7]Abe R, Donnelly SC, Peng T, Bucula R, Metz CN. Peripheral blood fibrocytes: differentiation pathway and migration to wound sites. J Immunol.2001.166:7556-7662
[8]Fathke C, Wilson L, Hutter J, Kapoor V, Smith A, Hocking A, Isik F. Contribution of bone marrow-derived cells to skin:collagen deposition and wound repair. Stem Cells.2004. 22:812-822.
[9]何觅春,李静,赵春华.低氧对间充质干细胞的影响.中国实验血液学杂志.2007,15(2)：433-436.
[10]Branski LK, Gauglitz GG, Herndon DN, Jeschke MG. A review of gene and stem cell therapy in cutaneous wound healing. Burns,2009,35(2):171-80.
[11]Nakagawa H, Akita S, Fukui M, Fujii T. Akino K. Human mesenchymal stem cells successfully improve skin substitute wound healing. Br J Dermatol 2005;153(1):29-36. [12] Hao L. Wang J, Zou Z. Yan G, Dong S, Deng J, Ran X, Feng Y, Luo C, Wang Y, Cheng T. Transplantation of BMSCs expressing hPDGF-A/hBD2 promotes wound healing in rats with combined radiation-wound injury. Gene Ther.2009,16(1):34-42.
[13]Falanga V, Iwamoto S, Chartier M, Yufit T, Butmarc J, Kouttab N, Shrayer D, Carson P. Autologous bone marrow-derived cultured mesenchymal stem cells delivered in a fibrin spray accelerate healing in murine and human cutaneous wounds. Tissue Eng,2007, 13(6):1299-312
[14]Chen L, Tredget EE, Wu PY, Wu Y. Paracrine factors of mesenchymal stem cells recruit macrophages and endothelial lineage cells and enhance wound healing. PLoS ONE. 2008,2;3(4):e1886.
[15]Velazquez OC. Angiogenesis and vasculogenesis:inducing the growth of new blood vessels and wound healing by stimulation of bone marrow-derived progenitor cell mobilization and homing. J Vasc Surg.2007,45 Suppl A:A39-47
[16]De Filippo RE, Atala A. Stretch and growth:the molecular and physiologic influences of tissue ex pansion. Plast Reconstr Surg.2002; 109(7):2450-62.
[17]Melis P, Noorlander ML, van der Horst CM, van Noorden CJ. Rapid alignment of collagen fibers in the dermis of undermined and not undermined skin stretched with a skin-stretching device. Plast Reconstr Surg.2002; 109(2):674-80.
[18]Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature.2002,418:41-49.
[19]Silva GV, Litovsky S, Assad JA, et al. Mesenchymal stem cells differentiate into an endothelial phenotype, enhance vascular density, and improve heart function in a canine chronic ischemia model. Circulation.2005,111(2):150-6.
[20]Aoto K. Shikata Y, Imai H.et al. Mouse Shh is required for prechordal plate maintenance during brain and craniofacial morphogenesis. Dev Biol.2009; 327(1):106-120.
[21]Hemmrich K, Meersch M, vonHeimburg D, et al. Applicability of the dyes CFSE, CM-Dil and PKH26 for tracking of human preadipocytes to evaluate adipose tissueen gineering [J]. Cells Tissues Organs,2006.184(3—4):117—127.
[22]Siegert R, Weerda H, Loffler JA, et al. Epidermal proliferation rate after skin expansion in the dog model. Laryng orhinootologie,1994,74:2062208.
[23]Badiavas EV, Abedi M, Butmarc J, Falanga V, Quesenberry P. Participation of bone marrow derived cells in cutaneous wound healing.J Cell Physiol.2003,196:245-250.
[24]Kataoka K, Medina RJ, Kageyama T, Miyazaki M, Yoshino T, Makino T, Huh NH. Participation of adult mouse bone marrow cells in reconstitution of skin. Am J Pathol.2003, 163:1227-1231.
