骨髓间充质干细胞分化为视网膜结构及其在重度视网膜损伤中的治疗作用
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摘要
视网膜疾病占重症眼病的一半以上，其中既有先天性视网膜缺损，如视网膜色素变性，也有后天因为外伤或其他疾病产生的视网膜并发症，如普通强光或激光造成的视网膜光损伤、糖尿病并发的眼病等。目前对于较轻的视网膜损伤主要采取抗炎治疗或营养因子治疗，而对重度损伤尚无很好的治疗方法，主要是玻璃体止血、促进凝血的吸收。许多病例因未能及时治疗或无药可救而导致视力下降甚至失明，因此寻找更好的治疗方法对于治疗视网膜疾病至关重要。
干细胞工程的研究为视网膜损伤的治疗提供了一种新的途径。研究者试用了多种干细胞，以期解决视网膜损伤的修复问题，如胚胎干细胞(embryonic stem cell，ESC)、胚胎视网膜祖细胞、成体哺乳动物眼干细胞等。但都存在着各种缺陷，如异体移植问题、伦理学问题及取材问题等。
骨髓中含有可分化为外周循环血细胞的造血干细胞早已为人所知。此外，人们还发现骨髓中有一群细胞可以分化成多种间质样的细胞，于是将它们称为骨髓间充质干细胞(即Mesenchymal Stem Cells，MSC)。在体外不同条件的作用下MSC可以定向地被诱导分化为成骨细胞、成软骨细胞、脂肪细胞、肌肉细胞、肌腱细胞、神经元等不同的细胞。MSC取材相对较容易，来源丰富，体外增殖能力强，适于作为细胞供体进行自体移植。
鉴于MSC便利的取材及其可向神经细胞分化的潜能，本研究期望通过视网膜下移植的方式研究MSC在正常视网膜及激光损伤视网膜中分化为视网膜的可能性，以及分化成的细胞是否具有神经细胞的特性，是否有利于视网膜激光损伤后的组织修复及视功能的恢复。
本研究首先分离、培养并鉴定了MSC。利用骨髓间充质干细胞的密度特性，用一定密度的Percoll对骨髓进行密度梯度分离，再利用骨髓间充质干细胞粘附在培养皿上的特性，去除单核／巨噬细胞，从而达到富集骨髓间充质干细胞的目的，体外传代可达15代以上。对富集纯化的细胞进行流式细胞术表型鉴定，并将MSC诱导分化为脂肪细胞，证明了其具有多向分化能力。自行建立的培养方法已申请
军事医学利学院博}学位论文
专利(专利受理号01141865.6)。如此培养出的MSC既具备增殖快的特点，又保
持了其做为干细胞的多向分化潜能，为进一步研究工作扫一下基础。
为证实MSC可以在正常视网膜分化为视网膜结构，本研究首先分离培养了
雄性大鼠的MSC，将其移植到雌性大鼠视网膜下，4周后利用雌雄标记法进行Y
染色体原位杂交，示踪MSC的分布。光学显微镜观察结果显示，阳性细胞在移
植区域周围与原来的视网膜细胞融合，并形成了典型的内、外核层及节细胞层的
细胞形态。同一视野下同时存在着阳胜和阴性细胞，阳性细胞并非单独存在，而
是形成一定的阳性细胞群，中间夹杂着阴性细胞。其中以外核层的阳性细胞较多，
由外到内逐渐减少，节细胞层最少。整个视网膜形态平滑，未见到玫瑰花结结构
少巨成。
一证明MSC在视网膜卜的分化至少需要3种示踪方法，因此本研究采用了体
外经AAV一加感染MSC的视网膜下移植观察。荧光显微镜观察结果表明，移植
4周后发现视网膜内存在GFP阳性细胞，其中以视网膜色素上皮层、视细胞层为
多，内、外网状层及节细)泡层也有}朴胜细胞的存在。阳性细胞相对较聚集，夕手与
阴性细胞相融合，在光学显微镜卜‘末发现视网膜形态的异常。这一结果不仅说明
MSC在视网膜下分化的定位，也说明MSC司一以被AJ叭z所感染，司一以作为基因
修饰细胞移植的载体细胞。AAV一gfr感染的结果与Y染色体原位杂交结果基木一
致。
以__}二不沙}究只观察了4周时MSC的变化情况，为观察MSC在移植后不同时问
的变化过程，同时用另一种方法再次确证MSC在视网膜下的存在，本研究进一
步采用荧光染料DAPJ对MSC进行标记，分别于移植后10、20、35和50天观察
了视网膜MSC一DAPI的分布情况。MSC在正常视网膜分化有以下特点:①卜lIJ卜l-
细胞随着移植时间的延长，先是顺着移植部位向周围打、一展，阳性细胞所在的部位
不断扩大，而后又有缩小的趋势，50大观察到的卜卜}胜细胞范围比35大明显绷小
(阳性红l!胞4个时I泊」点分布范围分别为960‘:n飞、1280抖m、640工Lm及160牛tm)，这
说明MSC的分化增殖是受到调控的，不会无限制增生;而且在正常视网膜条件
下似乎存在着一种抑制机制，使MSC趋向于逐渐减少;②阳性部位的细胞分布
第4臾共127
军事队学利学院博卜学位论文
较特殊:有的外核层阳性在外核层的最外层，内核层阳性在内核层的最内层。③
观察到50天为止，米发现DAPI阳性细胞参与或导致视网膜的增生，也未发现细
胞层的增厚一。
前一部分工作发现了MSC可以在视网膜下存在，并示踪了其变化过程。那
么形成的视网膜样结构是否具有视网膜各层的细胞特征呢?本研究对以上各时间
点的MSC一DAPI冰冻切片进行了神经特异胜蛋白NeuN及NSE、字中经胶质细胞特
异性蛋白GFAP及视网膜色素上皮标志CK的兔疫组化染色。结果表明卜[I性细胞
在移植之初己经有部分细胞表达了Ne、lN和NSE等神经细胞表面标志，同时也观
察到G队P和CK在视网膜的?
