诱导hUC-MSCs和VECs转化为角膜内皮样细胞的实验研究
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摘要
1.目的（1）提高体外分离hUC-MCs的成功率
（2）探讨hUC-MCs作为组织工程角膜种子细胞的可能性
（3）改良冻存VECs的方法
（4）探讨VECs作为组织工程角膜种子细胞的可能性
2.方法（1）用多片脐带组织片悬浮法联合Ⅳ型胶原酶消化法、单纯多片脐带组织片悬浮法及脐带组织片悬浮法这三种方法体外分离hUC-MCs，并比较其成功率，鉴定细胞表面标记并诱导其分化
（2）以猪角膜基质后板层为载体培养hUC-MCs构建组织工程角膜后板层并行动物移植，观察角膜透明情况，并行hUC-MCs表面特异内皮标志物NSE和ZO-1的检测
（3）采用改良及传统方法冻存VECs，4周后复苏分别比较其存活率、形态、生长曲线，流式细胞术检测其凋亡率
（4）以猪角膜基质后板层为载体培养VECs构建组织工程角膜后板层，扫描电镜观察其表面结构
3.结果（1）多片悬浮脐带片法联合Ⅳ型胶原酶消化法及单纯多片悬浮脐带片法成功率高于悬浮脐带片法
（2）种植hUC-MCs移植组角膜较非种植组透明，hUC-MCs表面特异内皮标志物NSE和ZO-1表达阳性
（3）改良的冻存法VECs复苏后形态上较传统组好，存活率、生长曲线及凋亡率与传统组无明显差异
(4）扫描电镜可见hUC-MCs在猪角膜基质后板层表面生长良好，连接成片
4.结论（1）本研究探讨了hUC-MCs体外分离的几种方法，提供了一种较为高效的培养方法
（2）hUC-MCs可向CECs方向转化，为组织工程后板层的构建提供了新的种子细胞来源
（3）改良的VECs冻存法能大幅度缩短冻存所需时间达到与传统方法类似的效果
(4）VECs或能为组织工程后板层的构建提供了新的种子细胞来源
Objective (1) To improve the success rate of hUC-MCs separation in vitro
(2) To explore the possibility of hUC-MCs as engineering corneal tissueseed cells
(3)To improve the VECs cryopreserving method
(4) To explore the possibility of VECs as engineering corneal tissue seedcells
Methods (1) Separate the human umbilical cord-mesenchymal stem cells in vitrowith three methods: Multi-chip of umbilical cord tissue suspended combine with Ⅳcollagenase digestion method,Simple multi-chip of umbilical cord tissue suspendedmethod, umbilical cord tissue suspended method. After isolation,the cells’ shape andsurface markers is observed and detectde through the use of flow cytometry,andinduced to be adipocaytes and osteoblasts.
(2)Cultivate the hUC-MSCs on porcine corneal stroma to build tissueengineering corneal lamellar， observe the corneal stroma whether it istransparent,detect it’s surface marker for NSE and ZO-1which is specific for cornealendothelial cells
(3)Cryopreserve the VECs with two kinds of methods,compare theiractivity after4weeks through their survival rates,growth curve and apoptosis rate byflow cytometry
(4)Cultivate the VECs on porcine corneal stroma to build tissueengineering corneal lamellar,scaned it’s surface structure by electron microscopy
Results (1)The success rate of the Multi-chip suspended umbilical cord slicescombined with type IV collagenase digestion method and Simple multi-chip ofumbilical cord tissue suspended method were higher than umbilical cord tissuesuspended method
(2)The corneas with hUC-MSCs are transparenter than withoutit,surface-specific endothelial markers NSE and ZO-1expression were positive on hUC-MSCs
(3)The reformed method is better than traditional method in morphology,andthere was no significant difference in thei survival rate,growth curve and the apoptosis ratebetween these two kinds of methods
(4) Scanning electron microscopy showed the surface of the corneas withVECs grew well
Conclusion (1) In this research,we study sever kinds of methods to isolatehUC-MSCs in vitro,and create a more effcient method
(2)The hUC-MSCs can converse into CECs
(3)The reformed cryopreserved method is convenient and feasible
(4)The VECs may be new seed cells to establish engineering corneal