[25]Takasak IY, Kaneda K, Takeuehi K, el al. Analysis Of the structure of proteasonm proliferating cell nuclear antigen(PCNA) multiprotein complex and its autoimmune response in lupus patients[J].Ann N Y Acad Sci,2003,987(3):316-318.
[26]Janes SM, Lowell S, Hutter C. Epidermal stem cells. J Pathol,2002,197(4):479. [27] Amado LC, Saliaris AP, Schuleri KH, et al. Cardiac repair with intramyocardial injection of allogeneic mesenchymal stem cells after myocardial infarction. Proc Natl Acad Sci,2005; 102(32):11474-11479.
[28]李海红,付小兵,王君,等.骨髓间充质干细胞分化为皮肤附属器细胞的初步实验研究。中国修复重建外科杂志.2006,6(20)：675-678.
[29]Bhora FY, Dunkin BJ, Batzri S, et al. Effect of growth factors on cell proliferation and epithlialization in human skin. J Surg Res,1995,59(2):236-244.
[30]Wang HJ, Wan HL, Yang TS et al. Acceleration of skin graft healing by growth factors. Burns,1996,22(1):10-14.
[31]Cohen S and Carpenter G. Human epidermal growth factor:isolation and chemical and biological properties. Proc Natl Acad Sci,1975,72(4):1317-1321.
[32]Esch F, Baird A, ling N. et al. Primary structure of bovine pituitary basic fibroblast growth factor (FGF) and comparison with the amino-terminal sequence of bovine brain acidic FGF. Proc Natl Acad Sci USA,1985,82(19):6507-6511.
[33]Abbott JD, Huang Y, Liu DG, et al. Stromal cell-derived factor-1α plays a critical role in stem cell recruitment to the heart after myocardial infarction but is not sufficient to induce homing in the absence of injury. Circulation 2004; 110(21):3300-3305.
[34]Bhakta S, Hong P, Koc O. The surface adhesion molecule CXCR4 stimulates mesenchymal stem cell migration to stromal cell-derived factor-1 in vitro but does not decrease apoptosis under serum deprivation. Cardiovasc Revase Med 2006; 7:19-24.
[35]Kraitchman DL, Tat sumi M, Gilson WD, et al. Dynamic imaging of allogeneic mesenchymal stem cells trafficking to myocardial infarction [J]. Circulation,2005,112: 1451-1461.
[1]鲁开化,郭树忠,韩岩：马显杰等.皮肤软组织扩张术临床应用经验总结.中国美容医学,2007,16(11)：1476-1480
[2]丁锐.谭敏,傅晓源,柳辉.皮肤扩张后修复过程的实验观察研究.国际医药卫生导报,2007, 13(20)：14-16
[3]Sellheyer K, Krahl D. Skin mesenchymal stem cells:prospects for clinical dermatology. J Am Acad Dermatol.2010,63(5):859-65.
[4]Zouboulis CC, Adjaye J, Akamatsu H, Moe-Behrens G, Niemann C. Human skin stem cells and the ageing process. Exp Gerontol.2008,43(11):986-97.
[5]孙志刚,郭树忠,鲁开化,韩岩.皮肤伸展术中新增皮肤来源的实验研究.中国美容医学,2003,12(3)：231-233
[6]De Filippo RE, Atala A. Stretch and growth:the molecular and physiologic influences of tissue expansion. Plast Reconstr Surg.2002;109(7):2450-62.
[7]Melis P, Noorlander ML, van der Horst CM, van Noorden CJ. Rapid alignment of collagen fibers in the dermis of undermined and not undermined skin stretched with a skin-stretching device. Plast Reconstr Surg.2002;109(2):674-80.
[8]刘学军,张海明,孙广慈,胡华新.超量扩张皮肤的血流量改变与安全性的实验研究.中华整形外科杂志,2005,21(6)：433-6
[9]刘文阁,季莹,杨佩瑛等.曲安缩松加速组织扩张的临床应用研究.中华整形烧伤外科杂志.1999；15(1)：41-42
[10]Tang Y, Luan J, Zhang X. Accelerating tissue expansion by application of topical papaverine cream. Plast Reconstr Surg.2004; 114(5):1166-9.