Within the bone marrow stroma there exists a subset of nonhematopoietic cells referred to as mesenchymal stem or mesenchymal progenitor cells. Mesenchymal stem cells (MSC) are a population of pluripotent cells within the human, bird or rodent bone marrow microenvironment defined by their ability, either in vitro or in vivo, to differentiate into the types of the osteogenic, chondrogenic, tendonogenic, adipogenic, neural cells and myogenic lineages. The methodologies to isolate and culture-expand MSC from human bone marrow for establishing the cellular or tissue aspects of differentiation model in vitro have been set up. The multipotential of these cells, the easy isolation and culture property, as well as their high ex vivo expansive potential makes these cells an attractive therapeutic tool. Mere we report that bone marrow mesenchymal stem cells, after subretinally transplanted into normal or Nd: YAG laser-injured rat eyes, can integrate into retina pigmented epithelial (RPE) , photoreceptor cell layer, bipolar cell layer and ganglion layer.
We tested the hypothesis that MSC can be differentiated into retinal cells by transplanting MSC in vivo into subretinal space. This hypothesis was tested by three approaches (i) subretinal transplantation of MSC (STM) from male rats into the females and the detection of donor cells in the recipients by means of DNA probes to the Y chromosome sry region and (ii) STM from male MSC transduced by recombinant adeno-associated virus (rAAV) carrying green fluorescence protein (GFP) into the females and the detection of the GFP fluorescence by means of fluorescence microscope, (iii) STM by DAPI labeling and the detection of the GFP fluorescence by means of fluorescence microscope. In situ hybridization and fluorescence microscope
observation were used to distinguish donor cells from recipient cells. Then we verified that the MSC-derived cells could express CK, NSE and glial fibrillary acidic protein (GFAP) respectively by immunohistochemistry analysis.
Female rats were subretinally injected with male MSC engrafts. 4 weeks later the rats were sacrificed and the eyes were enucleated and embedded in paraffin. Using polymerase chain reaction (PCR), a digoxigenin-labeled 459 base pair sized sry gene, which is a gene of the Y chromosome in rat, was synthesized for the sex determination. The probe was then used to detect male mouse cells in tissue sections by in situ hybridization. This constituted a system in which the presence of cells originating from the donor in the recipient retina could be easily detected. Y chromosome sry gene hybridization was finally changed into a color reaction. Some nuclei showed positive signal for the sry gene PCR probe. Under optical microscope observation the ivy-positive cells were found in the outer and inner nuclear layers and photoreceptor cells.
Another approach to examine the differentiation of MSC was GFP detection. AAW-gfp was transduced into MSC in vitro. Adenovirus was added into the culture to show that MSC can express GFP. STM was performed from the AAV-gfp-transduced male MSC into female retina. 4 weeks later the female rats were sacrificed and the eyes were enucleated and were made into frozen sections. Fluorescence microscope observation in 490nm wavelength showed that GFP positive cells were dispersed in RPE layer, photoreceptor layer and ganglion cell layer, whereas the untreated female retina has no GFP positive cells. This result showed that the STM has made the donor MSC integrated in the subretinal space without disturbing the retinal organization and lamination and AAV-gfp-MSC can be differentiated into retina-like cells, which have the same results as the ISH showed. We also found that some of the vessel endothelial cells in choroid showed GFP fluorescence, which means that the GFP-labeled cells might take part in the vessel construction (data not shown).