（2）探讨hUC-MCs作为组织工程角膜种子细胞的可能性
（3）改良冻存VECs的方法
（4）探讨VECs作为组织工程角膜种子细胞的可能性
2.方法（1）用多片脐带组织片悬浮法联合Ⅳ型胶原酶消化法、单纯多片脐带组织片悬浮法及脐带组织片悬浮法这三种方法体外分离hUC-MCs，并比较其成功率，鉴定细胞表面标记并诱导其分化
（2）以猪角膜基质后板层为载体培养hUC-MCs构建组织工程角膜后板层并行动物移植，观察角膜透明情况，并行hUC-MCs表面特异内皮标志物NSE和ZO-1的检测
（3）采用改良及传统方法冻存VECs，4周后复苏分别比较其存活率、形态、生长曲线，流式细胞术检测其凋亡率
（4）以猪角膜基质后板层为载体培养VECs构建组织工程角膜后板层，扫描电镜观察其表面结构
3.结果（1）多片悬浮脐带片法联合Ⅳ型胶原酶消化法及单纯多片悬浮脐带片法成功率高于悬浮脐带片法
（2）种植hUC-MCs移植组角膜较非种植组透明，hUC-MCs表面特异内皮标志物NSE和ZO-1表达阳性
（3）改良的冻存法VECs复苏后形态上较传统组好，存活率、生长曲线及凋亡率与传统组无明显差异
(4）扫描电镜可见hUC-MCs在猪角膜基质后板层表面生长良好，连接成片
4.结论（1）本研究探讨了hUC-MCs体外分离的几种方法，提供了一种较为高效的培养方法
（2）hUC-MCs可向CECs方向转化，为组织工程后板层的构建提供了新的种子细胞来源
（3）改良的VECs冻存法能大幅度缩短冻存所需时间达到与传统方法类似的效果
(4）VECs或能为组织工程后板层的构建提供了新的种子细胞来源
Objective (1) To improve the success rate of hUC-MCs separation in vitro
(2) To explore the possibility of hUC-MCs as engineering corneal tissueseed cells
(3)To improve the VECs cryopreserving method
(4) To explore the possibility of VECs as engineering corneal tissue seedcells
Methods (1) Separate the human umbilical cord-mesenchymal stem cells in vitrowith three methods: Multi-chip of umbilical cord tissue suspended combine with Ⅳcollagenase digestion method,Simple multi-chip of umbilical cord tissue suspendedmethod, umbilical cord tissue suspended method. After isolation,the cells’ shape andsurface markers is observed and detectde through the use of flow cytometry,andinduced to be adipocaytes and osteoblasts.
(2)Cultivate the hUC-MSCs on porcine corneal stroma to build tissueengineering corneal lamellar， observe the corneal stroma whether it istransparent,detect it’s surface marker for NSE and ZO-1which is specific for cornealendothelial cells
(3)Cryopreserve the VECs with two kinds of methods,compare theiractivity after4weeks through their survival rates,growth curve and apoptosis rate byflow cytometry
(4)Cultivate the VECs on porcine corneal stroma to build tissueengineering corneal lamellar,scaned it’s surface structure by electron microscopy
Results (1)The success rate of the Multi-chip suspended umbilical cord slicescombined with type IV collagenase digestion method and Simple multi-chip ofumbilical cord tissue suspended method were higher than umbilical cord tissuesuspended method
(2)The corneas with hUC-MSCs are transparenter than withoutit,surface-specific endothelial markers NSE and ZO-1expression were positive on hUC-MSCs
(3)The reformed method is better than traditional method in morphology,andthere was no significant difference in thei survival rate,growth curve and the apoptosis ratebetween these two kinds of methods
(4) Scanning electron microscopy showed the surface of the corneas withVECs grew well
Conclusion (1) In this research,we study sever kinds of methods to isolatehUC-MSCs in vitro,and create a more effcient method
(2)The hUC-MSCs can converse into CECs
(3)The reformed cryopreserved method is convenient and feasible
(4)The VECs may be new seed cells to establish engineering corneal
引文
[1]第二次全国残疾人抽样调查领导小组.2006年第二次全国残疾人抽样调查主要数据公报[J].时政文献辑览,2007:382-385.
[2]顾传龙,罗保国.角膜保存技术全国领先[J].中国医药报.2002,10.