[11]陕声国,张端莲,吴小蔚等.联合性快速皮肤软组织扩张法的研究.中华实验外科杂志,2002,19(4)：373-375
[12]Shi CM, Cheng TM, Su YP, et al. Effects of dermal multipotent cell transplantation on skin wound healing. J Surg Res,2004,121:13.
[13]Herzog EL, Chai L, and Krause DS. Plasticity of marrow-derived stem cells. Blood.2003,102:3483-3493
[14]Wu Y. Chen L, Scott PG, Tredget EE. Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis. Stem Cells.2007,25(10):2648-59
[15]李江,鲁开化,艾玉峰,郭树忠.持续恒压扩张和常规间断扩张后皮肤超微结构的改变.中华整形外科杂志,2002,18(6)：365-366
[16]Herzog EL. Chai L, and Krause DS. Plasticity of marrow-derived stem cells. Blood.2003,102:3483-3493
[17]Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science,1997.276:71-74
[18]Herzog EL, Chai L, and Krause DS. Plasticity of marrow-derived stem cells. Blood.2003,102:3483-3493
[19]Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science,1997.276:71-74
[20]Wu Y, Wang J, Scott PG, Tredget EE. Bone marrow-derived stem cells in wound healing:a review. Wound Repair Regen.2007,15 Suppl 1:S18-26
[21]Fox JM, Chamberlain G, Ashton BA, Middleton J. Recent advances into the understanding of mesenchymal stem cell trafficking. Br J Haematol.2007,137(6):491-502
[22]Bauer SM, Bauer RJ, Liu ZJ, Chen H, Goldstein L, Velazquez OC. Vascular endothelial growth factor-C promotes vasculogenesis, angiogenesis, and collagen constriction in three-dimensional collagen gels. J Vasc Surg.2005,41(4):699-707
[23]Abe R, Donnelly SC, Peng T, Bucula R, Metz CN. Peripheral blood fibrocytes: differentiation pathway and migration to wound sites. J Immunol.2001.166:7556-7662
[24]Fathke C, Wilson L, Hutter J, Kapoor V, Smith A, Hocking A, Isik F. Contribution of bone marrow-derived cells to skin:collagen deposition and wound repair. Stem Cells.2004.22:812-822.
[25]何觅春,李静,赵春华.低氧对间充质干细胞的影响.中国实验血液学杂志.2007,15(2)：433-436.
[26]Li C, Zheng Y, Xia W, Wang X, Gao H, Li D, Ma X. Bone marrow-derived stem cells contribute skin regeneration in skin and soft tissue expansion.J Cell Physiol. 2011 Feb 1 [Epub ahead of print]
[27]Branski LK, Gauglitz GG, Herndon DN, Jeschke MG. A review of gene and stem cell therapy in cutaneous wound healing. Burns,2009,35(2):171-80.
[28]Nakagawa H, Akita S, Fukui M, Fujii T, Akino K. Human mesenchymal stem cells successfully improve skin substitute wound healing. Br J Dermatol 2005;153(1):29-36.
[29]Hao L, Wang J, Zou Z, Yan G, Dong S, Deng J, Ran X, Feng Y, Luo C, Wang Y, Cheng T. Transplantation of BMSCs expressing hPDGF-A/hBD2 promotes wound healing in rats with combined radiation-wound injury. Gene Ther.2009,16(1):34-42.
[30]Falanga V, Iwamoto S, Chartier M, Yufit T, Butmarc J, Kouttab N, Shrayer D, Carson P. Autologous bone marrow-derived cultured mesenchymal stem cells delivered in a fibrin spray accelerate healing in murine and human cutaneous wounds. Tissue Eng.2007, 13(6):1299-312
[31]Wu Y, Chen L, Scott PG, Tredget EE. Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis. Stem Cells.2007, 25(10):2648-59.