DAPI labeling was used to trace how MSC change in the retina. We found that on day 10 DAPI positive cells were found mainly around the injected site in either
干细胞工程的研究为视网膜损伤的治疗提供了一种新的途径。研究者试用了多种干细胞，以期解决视网膜损伤的修复问题，如胚胎干细胞(embryonic stem cell，ESC)、胚胎视网膜祖细胞、成体哺乳动物眼干细胞等。但都存在着各种缺陷，如异体移植问题、伦理学问题及取材问题等。
骨髓中含有可分化为外周循环血细胞的造血干细胞早已为人所知。此外，人们还发现骨髓中有一群细胞可以分化成多种间质样的细胞，于是将它们称为骨髓间充质干细胞(即Mesenchymal Stem Cells，MSC)。在体外不同条件的作用下MSC可以定向地被诱导分化为成骨细胞、成软骨细胞、脂肪细胞、肌肉细胞、肌腱细胞、神经元等不同的细胞。MSC取材相对较容易，来源丰富，体外增殖能力强，适于作为细胞供体进行自体移植。
鉴于MSC便利的取材及其可向神经细胞分化的潜能，本研究期望通过视网膜下移植的方式研究MSC在正常视网膜及激光损伤视网膜中分化为视网膜的可能性，以及分化成的细胞是否具有神经细胞的特性，是否有利于视网膜激光损伤后的组织修复及视功能的恢复。
本研究首先分离、培养并鉴定了MSC。利用骨髓间充质干细胞的密度特性，用一定密度的Percoll对骨髓进行密度梯度分离，再利用骨髓间充质干细胞粘附在培养皿上的特性，去除单核／巨噬细胞，从而达到富集骨髓间充质干细胞的目的，体外传代可达15代以上。对富集纯化的细胞进行流式细胞术表型鉴定，并将MSC诱导分化为脂肪细胞，证明了其具有多向分化能力。自行建立的培养方法已申请
军事医学利学院博}学位论文
专利(专利受理号01141865.6)。如此培养出的MSC既具备增殖快的特点，又保
持了其做为干细胞的多向分化潜能，为进一步研究工作扫一下基础。
为证实MSC可以在正常视网膜分化为视网膜结构，本研究首先分离培养了
雄性大鼠的MSC，将其移植到雌性大鼠视网膜下，4周后利用雌雄标记法进行Y
染色体原位杂交，示踪MSC的分布。光学显微镜观察结果显示，阳性细胞在移
植区域周围与原来的视网膜细胞融合，并形成了典型的内、外核层及节细胞层的
细胞形态。同一视野下同时存在着阳胜和阴性细胞，阳性细胞并非单独存在，而
是形成一定的阳性细胞群，中间夹杂着阴性细胞。其中以外核层的阳性细胞较多，
由外到内逐渐减少，节细胞层最少。整个视网膜形态平滑，未见到玫瑰花结结构
少巨成。
一证明MSC在视网膜卜的分化至少需要3种示踪方法，因此本研究采用了体
外经AAV一加感染MSC的视网膜下移植观察。荧光显微镜观察结果表明，移植
4周后发现视网膜内存在GFP阳性细胞，其中以视网膜色素上皮层、视细胞层为
多，内、外网状层及节细)泡层也有}朴胜细胞的存在。阳性细胞相对较聚集，夕手与
阴性细胞相融合，在光学显微镜卜‘末发现视网膜形态的异常。这一结果不仅说明
MSC在视网膜下分化的定位，也说明MSC司一以被AJ叭z所感染，司一以作为基因
修饰细胞移植的载体细胞。AAV一gfr感染的结果与Y染色体原位杂交结果基木一
致。
以__}二不沙}究只观察了4周时MSC的变化情况，为观察MSC在移植后不同时问
的变化过程，同时用另一种方法再次确证MSC在视网膜下的存在，本研究进一
步采用荧光染料DAPJ对MSC进行标记，分别于移植后10、20、35和50天观察
了视网膜MSC一DAPI的分布情况。MSC在正常视网膜分化有以下特点:①卜lIJ卜l-
细胞随着移植时间的延长，先是顺着移植部位向周围打、一展，阳性细胞所在的部位
不断扩大，而后又有缩小的趋势，50大观察到的卜卜}胜细胞范围比35大明显绷小
(阳性红l!胞4个时I泊」点分布范围分别为960‘:n飞、1280抖m、640工Lm及160牛tm)，这
说明MSC的分化增殖是受到调控的，不会无限制增生;而且在正常视网膜条件
下似乎存在着一种抑制机制，使MSC趋向于逐渐减少;②阳性部位的细胞分布
第4臾共127
军事队学利学院博卜学位论文
较特殊:有的外核层阳性在外核层的最外层，内核层阳性在内核层的最内层。③
观察到50天为止，米发现DAPI阳性细胞参与或导致视网膜的增生，也未发现细
胞层的增厚一。
前一部分工作发现了MSC可以在视网膜下存在，并示踪了其变化过程。那
么形成的视网膜样结构是否具有视网膜各层的细胞特征呢?本研究对以上各时间
点的MSC一DAPI冰冻切片进行了神经特异胜蛋白NeuN及NSE、字中经胶质细胞特
异性蛋白GFAP及视网膜色素上皮标志CK的兔疫组化染色。结果表明卜[I性细胞
在移植之初己经有部分细胞表达了Ne、lN和NSE等神经细胞表面标志，同时也观
察到G队P和CK在视网膜的?
Within the bone marrow stroma there exists a subset of nonhematopoietic cells referred to as mesenchymal stem or mesenchymal progenitor cells. Mesenchymal stem cells (MSC) are a population of pluripotent cells within the human, bird or rodent bone marrow microenvironment defined by their ability, either in vitro or in vivo, to differentiate into the types of the osteogenic, chondrogenic, tendonogenic, adipogenic, neural cells and myogenic lineages. The methodologies to isolate and culture-expand MSC from human bone marrow for establishing the cellular or tissue aspects of differentiation model in vitro have been set up. The multipotential of these cells, the easy isolation and culture property, as well as their high ex vivo expansive potential makes these cells an attractive therapeutic tool. Mere we report that bone marrow mesenchymal stem cells, after subretinally transplanted into normal or Nd: YAG laser-injured rat eyes, can integrate into retina pigmented epithelial (RPE) , photoreceptor cell layer, bipolar cell layer and ganglion layer.