[3] Laing RA,Neubauer L,Oak SS..Evidence for mitosis in the adult cornealendothelium[J].Ophthalmology.1984,91(10):1129-34.
[4]周道伐，朱志忠.角膜内皮细胞的研究进展[J].国际眼科学纵览，1980(05).
[5] Senoo T,Joyce NC.Cell cycle kinetics in corneal endothelium from old andyoung donors[J].Invest Ophthalmol Vis Sci.2000,41(3):660-7.
[6] Joyce NC.Cell cycle status in human corneal endothelium[J].Exp EyeRes.2005,81(6):629-38.
[7] Joyce NC, Zhu CC,Harris DL. Relationship among oxidative stress, DNAdamage, and proliferative capacity in human corneal endothelium[J].InvestOphthalmol Vis Sci.2009,50(5):2116-22.
[8] Hatou S, Yamada M, Akune Y. Role of insulin in regulation of Na+-/K+-dependent ATPaseactivity and pump function in corneal endothelial cells[J]. Division for VisionResearch.2010,51(8):3935-42
[9]樊廷俊,赵君,王晶.体外重建组织工程人角膜内皮在新西兰兔角膜内皮移植中的应用[J].国际眼科杂志.2009,9(12):2278-82.
[10]曹谊林，崔磊，高庆新，等.组织工程的研究现状与应用展望[J].中国医疗器械信息，2002,6(4):11-13.
[11] Gospodarowicz D, Greenburg G..The coating of bovine and rabbit corneasdenuded of their endothelium with bovine corneal endothelial cells[J].Exp EyeRes.1979,28(3):249-65.
[12] Zavos PM. Stem cells and cellular theraPy: Potential treatment for cardiovaseulardiseases[J]. Int J Cardiol,2006,107(1):1-6.
[13] Liu Qi, Chen WP. Research progress in the application of embryonic stem cellsin vitro culture system in ophthalmology. Yanke Yanjiu.2009;27(5):420-433.
[14] Osakada F, Ikeda H, Mandai M.Toward the generation of rod and conephotoreceptors from mouse, monkey and human embryonic stem cells[J].NatBiotechnol.2008,26(2):215-24.
[15] Notara M, Hernandez D, Mason C. Characterization of the phenotype and functionality ofcorneal epithelial cells derived from mouse embryonic stem cells[J].RegenMed.2012,7(2):167-78.
[16]张玉媛,王取南,王允.镉对小鼠胚胎神经干细胞向胶质细胞分化的影响[J].癌变畸变突变,2012,24(1):39-45.
[17]张开刚,张月东,王玫.脂肪干细胞与小肠黏膜下层构建仿生骨膜的体内成骨[J].中国组织工程研究,2012,16(3):426-30.
[18] Cheng X, Chen S, Yu X. BMP15Gene Is Activated During Human AmnioticFluid Stem Cell Differentiation into Oocyte-Like Cells [J]. DNA Cell Biol,2012.
[19]陈义.脐带间充质干细胞培养及体外重建角膜后板层的初步研究[J].暨南大学.2009
[20] Gospodarowicz D, Greenburg G, Alarado J. Transplantation of cultured bovinecorneal endothelial cell to species with nonregenerative endothelium. The cat as anexperimental model[J]. Arch ophthalmol,1979,97(11):2163-2169.
[21]吴小莉,赵英贤,宋虎平.血管内皮细胞替代角膜内皮细胞的可行性[J].国际眼科杂志.2010,10(10):1885-7.
[22]罗哲文,胡竹林,徐岩泽.自体血管内皮细胞移植替代角膜内皮层的实验研究[J].眼科研究.2008,26(4):249-52.
[23] Burman S, Tejwani S, Vemuganti GK, et al. Ophthalmic applications ofpreserved human amniotic membrane: a review of current indications. Cell TissueBank.2004;5(3):161-175.
[24]张米,尹天泉,刘源.复合自体脂肪干细胞的脱细胞猪角膜基质移植治疗兔角膜碱烧伤[J].国际眼科杂志.2009,9(4):650-2.
[25]邵春益,傅瑶,陆雯娟.以猪角膜脱细胞基质为载体培养猫骨髓内皮祖细胞构建角膜后板层的实验研究[J].眼科新进展.2008,28(4):249-58.