[32]Chen L, Tredget EE, Wu PY, Wu Y. Paracrine factors of mesenchymal stem cells recruit macrophages and endothelial lineage cells and enhance wound healing. PLoS ONE.2008,2;3(4):e1886.
[33]Velazquez OC. Angiogenesis and vasculogenesis:inducing the growth of new blood vessels and wound healing by stimulation of bone marrow-derived progenitor cell mobilization and homing. J Vasc Surg.2007,45 Suppl A:A39-47
[34]王晓燕,郑岩,李翅翅,等.异体骨髓间充质干细胞促进扩张皮肤新生的研究.中华医学美学与美容杂志.2011,待发表.
[35]Shi C, Zhu Y, Su Y, Cheng T. Stem cells and their applications in skin-cell therapy. Trends Biotechnol.2006,24(1):48-52
[36]Brouard M, Barrandon Y. Controlling skin morphogenesis:hope and despair. Curr Opin Biotechnol,2003,14:520.
[37]金岩,董蕊.皮肤干细胞的生物学特点与应用研究.基础医学与临床,2005,,25(10)：878-882
[38]刘洋,李建福,付小兵.皮肤软组织扩张术对大鼠表皮细胞增殖分化的影响.第三军医大学学报,2006,28(15)：1557-]559
[39]李幼忱,刘杰,王德文.毛囊真皮鞘细胞的生物学特性研究进展.生物医学工程学杂志,2007,24(3)：694-696
[40]祁少海,柯昌能,利天增等.真皮源干细胞复合人血浆促进皮肤创面神经修复的实验研究.中华显微外科杂志,2006,29(4)：282-283
[41]Toma JG, McKenzie IA, Bagli D, Miller FD. Isolation and characterization of multipotent skin-derived precursors from human skin. Stem Cells.2005,23(6):727-37
[42]Gorio A, Torrente Y, Madaschi L, et al. Fate of autologous dermal stem cells transplanted into the spinal cord after traumatic injury (TSCI). Neuroscience,2004, 25:179
[43]Gharzi A, Reynolds AJ, Jahoda CA. Plasticity of hair follicle dermal cells in wound healing and induction. Exp Dermatol.2003,12(2):126-36.
[44]Hoogduijn M.T, Gorjup E, Genever PG. Comparative Characterization of Hair Follicle Dermal Stem Cells and Bone Marrow Mesenchymal Stem Cells. Stem Cells Dev,2006,15(1):49-60.
[45]Chunmeng S, Tianmin C. Effects of plastic-adherent dermal multipotent cells on peripheral blood leukocytes and CFU-GM in rats. Transplantat Proceedings,2004,36: 1578.
[46]史春梦,程天民.创伤局部微环境对大鼠真皮多能干细胞生物学性状的影响.中华创伤杂志,2003, 19(4)：199-202
[47]Richardson GD, Arnott EC, Whitehouse CJ, et al. Plasticity of rodent and human hair follicle dermal cells:implications for cell therapy and tissue engineering. J Investig Dermatol Symp Proc.2005,10(3):180-3
[48]Qi SH, Liu P, Xie JL, et al. Experimental study on repairing of nude mice skin defects with composite skin consisting of xenogeneic dermis and epidermal stem cells and hair follicle dermal papilla cells. Burns.2007, Sep 10
[49]Jahoda CA, Reynolds AJ. Hair follicle dermal sheath cells:unsung participants in wound healing. Lancet,2001,358 (9291):1445
[2]Herzog EL, Chai L, and Krause DS. Plasticity of marrow-derived stem cells. Blood. 2003,102:3483-3493
[3]Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science,1997,276:71-74
[4]Wu Y, Wang J, Scott PG, Tredget EE. Bone marrow-derived stem cells in wound healing:a review. Wound Repair Regen.2007,15 Suppl 1:S 18-26
[5]Fox JM, Chamberlain G, Ashton BA, Middleton J. Recent advances into the understanding of mesenchymal stem cell trafficking. Br J Haematol.2007,137(6):491-502
[6]Bauer SM, Bauer RJ, Liu ZJ, Chen H, Goldstein L, Velazquez OC. Vascular endothelial growth factor-C promotes vasculogenesis, angiogenesis, and collagen constriction in three-dimensional collagen gels. J Vasc Surg.2005,41(4):699-707
[7]Abe R, Donnelly SC, Peng T, Bucula R, Metz CN. Peripheral blood fibrocytes: differentiation pathway and migration to wound sites. J Immunol.2001.166:7556-7662
[8]Fathke C, Wilson L, Hutter J, Kapoor V, Smith A, Hocking A, Isik F. Contribution of bone marrow-derived cells to skin:collagen deposition and wound repair. Stem Cells.2004. 22:812-822.