We tested the hypothesis that MSC can be differentiated into retinal cells by transplanting MSC in vivo into subretinal space. This hypothesis was tested by three approaches (i) subretinal transplantation of MSC (STM) from male rats into the females and the detection of donor cells in the recipients by means of DNA probes to the Y chromosome sry region and (ii) STM from male MSC transduced by recombinant adeno-associated virus (rAAV) carrying green fluorescence protein (GFP) into the females and the detection of the GFP fluorescence by means of fluorescence microscope, (iii) STM by DAPI labeling and the detection of the GFP fluorescence by means of fluorescence microscope. In situ hybridization and fluorescence microscope
observation were used to distinguish donor cells from recipient cells. Then we verified that the MSC-derived cells could express CK, NSE and glial fibrillary acidic protein (GFAP) respectively by immunohistochemistry analysis.
Female rats were subretinally injected with male MSC engrafts. 4 weeks later the rats were sacrificed and the eyes were enucleated and embedded in paraffin. Using polymerase chain reaction (PCR), a digoxigenin-labeled 459 base pair sized sry gene, which is a gene of the Y chromosome in rat, was synthesized for the sex determination. The probe was then used to detect male mouse cells in tissue sections by in situ hybridization. This constituted a system in which the presence of cells originating from the donor in the recipient retina could be easily detected. Y chromosome sry gene hybridization was finally changed into a color reaction. Some nuclei showed positive signal for the sry gene PCR probe. Under optical microscope observation the ivy-positive cells were found in the outer and inner nuclear layers and photoreceptor cells.
Another approach to examine the differentiation of MSC was GFP detection. AAW-gfp was transduced into MSC in vitro. Adenovirus was added into the culture to show that MSC can express GFP. STM was performed from the AAV-gfp-transduced male MSC into female retina. 4 weeks later the female rats were sacrificed and the eyes were enucleated and were made into frozen sections. Fluorescence microscope observation in 490nm wavelength showed that GFP positive cells were dispersed in RPE layer, photoreceptor layer and ganglion cell layer, whereas the untreated female retina has no GFP positive cells. This result showed that the STM has made the donor MSC integrated in the subretinal space without disturbing the retinal organization and lamination and AAV-gfp-MSC can be differentiated into retina-like cells, which have the same results as the ISH showed. We also found that some of the vessel endothelial cells in choroid showed GFP fluorescence, which means that the GFP-labeled cells might take part in the vessel construction (data not shown).
DAPI labeling was used to trace how MSC change in the retina. We found that on day 10 DAPI positive cells were found mainly around the injected site in either
引文
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19． Fischer AJ, Dierks BD, Reh TA. Exogenous growth factors induce the production of ganglion cells at the retinal margin. Development, 2002, 129: 2283-2291．
20． Grant MB, May WS, Caballero S, et al. Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization. Nat Med, 2002, 8: 607-612．
21． Uckermann O, Grosche J, Reichenbach A, et al. ATP-evoked calcium responses of radial glial (Muller) cells in the postnatal rabbit retina. J Neurosci Res, 2002, 70: 209-218．
22． Silverman MS, Hughes SE. Transplantation of photoreceptors to light-damaged retina. Invest Ophthalmol Visual Sci, 1989, 30: 1684-1690．
23． Chen S, Samuel W, Fariss RN, et al. Differentiation of human retinal pigment epithelial cells into neuronal phenotype by N-(4-hydroxyphenyl)retinamide. J Neurochem, 2003, 84: 972-981．
24． Lu B, Kwan T, Kurimoto Y, et al. Transplantation of EGF-responsive neurospheres from GFP transgenic mice into the eyes of rd mice. Brain Res, 2002, 943: 292-300．
25． Wojciechowski AB, Englund U, Lundberg C, et al. Long-term survival and glial differentiation of the brain-derived precursor cell line RN33B after subretinal transplantation to adult normal rats. Stem Cells, 2002, 20: 163-173．
26． Kathyjo AJ, Tiejuan M, Margret AG. Hematopoietic potential of stem cells isolated from murine skeletal muscle. PNAS, 1999, 96: 14482-14486．
27． Reyes M., Verfaillie C.M. Turning marrow into brain: Generation of glial and neuronal cells from adult bone marrow mesenchymal stem cells. Blood, 1999, 94: 377a-.