[26] Lin Q, Lü L, Han B, et al. Zhonghua Yanke Zazhi. Transplantation ofbioengineered corneal epithelium for the treatment of total limbal stem cell deficiencyin rabbit.2006;42(8):679-685.
[27] Ibrahim-Elzembely HA, Kaufman SC, Kaufinan HE. Human fibrin tissue gluefor corneal lamellar adhesion in rabbits:a preliminary study. Cornea.2003;22(8):735-739.
[28]李晓霞,陈建苏,李沁华.模拟微重力条件下兔角膜基质细胞在复合材料上的三维培养[J].眼科研究.2008,26(4):241-4.
[29] Qian YX,Shu Q,Cai HX, et al.Surface marker changes in humanumbilical cord-derived mesenchymal stem cells after cryopreservation andresuscitation.J Clin Rehabilit Tissue Eng Res.2011；15(1):187-190.
[30]柳菁,宇丽,许超.长期培养人脐带间充质干细胞分化能力改变的研究[J].中国生物工程杂志.2012,32(2):8-16
[31]程欣,皮婷,肖飞.人脐带间充质干细胞诱导分化为神经元样细胞[J].基础医学与临床.2011,31(3):269-74.
[32]许永安,黄沙,付小兵.特定微环境诱导人脐带沃顿胶间充质干细胞向汗腺样细胞分化的实验研究[J].解放军医学之.2011,36(3):211-4.
[33]荣靖,彭鑫磊,马岩岩.人脐带间充质干细胞向成骨细胞及脂肪细胞分化过程中nm23-H1基因的表达[J].中国组织工程与临床康复.2011,15(19):3467-71.
[34]季凤清,王屹,孙海梅.人脐血非造血干细胞免疫原性的实验研究[J].解剖学报.2008,39(1):55-9.
[35]陈恩,祝加学,秦金保.人脐带间充质干细胞向血管内皮祖细胞诱导分化的实验研究[J].组织工程与重建外科杂志.2009,5:310-3.
[36] Nilius B,Droogmans G..Ion channels and their functional role in vascularendothelium[J].Physiol Rev.2001,81(4):1415-59.
[37]吴小莉,赵英贤,宋虎平.血管内皮细胞替代角膜内皮细胞的可行性[J].国际眼科杂志.2010,10(10):1885-7.
[38]罗哲文,胡竹林,徐岩泽.自体血管内皮细胞移植替代角膜内皮层的实验研究[J].眼科研究.2008,26(4):249-52.
[39]何绍清,罗振宇,刘秋英.人脐带间充质干细胞分离培养及向脂肪与成骨细胞的分化[J].中国组织工程研究与临床康复.2010,14(14):2492-6.
[40]蒋洁,谭灿,张李洋.脐带沃顿胶间充质干细胞的分离培养及其诱导分化[J].中国组织工程研究与临床康复.2010,14(10):1734-8.
[41]杨卿,杨扬,刘剑戎.人脐带间充质干细胞分离扩增方法的优化[J].中山大学学报（医学科学版）.2011,32(6):818-23.
[42] Fernandez MM, Afshari NA.Endothelial Keratoplasty: From DLEK toDMEK[J].Middle East Afr J Ophthalmol.2010,17(1):5-8.
[43]韦春玲，恒河猴骨髓间充质干细胞移植替代损伤的角膜内皮细胞的实验研究[M].昆明医学院.2010.
[44]肖玉翠,王吉波,董静.脐带间充质干细胞移植对系统性红斑狼疮免疫系统的影响[J].中国组织工程研究与临床康复.2011,15(19):3489-93.
[45] Zenclussen AC,Ki,k6ny G,Thimm O,et al.Mechanisms behind flare of renal lupusduring murine pregnancy.Reprod Biomed0nline.2008;17:114-126.
[46]颜静,高岩,王清.明胶/聚乳酸复合纤维膜的制备、表征及作为角膜细胞载体的评价[J].高等学校化学学报.2012,33:389-94.
[47]唐光霞,陈建苏,徐锦堂.肾纤维囊为载体培养角膜内皮细胞的实验研究[J].眼科新进展.2011,31:304-7.
[48]阿衣努尔.吐尔地,阿依努尔.艾买尔.人羊膜作为载体体外培养兔角膜上皮细胞移植治疗碱烧伤动物的实验研究[J].眼外伤职业眼病杂志.2010,32(4):251-5.