[9]何觅春,李静,赵春华.低氧对间充质干细胞的影响.中国实验血液学杂志.2007,15(2)：433-436.
[10]Branski LK, Gauglitz GG, Herndon DN, Jeschke MG. A review of gene and stem cell therapy in cutaneous wound healing. Burns,2009,35(2):171-80.
[11]Nakagawa H, Akita S, Fukui M, Fujii T. Akino K. Human mesenchymal stem cells successfully improve skin substitute wound healing. Br J Dermatol 2005;153(1):29-36. [12] Hao L. Wang J, Zou Z. Yan G, Dong S, Deng J, Ran X, Feng Y, Luo C, Wang Y, Cheng T. Transplantation of BMSCs expressing hPDGF-A/hBD2 promotes wound healing in rats with combined radiation-wound injury. Gene Ther.2009,16(1):34-42.
[13]Falanga V, Iwamoto S, Chartier M, Yufit T, Butmarc J, Kouttab N, Shrayer D, Carson P. Autologous bone marrow-derived cultured mesenchymal stem cells delivered in a fibrin spray accelerate healing in murine and human cutaneous wounds. Tissue Eng,2007, 13(6):1299-312
[14]Chen L, Tredget EE, Wu PY, Wu Y. Paracrine factors of mesenchymal stem cells recruit macrophages and endothelial lineage cells and enhance wound healing. PLoS ONE. 2008,2;3(4):e1886.
[15]Velazquez OC. Angiogenesis and vasculogenesis:inducing the growth of new blood vessels and wound healing by stimulation of bone marrow-derived progenitor cell mobilization and homing. J Vasc Surg.2007,45 Suppl A:A39-47
[16]De Filippo RE, Atala A. Stretch and growth:the molecular and physiologic influences of tissue ex pansion. Plast Reconstr Surg.2002; 109(7):2450-62.
[17]Melis P, Noorlander ML, van der Horst CM, van Noorden CJ. Rapid alignment of collagen fibers in the dermis of undermined and not undermined skin stretched with a skin-stretching device. Plast Reconstr Surg.2002; 109(2):674-80.
[18]Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature.2002,418:41-49.
[19]Silva GV, Litovsky S, Assad JA, et al. Mesenchymal stem cells differentiate into an endothelial phenotype, enhance vascular density, and improve heart function in a canine chronic ischemia model. Circulation.2005,111(2):150-6.
[20]Aoto K. Shikata Y, Imai H.et al. Mouse Shh is required for prechordal plate maintenance during brain and craniofacial morphogenesis. Dev Biol.2009; 327(1):106-120.
[21]Hemmrich K, Meersch M, vonHeimburg D, et al. Applicability of the dyes CFSE, CM-Dil and PKH26 for tracking of human preadipocytes to evaluate adipose tissueen gineering [J]. Cells Tissues Organs,2006.184(3—4):117—127.
[22]Siegert R, Weerda H, Loffler JA, et al. Epidermal proliferation rate after skin expansion in the dog model. Laryng orhinootologie,1994,74:2062208.