28． Hansel DE, Eipper BA, Ronnett GV. Neuropeptide Y functions as a neuroproliferative factor. Nature, 2001, 410: 940-943．
29． Zohar R, Sodek J, McCulloch CA. Characterization of stromal progenitor cells enriched by flow cytometry. Blood, 1997, 90: 3471-3481．
30． Ose JM, Alejandro E, Paulette C. Mesenchymal stem cells. Experimental Biology and Medicine, 2001, 226: 507-520．
31． Fortier LA, Nixon AJ, Williams J. Isolation and chondrocytic differentiation of equine bone marrow-derived mesenchymal stem cells. Am J Vet Res, 1998, 59: 1182-1187．
32． Hu Y, Ma L, Ma G, et al. [Comparative study of human fetal and adult bone marrow derived mesenchymal stem cells]. Zhonghua Xue Ye Xue Za Zhi, 2002, 23: 645-648．
33． Anselme K, Broux O, Noel B, et al. In Vitro Control of Human Bone Marrow Stromal Cells for Bone Tissue Engineering. Tissue Eng, 2002, 8: 941-953．
34． Minguell JJ, Erices A, Conget P. Mesenchymal stem cells . Exp Biol Med, 2001, 226: 507-520．
35． Kopen G.C., Prockop D.J. Marrow stroma cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc Natl Acad Sci USA, 1999, 96: 10711-10716．
36． Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science, 1997,276:71-71．
37． Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science, 1999, 284: 143-147．
38． Caplan AI Bruder SP. Mesenchymal stem cells: building blocks for molecular medicine in the 21st century. Trends Mol Med, 2001, 7: 259-264．
39． Claudio Bordignon, Carmelo Carlo-Stella, Mario Paolo Colombo, et al. Cell therapy: achievements and perspectives. Haematologica, 1999, 84: 1110-1149．
40． Campagnoli C, Roberts IA, Kumar S, et al. Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow. Blood, 2001,98:2396-2402．
41． Seshi B, Kumar S, Sellers D. Human bone marrow stromal cell: coexpression of markers specific for multiple mesenchymal cell lineages. Blood Cells Mol Dis, 2000, 26: 234-246．
42． Goodell MA, Brose K, Paradis G, et al. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med, 1996, 183: 1979-1806．
43． Tomita M, Adachi Y, Yamada H, et al. Bone marrow-derived stem cells can differentiate into retinal cells in injured rat retina. Stem Cells, 2002, 20: 279-283．
44． Guo Z, Tang P, Liu X, et al. Mesenchymal Stem Cells Derived from Human Bone Marrow Support Hematopoiesis in Vitro. Zhongguo Shi Yan Xue Ye Xue Za Zhi, 2000, 8: 93-96．
45． Robert JD, Annemarie BM. Mesenchymal stem cells: biology and potential clinical uses. Experimental Hematology, 2000, 28: 875-884．
46． Justesen J, Stenderup K, Eriksen EF, et al. Maintenance of osteoblastic and adipocytic differentiation potential with age and osteoporosis in human marrow stromal cell cultures. Calcif Tissue Int, 2002, 71: 36-44．
47． Devine SM, Hoffman R. Role of mesenchymal stem cells in hematopoietic stem cell transplantation. Curr Opin Hematol, 2000, 7: 358-363．
48． Devine SM. Mesenchymal stem cells: will they have a role in the clinic? J Cell Biochem Suppl, 2002, 38: 73-79．
49． Devine SM, Peter S, Martin BJ, et al. Mesenchymal stem cells: stealth and suppression. Cancer J, 2001, 7 Suppl 2: S76-S82．
50． Minguell JJ, Conget P, Erices A. Biology and clinical utilization of mesenchymal progenitor cells. Braz J Med Biol Res, 2000, 33: 881-887．
51． Deans RJ, Moseley AB. Mesenchymal stem cells: biology and potential clinical uses. Exp Hematol, 2000, 28: 875-884．
52． Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science, 1999, 284: 143-147．
53． Pittenger MF, Thiede MA, Mosca JD. Phenotypic and functional comparision of cultures of marrow-derived mesenchymal stem cells(MSC) and stromal cell. J Cell Physio, 1998, 176: 57-66．
54． Bruder SP, Ricalton NS, Boynton RE. Mesenchymal stem cell surface antigen SB-10corresponds to activated leukocyte cell adhesion molecule and is involved in osteogenic differentiation. J Bone Miner Res, 1998, 13: 655-663．
55． Williams JT, Southerland SS, Souza J. Cells isolate from adulthuman skeletal muscle capable of differentiation into multiple mesodermal phenotype. Am Surg, 1999,65:22-26．
56． Nakahara H, Dennis JE. Bruder SP. In vitro differentiation of bone and hypertrophic cartilage from periosteal-derived cell. ExpCell Re, 1991, 195: 492-503．
57． Nuttall ME, Patton AJ, Patton AJ, et al. Human trabecular bone cells are able to expressboth osteoblastic and adipocytic phenotype:implications for osteopenic disorders. J Bone Miner Res, 1998, 13 : 371-382．
58． Ghilzon R, McCulloch CA, Zohar R. Stromal mesenchymal progenitor cells in process citation. Leuk Lymphoma, 1999, 32: 211-222．
59． Kicic A, Shen W, Rakoczy PE. The potential of marrow stromal cells in stem cell therapy. Eye, 2001, 15: 695-707．
60． Justesen J, Stenderup K, Kassem MS. [Mesenchymal stem cells. Potential use in cell and gene therapy of bone loss caused by aging and osteoporosis]. Ugeskr Laeger, 2001, 163: 5491-5495．
61． Hoerstrup SP, Kadner A, Melnitchouk S, et al. Tissue engineering of functional trileaflet heart valves from human marrow stromal cells. Circulation, 2002, 106: 1143-1150．
62． Jose J.M., Alejandro E.. Paillette C. Mesenchymal stem cells. Exp Bio Med, 2001, 226: 507-520．
63． Le Blanc K. [Mesenchymal stem cells. Basic science and future clinical use]. Lakartidningen, 2002, 99: 1318-21, 1324．
64． Peterson BE, Bowen WC, Partrene KD, et al. Bone Marrow as a potential source of hepatic oval cells. Science, 2000, 284: 1168-1170．
65． Sambook J. Molecular Cloning, a laboratary manual. 2nd edition. 1989．
66． Grant C A, Ponnazhagan S, Wang XS. Evaluation of recombinant adeno-associated virus as a gene transfer vector for the retina. Curr Eye Res, 1997, 16: 956-.