[49]赵博,马蕾,黄晓梅.兔晶状体前囊膜为角膜缘干细胞培养载体的初步研究[J].中国医疗前沿.2011,6:11-2.
[50]施文建，侯光辉，吴静.种植人骨髓间充质干细胞的猪角膜板层移植治疗兔角膜损伤的初步研究[J].眼科研究,2010,28(4):300-5.
[51]刘先宁,朱秀萍,银勇.以干燥脱水法保存的异种角膜基质为载体构建人工生物角膜上皮组织[J].国际眼科杂志.2008,8(11):2214-6.
[52]范子扬,宋振顺.骨髓间充质干细胞多向分化潜能和微环境关系的研究[J].现代肿瘤医学.2011,19(2):211-4.
[53]汪映,张立行,王共先.后肾细胞微环境诱导胚胎干细胞向肾系细胞分化的实验研究[J].中国病理生理杂志，2011,27（1）：129-133
[54]秦佳佳,冼绍祥,黄习文.体外诱导间充质干细胞向心肌样细胞分化的研究：药物、微环境及方法[J].中国组织工程研究与临床康复,2011,15(1):139-42
[55]许永安,黄沙,付小兵.特定微环境诱导人脐带沃顿胶间充质干细胞向汗腺样细胞分化的实验研究[J].解放军医学杂志,2011,36(3):211-4
[56] B hnke M，Vogelberg K，Engelmann K.Detection of neurone-specific enolase inlong-term cultures of human corneal endothelium[J].Gradefes Arch Clin ExpOphthalmol，1998，236(7)：522-526．
[57] Zhu C, Joyce NC.Proliferative response of corneal endothelial cells from youngand older donors[J].Invest Ophthalmol Vis Sci.2004,45(6):1743-51.
[58] Kehat I, Kenyagin-Karsenti D,Snir M, et al.Human embryonic stem cells candifferentiate into myocytes with structural and functional properties ofcardiomyocytes[J].J.Clin.Invest,2001，108:407-414.
[59] Sethe S, Scutt A, Stolzing A. Aging of mesenchymal stem cells. Ageing ResRev,2006,5(1):91-116
[60] Qian YX,Shu Q,Cai HX, et al.Surface marker changes in human umbilicalcord-derived mesenchymal stem cells after cryopreservation and resuscitation.J ClinRehabilit Tissue Eng Res.2011；15(1):187-190.
[61] Kusano T,Aoki T,Yasuda D,et al.Microencapsule technique protectshepatocytes from cryoinjury.Gastroent Hepatol.2008;38:593-600.
[62]贾光,刘世杰.应用台盼兰拒染法与MTT法判断Cr（VI）细胞毒性的比较[J].中国职业医学,2000,27(3):49.
[63]杨景山.医学细胞化学与细胞生物学技术[M].北京:北京医科大学、中国协和医科大学联合出版社,1990.
[64] Kuczynski W,Dhont M,Grygoruk C,et al.The outcome ofintracytoplasmic injection of fresh and cryopreserved ejaculated spermatozoa--aprospective randomized study.Hum Reproduction.2001;16:2109-2113.
[65] Spurr EE,Wiggins NE,Marsden KA,et al.Cryopreserved humanhaematopoietic stem cells retain engraftment potential after extended (5-14years) cryostorage.Cryobiology.2002;44:210-217.
[66] Zaman GJ,de Roos JA,Blomenrohr M,et al.Cryopreserved cellsfacilitate cell-based drug discovery. Drug DiscovToday.2007;12:521-526.
[67]薛庆善.体外培养的原理与技术[M].北京：科学出版社，2001.
[68] Sarkis R,Benoist S,Honiger J,et al.Transplanted cryopreservedencapsulated porcine hepatocytes are as effective as fresh hepatocytes inpreventing death from acute liver failure in rats. Transplantation.2000;70:58-64.
[69]张延辉,李伟,李永香,等.人胚胎冻存时间对妊娠结局的影响[J].中国优生与遗传杂志,2011,19(4):98.
[70]Rigol M,Heras M,Martinez A,et al.Changes in the cooling rate and mediumimprove the vascular function in cryopreserved porcine femoral arteries.VasSurg.2000;31:1018-1025..
[2]顾传龙,罗保国.角膜保存技术全国领先[J].中国医药报.2002,10.