[23]Badiavas EV, Abedi M, Butmarc J, Falanga V, Quesenberry P. Participation of bone marrow derived cells in cutaneous wound healing.J Cell Physiol.2003,196:245-250.
[24]Kataoka K, Medina RJ, Kageyama T, Miyazaki M, Yoshino T, Makino T, Huh NH. Participation of adult mouse bone marrow cells in reconstitution of skin. Am J Pathol.2003, 163:1227-1231.
[25]Takasak IY, Kaneda K, Takeuehi K, el al. Analysis Of the structure of proteasonm proliferating cell nuclear antigen(PCNA) multiprotein complex and its autoimmune response in lupus patients[J].Ann N Y Acad Sci,2003,987(3):316-318.
[26]Janes SM, Lowell S, Hutter C. Epidermal stem cells. J Pathol,2002,197(4):479. [27] Amado LC, Saliaris AP, Schuleri KH, et al. Cardiac repair with intramyocardial injection of allogeneic mesenchymal stem cells after myocardial infarction. Proc Natl Acad Sci,2005; 102(32):11474-11479.
[28]李海红,付小兵,王君,等.骨髓间充质干细胞分化为皮肤附属器细胞的初步实验研究。中国修复重建外科杂志.2006,6(20)：675-678.
[29]Bhora FY, Dunkin BJ, Batzri S, et al. Effect of growth factors on cell proliferation and epithlialization in human skin. J Surg Res,1995,59(2):236-244.
[30]Wang HJ, Wan HL, Yang TS et al. Acceleration of skin graft healing by growth factors. Burns,1996,22(1):10-14.
[31]Cohen S and Carpenter G. Human epidermal growth factor:isolation and chemical and biological properties. Proc Natl Acad Sci,1975,72(4):1317-1321.
[32]Esch F, Baird A, ling N. et al. Primary structure of bovine pituitary basic fibroblast growth factor (FGF) and comparison with the amino-terminal sequence of bovine brain acidic FGF. Proc Natl Acad Sci USA,1985,82(19):6507-6511.
[33]Abbott JD, Huang Y, Liu DG, et al. Stromal cell-derived factor-1α plays a critical role in stem cell recruitment to the heart after myocardial infarction but is not sufficient to induce homing in the absence of injury. Circulation 2004; 110(21):3300-3305.
[34]Bhakta S, Hong P, Koc O. The surface adhesion molecule CXCR4 stimulates mesenchymal stem cell migration to stromal cell-derived factor-1 in vitro but does not decrease apoptosis under serum deprivation. Cardiovasc Revase Med 2006; 7:19-24.
[35]Kraitchman DL, Tat sumi M, Gilson WD, et al. Dynamic imaging of allogeneic mesenchymal stem cells trafficking to myocardial infarction [J]. Circulation,2005,112: 1451-1461.
[1]鲁开化,郭树忠,韩岩：马显杰等.皮肤软组织扩张术临床应用经验总结.中国美容医学,2007,16(11)：1476-1480
[2]丁锐.谭敏,傅晓源,柳辉.皮肤扩张后修复过程的实验观察研究.国际医药卫生导报,2007, 13(20)：14-16
[3]Sellheyer K, Krahl D. Skin mesenchymal stem cells:prospects for clinical dermatology. J Am Acad Dermatol.2010,63(5):859-65.
[4]Zouboulis CC, Adjaye J, Akamatsu H, Moe-Behrens G, Niemann C. Human skin stem cells and the ageing process. Exp Gerontol.2008,43(11):986-97.
[5]孙志刚,郭树忠,鲁开化,韩岩.皮肤伸展术中新增皮肤来源的实验研究.中国美容医学,2003,12(3)：231-233
[6]De Filippo RE, Atala A. Stretch and growth:the molecular and physiologic influences of tissue expansion. Plast Reconstr Surg.2002;109(7):2450-62.
[7]Melis P, Noorlander ML, van der Horst CM, van Noorden CJ. Rapid alignment of collagen fibers in the dermis of undermined and not undermined skin stretched with a skin-stretching device. Plast Reconstr Surg.2002;109(2):674-80.