67． Wu ZJ, Wu XB, Cao H, et al. A new high-efficiency method for generating recombinant adeno-associated virus. SCIENCE IN CHINA ( Series C ), 2001, 31: 423-430．
68． Zacks DN, Samson CM, Loewenstein J, et al. Electroretinograms as an indicator of disease activity in birdshot retinochoroidopathy. Graefes Arch Clin Exp Ophthalmol, 2002, 240: 601-607．
69． Garway-Heath DF, Holder GE, Fitzke FW, et al. Relationship between electrophysiological, psychophysical, and anatomical measurements in glaucoma. Invest Ophthalmol Vis Sci, 2002, 43: 2213-2220．
70． Qiao C, Wang B, Zhu X, et al. A novel gene expression control system and its use in stable, high-titer 293 cell-based adeno-associated virus packaging cell lines. J Virol, 2002, 76: 13015-13027．
71． Yuasa K, Sakamoto M, Miyagoe-Suzuki Y, et al. Adeno-associated virus veclor-medialed gene transfer into dystrophin-deficient skeletal muscles evokes enhanced immune response againsl the transgene product. Gene Ther, 2002, 9: 1576-1588．
72． Tsai TH, Chen SL, Xiao X, et al. Gene therapy for trealment of cerebral ischemia using defective recombinanl adeno-associated virus vectors. Methods, 2002, 28: 253-258．
73． Wu WC, Lai CC, Chen SL, et al. Gene therapy for delached relina by adeno-associated virus vector expressing glial cell line-derived neurotrophic factor. Invest Ophthalmol Vis Sci, 2002, 43: 3480-3488．
74． Pelled G, G T, Asian H, el al. Mesenchymal stem cells for bone gene therapy and tissue engineering. Curr Pharm Des, 2002, 8: 1917-1928．
75． Van Damme A, Vanden Driessche T, Collen D, et al. Bone marrow stromal cells as targets for gene therapy. Curr Gene Ther, 2002, 2: 195-209．
76． Pei X. Stem cell engineering: the new generalion of cellular therapeutics. Int J Hemalol, 2002, 76: 155-156．
77． Hochedlinger K, Jaenisch R. Monoclonal mice generated by nuclear Iransfer from malure B and T donor cells. Nature, 2002, 415: 1035-1038．
78． Koc ON, Lazarus HM. Mesenchymal stem cells: heading into the clinic. Bone Marrow Transplant, 2001, 27: 235-239．
79． Wu P, Ye Y, Svendsen CN. Transduction of human neural progenitor cells using recombinanl adeno-associaled viral vectors. Gene Ther, 2002, 9: 245-255．
80． Owens RA. Second generalion adeno-associated virus lype 2-based gene Iherapy systems with the potential for preferential integration into AAVS1． Curr Gene Ther, 2002, 2: 145-159．
81． Lian JB, Stein GS, Stein JL, et al. Marrow transplanlalion and targeted gene therapy to the skeleton. Clin Orthop, 2000, 379 (Suppl): S146-155．
82． Wolf HK, Buslei R, Schmidt-Kaslner R, et al. NeuN: a useful neuronal marker for diagnostic histopathology. J Histochem Cytochem, 1996, 44: 1167-1171．
83． Nolte C, Malyash M, Pivneva T, el al. GFAP promoler-conlrolled EGFP-expressing transgenic mice: a tool to visualize astrocyles and aslrogliosis in living brain lissue. Glia, 2001, 33: 72-86．
84． Wolf HK, Buslei R, Schmidl-Kaslner R, el al. NeuN: a useful neuronal marker for diagnoslic hislopalhology. J Histochem Cytochem, 1996, 44: 1167-1171．
85． Pleines UE, Morganti-Kossmann MC, Rancan M, el al. S-100 beta reflecls the extenl of injury and oulcome, whereas neuronal specific enolase is a belter indicator of neuroinflammation in patienls with severe Iraumatic brain injury. J Neurotrauma, 2001, 18: 491-498．
86． Satoh F, Umemura S, Yasuda M, et al. Neuroendocrine Marker expression in thyroid epithelial lumors. Endocr Parhol, 2001, 12: 291-299．
87． Yuge K, Nakajima M, Uemura Y, et al. Immunohistochemical features of the human retina and retinoblastoma. Virchows Arch, 1995, 426: 571-575．
88． Eng LF, Vanderhaeghen JJ, Bignami A, et al. An acidic protein isolated from fibrous astrocytes. brain research, 1971, 28: 351-354．
89． Lazarides E. Intermediate filaments as mechanical integrators of cellular space. Nature, 1980,28:249-256．
90． Pei RJ, Liu YH, Su B, et al. Do the CK18 related proteins change in general in epithelial cancers? Res Commun Mol Pathol Pharmacol, 2000, 108: 253-260．
91． Kurzen H, Esposito L, Langbein L, et al. Cytokeratins as markers of follicular differentiation: an immunohistochemical study of trichoblastoma and basal cell carainoma. Am J Dermatopathol, 2001, 23: 501-509．
2． Angelov DN, Arnhold S, Andressen C, et al. Temporospatial relationships between macroglia and microglia during in vitro differentiation of murine stem cells. Dev Neurosci, 1998,20: 42-51．
3． Li Y, Zhong X, Yi Y. Differentiation of embryonic stem cells into neurons and retina-like structure in nude mice. Chin J Ocul Fundus Dis, 2000, 16: 259-.