[3] Laing RA,Neubauer L,Oak SS..Evidence for mitosis in the adult cornealendothelium[J].Ophthalmology.1984,91(10):1129-34.
[4]周道伐，朱志忠.角膜内皮细胞的研究进展[J].国际眼科学纵览，1980(05).
[5] Senoo T,Joyce NC.Cell cycle kinetics in corneal endothelium from old andyoung donors[J].Invest Ophthalmol Vis Sci.2000,41(3):660-7.
[6] Joyce NC.Cell cycle status in human corneal endothelium[J].Exp EyeRes.2005,81(6):629-38.
[7] Joyce NC, Zhu CC,Harris DL. Relationship among oxidative stress, DNAdamage, and proliferative capacity in human corneal endothelium[J].InvestOphthalmol Vis Sci.2009,50(5):2116-22.
[8] Hatou S, Yamada M, Akune Y. Role of insulin in regulation of Na+-/K+-dependent ATPaseactivity and pump function in corneal endothelial cells[J]. Division for VisionResearch.2010,51(8):3935-42
[9]樊廷俊,赵君,王晶.体外重建组织工程人角膜内皮在新西兰兔角膜内皮移植中的应用[J].国际眼科杂志.2009,9(12):2278-82.
[10]曹谊林，崔磊，高庆新，等.组织工程的研究现状与应用展望[J].中国医疗器械信息，2002,6(4):11-13.
[11] Gospodarowicz D, Greenburg G..The coating of bovine and rabbit corneasdenuded of their endothelium with bovine corneal endothelial cells[J].Exp EyeRes.1979,28(3):249-65.
[12] Zavos PM. Stem cells and cellular theraPy: Potential treatment for cardiovaseulardiseases[J]. Int J Cardiol,2006,107(1):1-6.
[13] Liu Qi, Chen WP. Research progress in the application of embryonic stem cellsin vitro culture system in ophthalmology. Yanke Yanjiu.2009;27(5):420-433.
[14] Osakada F, Ikeda H, Mandai M.Toward the generation of rod and conephotoreceptors from mouse, monkey and human embryonic stem cells[J].NatBiotechnol.2008,26(2):215-24.
[15] Notara M, Hernandez D, Mason C. Characterization of the phenotype and functionality ofcorneal epithelial cells derived from mouse embryonic stem cells[J].RegenMed.2012,7(2):167-78.
[16]张玉媛,王取南,王允.镉对小鼠胚胎神经干细胞向胶质细胞分化的影响[J].癌变畸变突变,2012,24(1):39-45.
[17]张开刚,张月东,王玫.脂肪干细胞与小肠黏膜下层构建仿生骨膜的体内成骨[J].中国组织工程研究,2012,16(3):426-30.
[18] Cheng X, Chen S, Yu X. BMP15Gene Is Activated During Human AmnioticFluid Stem Cell Differentiation into Oocyte-Like Cells [J]. DNA Cell Biol,2012.
[19]陈义.脐带间充质干细胞培养及体外重建角膜后板层的初步研究[J].暨南大学.2009
[20] Gospodarowicz D, Greenburg G, Alarado J. Transplantation of cultured bovinecorneal endothelial cell to species with nonregenerative endothelium. The cat as anexperimental model[J]. Arch ophthalmol,1979,97(11):2163-2169.
[21]吴小莉,赵英贤,宋虎平.血管内皮细胞替代角膜内皮细胞的可行性[J].国际眼科杂志.2010,10(10):1885-7.
[22]罗哲文,胡竹林,徐岩泽.自体血管内皮细胞移植替代角膜内皮层的实验研究[J].眼科研究.2008,26(4):249-52.
[23] Burman S, Tejwani S, Vemuganti GK, et al. Ophthalmic applications ofpreserved human amniotic membrane: a review of current indications. Cell TissueBank.2004;5(3):161-175.
[24]张米,尹天泉,刘源.复合自体脂肪干细胞的脱细胞猪角膜基质移植治疗兔角膜碱烧伤[J].国际眼科杂志.2009,9(4):650-2.
[25]邵春益,傅瑶,陆雯娟.以猪角膜脱细胞基质为载体培养猫骨髓内皮祖细胞构建角膜后板层的实验研究[J].眼科新进展.2008,28(4):249-58.