[8]刘学军,张海明,孙广慈,胡华新.超量扩张皮肤的血流量改变与安全性的实验研究.中华整形外科杂志,2005,21(6)：433-6
[9]刘文阁,季莹,杨佩瑛等.曲安缩松加速组织扩张的临床应用研究.中华整形烧伤外科杂志.1999；15(1)：41-42
[10]Tang Y, Luan J, Zhang X. Accelerating tissue expansion by application of topical papaverine cream. Plast Reconstr Surg.2004; 114(5):1166-9.
[11]陕声国,张端莲,吴小蔚等.联合性快速皮肤软组织扩张法的研究.中华实验外科杂志,2002,19(4)：373-375
[12]Shi CM, Cheng TM, Su YP, et al. Effects of dermal multipotent cell transplantation on skin wound healing. J Surg Res,2004,121:13.
[13]Herzog EL, Chai L, and Krause DS. Plasticity of marrow-derived stem cells. Blood.2003,102:3483-3493
[14]Wu Y. Chen L, Scott PG, Tredget EE. Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis. Stem Cells.2007,25(10):2648-59
[15]李江,鲁开化,艾玉峰,郭树忠.持续恒压扩张和常规间断扩张后皮肤超微结构的改变.中华整形外科杂志,2002,18(6)：365-366
[16]Herzog EL. Chai L, and Krause DS. Plasticity of marrow-derived stem cells. Blood.2003,102:3483-3493
[17]Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science,1997.276:71-74
[18]Herzog EL, Chai L, and Krause DS. Plasticity of marrow-derived stem cells. Blood.2003,102:3483-3493
[19]Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science,1997.276:71-74
[20]Wu Y, Wang J, Scott PG, Tredget EE. Bone marrow-derived stem cells in wound healing:a review. Wound Repair Regen.2007,15 Suppl 1:S18-26
[21]Fox JM, Chamberlain G, Ashton BA, Middleton J. Recent advances into the understanding of mesenchymal stem cell trafficking. Br J Haematol.2007,137(6):491-502
[22]Bauer SM, Bauer RJ, Liu ZJ, Chen H, Goldstein L, Velazquez OC. Vascular endothelial growth factor-C promotes vasculogenesis, angiogenesis, and collagen constriction in three-dimensional collagen gels. J Vasc Surg.2005,41(4):699-707
[23]Abe R, Donnelly SC, Peng T, Bucula R, Metz CN. Peripheral blood fibrocytes: differentiation pathway and migration to wound sites. J Immunol.2001.166:7556-7662
[24]Fathke C, Wilson L, Hutter J, Kapoor V, Smith A, Hocking A, Isik F. Contribution of bone marrow-derived cells to skin:collagen deposition and wound repair. Stem Cells.2004.22:812-822.
[25]何觅春,李静,赵春华.低氧对间充质干细胞的影响.中国实验血液学杂志.2007,15(2)：433-436.
[26]Li C, Zheng Y, Xia W, Wang X, Gao H, Li D, Ma X. Bone marrow-derived stem cells contribute skin regeneration in skin and soft tissue expansion.J Cell Physiol. 2011 Feb 1 [Epub ahead of print]
[27]Branski LK, Gauglitz GG, Herndon DN, Jeschke MG. A review of gene and stem cell therapy in cutaneous wound healing. Burns,2009,35(2):171-80.
[28]Nakagawa H, Akita S, Fukui M, Fujii T, Akino K. Human mesenchymal stem cells successfully improve skin substitute wound healing. Br J Dermatol 2005;153(1):29-36.
[29]Hao L, Wang J, Zou Z, Yan G, Dong S, Deng J, Ran X, Feng Y, Luo C, Wang Y, Cheng T. Transplantation of BMSCs expressing hPDGF-A/hBD2 promotes wound healing in rats with combined radiation-wound injury. Gene Ther.2009,16(1):34-42.