4． David MC, Jim AR, James ET. Survival and differentation of cultured retinal progenitors transplanted in the subretinal space of the rat. BBRC, 2000, 268: 842-846．
5． Vincent T, Brenda LKC, BJ, et al. Retinal stem cells in the adult mammalian eye. Science, 2000, 287: 2032-2036．
6． Arnhold S, Fassbender A, Klinz FJ, et al. NOS-Ⅱ is involved in early differentiation of murine cortical, retinal and ES cell-derived neurons-an immunocytochemical and functional approach. Int J Dev Neurosci, 2002, 20: 83-92．
7． Kawasaki H, Suemori H, Mizuseki K, et al. Generation of dopaminergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity. Proc Natl Acad Sci USA, 2002, 99: 1580-1585．
8． Zhao X, Liu J, Ahmad I. Differentiation of embryonic stem cells into retinal neurons. Biochem Biophys Res Commun, 2002, 297: 177-184．
9． Schraermeyer U, Thumann G, Luther T, et al. Subretinally transplanted embryonic stem cells rescue photoreceptor cells from degeneration in the RCS rats. Cell Transplant, 2001, 10: 673-680．
10． Kubo F, Takeichi M, Nakagawa S. Wnt2b controls retinal cell differentiation at the ciliary marginal zone. Development, 2003, 130: 587-598．
11． Perron M, Harris WA. Retinal stem cells in vertebrates. Bioessays, 2000, 22: 685-688．
12． Reh TA, Fischer AJ. Stem cells in the vertebrate retina. Brain Behav Evol, 2001, 58: 296-305．
13． Harris WA, Perron M. Molecular recapitulation: the growth of the vertebrate retina. Int J Dev Biol, 1998, 42: 299-304．
14． Li X, Perissi V, Liu F, et al. Tissue-specific regulation of retinal and pituitary precursor cell proliferation. Science. 2002, 297: 1180-1183．
15． Yang P, Seiler MJ, Aramant RB, et al. In vitro isolation and expansion of human retinal progenitor cells. Exp Neurol, 2002, 177: 326-331．
16． Yang P, Seiler MJ, Aramant RB, et al. Differential lineage restriction of rat retinal progenitor cells in vitro and in vivo. J Neurosci Res, 2002, 69: 466-476．
17． Fischer AJ, Reh TA. Identification of a proliferating marginal zone of retinal progenitors in postnatal chickens. Dev Biol, 2000, 220: 197-210．
18． Fischer AJ, McGuire CR, Dierks BD, et al. Insulin and fibroblast growth factor 2 activate a neurogenic program in Muller glia of the chicken retina. J Neurosci, 2002, 22: 9387-9398．
19． Fischer AJ, Dierks BD, Reh TA. Exogenous growth factors induce the production of ganglion cells at the retinal margin. Development, 2002, 129: 2283-2291．
20． Grant MB, May WS, Caballero S, et al. Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization. Nat Med, 2002, 8: 607-612．
21． Uckermann O, Grosche J, Reichenbach A, et al. ATP-evoked calcium responses of radial glial (Muller) cells in the postnatal rabbit retina. J Neurosci Res, 2002, 70: 209-218．
22． Silverman MS, Hughes SE. Transplantation of photoreceptors to light-damaged retina. Invest Ophthalmol Visual Sci, 1989, 30: 1684-1690．
23． Chen S, Samuel W, Fariss RN, et al. Differentiation of human retinal pigment epithelial cells into neuronal phenotype by N-(4-hydroxyphenyl)retinamide. J Neurochem, 2003, 84: 972-981．
24． Lu B, Kwan T, Kurimoto Y, et al. Transplantation of EGF-responsive neurospheres from GFP transgenic mice into the eyes of rd mice. Brain Res, 2002, 943: 292-300．
25． Wojciechowski AB, Englund U, Lundberg C, et al. Long-term survival and glial differentiation of the brain-derived precursor cell line RN33B after subretinal transplantation to adult normal rats. Stem Cells, 2002, 20: 163-173．
26． Kathyjo AJ, Tiejuan M, Margret AG. Hematopoietic potential of stem cells isolated from murine skeletal muscle. PNAS, 1999, 96: 14482-14486．
27． Reyes M., Verfaillie C.M. Turning marrow into brain: Generation of glial and neuronal cells from adult bone marrow mesenchymal stem cells. Blood, 1999, 94: 377a-.