[26] Lin Q, Lü L, Han B, et al. Zhonghua Yanke Zazhi. Transplantation ofbioengineered corneal epithelium for the treatment of total limbal stem cell deficiencyin rabbit.2006;42(8):679-685.
[27] Ibrahim-Elzembely HA, Kaufman SC, Kaufinan HE. Human fibrin tissue gluefor corneal lamellar adhesion in rabbits:a preliminary study. Cornea.2003;22(8):735-739.
[28]李晓霞,陈建苏,李沁华.模拟微重力条件下兔角膜基质细胞在复合材料上的三维培养[J].眼科研究.2008,26(4):241-4.
[29] Qian YX,Shu Q,Cai HX, et al.Surface marker changes in humanumbilical cord-derived mesenchymal stem cells after cryopreservation andresuscitation.J Clin Rehabilit Tissue Eng Res.2011；15(1):187-190.
[30]柳菁,宇丽,许超.长期培养人脐带间充质干细胞分化能力改变的研究[J].中国生物工程杂志.2012,32(2):8-16
[31]程欣,皮婷,肖飞.人脐带间充质干细胞诱导分化为神经元样细胞[J].基础医学与临床.2011,31(3):269-74.
[32]许永安,黄沙,付小兵.特定微环境诱导人脐带沃顿胶间充质干细胞向汗腺样细胞分化的实验研究[J].解放军医学之.2011,36(3):211-4.
[33]荣靖,彭鑫磊,马岩岩.人脐带间充质干细胞向成骨细胞及脂肪细胞分化过程中nm23-H1基因的表达[J].中国组织工程与临床康复.2011,15(19):3467-71.
[34]季凤清,王屹,孙海梅.人脐血非造血干细胞免疫原性的实验研究[J].解剖学报.2008,39(1):55-9.
[35]陈恩,祝加学,秦金保.人脐带间充质干细胞向血管内皮祖细胞诱导分化的实验研究[J].组织工程与重建外科杂志.2009,5:310-3.
[36] Nilius B,Droogmans G..Ion channels and their functional role in vascularendothelium[J].Physiol Rev.2001,81(4):1415-59.
[37]吴小莉,赵英贤,宋虎平.血管内皮细胞替代角膜内皮细胞的可行性[J].国际眼科杂志.2010,10(10):1885-7.
[38]罗哲文,胡竹林,徐岩泽.自体血管内皮细胞移植替代角膜内皮层的实验研究[J].眼科研究.2008,26(4):249-52.
[39]何绍清,罗振宇,刘秋英.人脐带间充质干细胞分离培养及向脂肪与成骨细胞的分化[J].中国组织工程研究与临床康复.2010,14(14):2492-6.
[40]蒋洁,谭灿,张李洋.脐带沃顿胶间充质干细胞的分离培养及其诱导分化[J].中国组织工程研究与临床康复.2010,14(10):1734-8.
[41]杨卿,杨扬,刘剑戎.人脐带间充质干细胞分离扩增方法的优化[J].中山大学学报（医学科学版）.2011,32(6):818-23.
[42] Fernandez MM, Afshari NA.Endothelial Keratoplasty: From DLEK toDMEK[J].Middle East Afr J Ophthalmol.2010,17(1):5-8.
[43]韦春玲，恒河猴骨髓间充质干细胞移植替代损伤的角膜内皮细胞的实验研究[M].昆明医学院.2010.
[44]肖玉翠,王吉波,董静.脐带间充质干细胞移植对系统性红斑狼疮免疫系统的影响[J].中国组织工程研究与临床康复.2011,15(19):3489-93.
[45] Zenclussen AC,Ki,k6ny G,Thimm O,et al.Mechanisms behind flare of renal lupusduring murine pregnancy.Reprod Biomed0nline.2008;17:114-126.
[46]颜静,高岩,王清.明胶/聚乳酸复合纤维膜的制备、表征及作为角膜细胞载体的评价[J].高等学校化学学报.2012,33:389-94.
[47]唐光霞,陈建苏,徐锦堂.肾纤维囊为载体培养角膜内皮细胞的实验研究[J].眼科新进展.2011,31:304-7.
[48]阿衣努尔.吐尔地,阿依努尔.艾买尔.人羊膜作为载体体外培养兔角膜上皮细胞移植治疗碱烧伤动物的实验研究[J].眼外伤职业眼病杂志.2010,32(4):251-5.