[30]Falanga V, Iwamoto S, Chartier M, Yufit T, Butmarc J, Kouttab N, Shrayer D, Carson P. Autologous bone marrow-derived cultured mesenchymal stem cells delivered in a fibrin spray accelerate healing in murine and human cutaneous wounds. Tissue Eng.2007, 13(6):1299-312
[31]Wu Y, Chen L, Scott PG, Tredget EE. Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis. Stem Cells.2007, 25(10):2648-59.
[32]Chen L, Tredget EE, Wu PY, Wu Y. Paracrine factors of mesenchymal stem cells recruit macrophages and endothelial lineage cells and enhance wound healing. PLoS ONE.2008,2;3(4):e1886.
[33]Velazquez OC. Angiogenesis and vasculogenesis:inducing the growth of new blood vessels and wound healing by stimulation of bone marrow-derived progenitor cell mobilization and homing. J Vasc Surg.2007,45 Suppl A:A39-47
[34]王晓燕,郑岩,李翅翅,等.异体骨髓间充质干细胞促进扩张皮肤新生的研究.中华医学美学与美容杂志.2011,待发表.
[35]Shi C, Zhu Y, Su Y, Cheng T. Stem cells and their applications in skin-cell therapy. Trends Biotechnol.2006,24(1):48-52
[36]Brouard M, Barrandon Y. Controlling skin morphogenesis:hope and despair. Curr Opin Biotechnol,2003,14:520.
[37]金岩,董蕊.皮肤干细胞的生物学特点与应用研究.基础医学与临床,2005,,25(10)：878-882
[38]刘洋,李建福,付小兵.皮肤软组织扩张术对大鼠表皮细胞增殖分化的影响.第三军医大学学报,2006,28(15)：1557-]559
[39]李幼忱,刘杰,王德文.毛囊真皮鞘细胞的生物学特性研究进展.生物医学工程学杂志,2007,24(3)：694-696
[40]祁少海,柯昌能,利天增等.真皮源干细胞复合人血浆促进皮肤创面神经修复的实验研究.中华显微外科杂志,2006,29(4)：282-283
[41]Toma JG, McKenzie IA, Bagli D, Miller FD. Isolation and characterization of multipotent skin-derived precursors from human skin. Stem Cells.2005,23(6):727-37
[42]Gorio A, Torrente Y, Madaschi L, et al. Fate of autologous dermal stem cells transplanted into the spinal cord after traumatic injury (TSCI). Neuroscience,2004, 25:179
[43]Gharzi A, Reynolds AJ, Jahoda CA. Plasticity of hair follicle dermal cells in wound healing and induction. Exp Dermatol.2003,12(2):126-36.
[44]Hoogduijn M.T, Gorjup E, Genever PG. Comparative Characterization of Hair Follicle Dermal Stem Cells and Bone Marrow Mesenchymal Stem Cells. Stem Cells Dev,2006,15(1):49-60.
[45]Chunmeng S, Tianmin C. Effects of plastic-adherent dermal multipotent cells on peripheral blood leukocytes and CFU-GM in rats. Transplantat Proceedings,2004,36: 1578.
[46]史春梦,程天民.创伤局部微环境对大鼠真皮多能干细胞生物学性状的影响.中华创伤杂志,2003, 19(4)：199-202
[47]Richardson GD, Arnott EC, Whitehouse CJ, et al. Plasticity of rodent and human hair follicle dermal cells:implications for cell therapy and tissue engineering. J Investig Dermatol Symp Proc.2005,10(3):180-3
[48]Qi SH, Liu P, Xie JL, et al. Experimental study on repairing of nude mice skin defects with composite skin consisting of xenogeneic dermis and epidermal stem cells and hair follicle dermal papilla cells. Burns.2007, Sep 10
[49]Jahoda CA, Reynolds AJ. Hair follicle dermal sheath cells:unsung participants in wound healing. Lancet,2001,358 (9291):1445