[49]赵博,马蕾,黄晓梅.兔晶状体前囊膜为角膜缘干细胞培养载体的初步研究[J].中国医疗前沿.2011,6:11-2.
[50]施文建，侯光辉，吴静.种植人骨髓间充质干细胞的猪角膜板层移植治疗兔角膜损伤的初步研究[J].眼科研究,2010,28(4):300-5.
[51]刘先宁,朱秀萍,银勇.以干燥脱水法保存的异种角膜基质为载体构建人工生物角膜上皮组织[J].国际眼科杂志.2008,8(11):2214-6.
[52]范子扬,宋振顺.骨髓间充质干细胞多向分化潜能和微环境关系的研究[J].现代肿瘤医学.2011,19(2):211-4.
[53]汪映,张立行,王共先.后肾细胞微环境诱导胚胎干细胞向肾系细胞分化的实验研究[J].中国病理生理杂志，2011,27（1）：129-133
[54]秦佳佳,冼绍祥,黄习文.体外诱导间充质干细胞向心肌样细胞分化的研究：药物、微环境及方法[J].中国组织工程研究与临床康复,2011,15(1):139-42
[55]许永安,黄沙,付小兵.特定微环境诱导人脐带沃顿胶间充质干细胞向汗腺样细胞分化的实验研究[J].解放军医学杂志,2011,36(3):211-4
[56] B hnke M，Vogelberg K，Engelmann K.Detection of neurone-specific enolase inlong-term cultures of human corneal endothelium[J].Gradefes Arch Clin ExpOphthalmol，1998，236(7)：522-526．
[57] Zhu C, Joyce NC.Proliferative response of corneal endothelial cells from youngand older donors[J].Invest Ophthalmol Vis Sci.2004,45(6):1743-51.
[58] Kehat I, Kenyagin-Karsenti D,Snir M, et al.Human embryonic stem cells candifferentiate into myocytes with structural and functional properties ofcardiomyocytes[J].J.Clin.Invest,2001，108:407-414.
[59] Sethe S, Scutt A, Stolzing A. Aging of mesenchymal stem cells. Ageing ResRev,2006,5(1):91-116
[60] Qian YX,Shu Q,Cai HX, et al.Surface marker changes in human umbilicalcord-derived mesenchymal stem cells after cryopreservation and resuscitation.J ClinRehabilit Tissue Eng Res.2011；15(1):187-190.
[61] Kusano T,Aoki T,Yasuda D,et al.Microencapsule technique protectshepatocytes from cryoinjury.Gastroent Hepatol.2008;38:593-600.
[62]贾光,刘世杰.应用台盼兰拒染法与MTT法判断Cr（VI）细胞毒性的比较[J].中国职业医学,2000,27(3):49.
[63]杨景山.医学细胞化学与细胞生物学技术[M].北京:北京医科大学、中国协和医科大学联合出版社,1990.
[64] Kuczynski W,Dhont M,Grygoruk C,et al.The outcome ofintracytoplasmic injection of fresh and cryopreserved ejaculated spermatozoa--aprospective randomized study.Hum Reproduction.2001;16:2109-2113.
[65] Spurr EE,Wiggins NE,Marsden KA,et al.Cryopreserved humanhaematopoietic stem cells retain engraftment potential after extended (5-14years) cryostorage.Cryobiology.2002;44:210-217.
[66] Zaman GJ,de Roos JA,Blomenrohr M,et al.Cryopreserved cellsfacilitate cell-based drug discovery. Drug DiscovToday.2007;12:521-526.
[67]薛庆善.体外培养的原理与技术[M].北京：科学出版社，2001.
[68] Sarkis R,Benoist S,Honiger J,et al.Transplanted cryopreservedencapsulated porcine hepatocytes are as effective as fresh hepatocytes inpreventing death from acute liver failure in rats. Transplantation.2000;70:58-64.
[69]张延辉,李伟,李永香,等.人胚胎冻存时间对妊娠结局的影响[J].中国优生与遗传杂志,2011,19(4):98.
[70]Rigol M,Heras M,Martinez A,et al.Changes in the cooling rate and mediumimprove the vascular function in cryopreserved porcine femoral arteries.VasSurg.2000;31:1018-1025..
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