28． Hansel DE, Eipper BA, Ronnett GV. Neuropeptide Y functions as a neuroproliferative factor. Nature, 2001, 410: 940-943．
29． Zohar R, Sodek J, McCulloch CA. Characterization of stromal progenitor cells enriched by flow cytometry. Blood, 1997, 90: 3471-3481．
30． Ose JM, Alejandro E, Paulette C. Mesenchymal stem cells. Experimental Biology and Medicine, 2001, 226: 507-520．
31． Fortier LA, Nixon AJ, Williams J. Isolation and chondrocytic differentiation of equine bone marrow-derived mesenchymal stem cells. Am J Vet Res, 1998, 59: 1182-1187．
32． Hu Y, Ma L, Ma G, et al. [Comparative study of human fetal and adult bone marrow derived mesenchymal stem cells]. Zhonghua Xue Ye Xue Za Zhi, 2002, 23: 645-648．
33． Anselme K, Broux O, Noel B, et al. In Vitro Control of Human Bone Marrow Stromal Cells for Bone Tissue Engineering. Tissue Eng, 2002, 8: 941-953．
34． Minguell JJ, Erices A, Conget P. Mesenchymal stem cells . Exp Biol Med, 2001, 226: 507-520．
35． Kopen G.C., Prockop D.J. Marrow stroma cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc Natl Acad Sci USA, 1999, 96: 10711-10716．
36． Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science, 1997,276:71-71．
37． Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science, 1999, 284: 143-147．
38． Caplan AI Bruder SP. Mesenchymal stem cells: building blocks for molecular medicine in the 21st century. Trends Mol Med, 2001, 7: 259-264．
39． Claudio Bordignon, Carmelo Carlo-Stella, Mario Paolo Colombo, et al. Cell therapy: achievements and perspectives. Haematologica, 1999, 84: 1110-1149．
40． Campagnoli C, Roberts IA, Kumar S, et al. Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow. Blood, 2001,98:2396-2402．
41． Seshi B, Kumar S, Sellers D. Human bone marrow stromal cell: coexpression of markers specific for multiple mesenchymal cell lineages. Blood Cells Mol Dis, 2000, 26: 234-246．
42． Goodell MA, Brose K, Paradis G, et al. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med, 1996, 183: 1979-1806．
43． Tomita M, Adachi Y, Yamada H, et al. Bone marrow-derived stem cells can differentiate into retinal cells in injured rat retina. Stem Cells, 2002, 20: 279-283．
44． Guo Z, Tang P, Liu X, et al. Mesenchymal Stem Cells Derived from Human Bone Marrow Support Hematopoiesis in Vitro. Zhongguo Shi Yan Xue Ye Xue Za Zhi, 2000, 8: 93-96．
45． Robert JD, Annemarie BM. Mesenchymal stem cells: biology and potential clinical uses. Experimental Hematology, 2000, 28: 875-884．
46． Justesen J, Stenderup K, Eriksen EF, et al. Maintenance of osteoblastic and adipocytic differentiation potential with age and osteoporosis in human marrow stromal cell cultures. Calcif Tissue Int, 2002, 71: 36-44．
47． Devine SM, Hoffman R. Role of mesenchymal stem cells in hematopoietic stem cell transplantation. Curr Opin Hematol, 2000, 7: 358-363．
48． Devine SM. Mesenchymal stem cells: will they have a role in the clinic? J Cell Biochem Suppl, 2002, 38: 73-79．
49． Devine SM, Peter S, Martin BJ, et al. Mesenchymal stem cells: stealth and suppression. Cancer J, 2001, 7 Suppl 2: S76-S82．
50． Minguell JJ, Conget P, Erices A. Biology and clinical utilization of mesenchymal progenitor cells. Braz J Med Biol Res, 2000, 33: 881-887．
51． Deans RJ, Moseley AB. Mesenchymal stem cells: biology and potential clinical uses. Exp Hematol, 2000, 28: 875-884．
52． Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science, 1999, 284: 143-147．
53． Pittenger MF, Thiede MA, Mosca JD. Phenotypic and functional comparision of cultures of marrow-derived mesenchymal stem cells(MSC) and stromal cell. J Cell Physio, 1998, 176: 57-66．
54． Bruder SP, Ricalton NS, Boynton RE. Mesenchymal stem cell surface antigen SB-10corresponds to activated leukocyte cell adhesion molecule and is involved in osteogenic differentiation. J Bone Miner Res, 1998, 13: 655-663．
55． Williams JT, Southerland SS, Souza J. Cells isolate from adulthuman skeletal muscle capable of differentiation into multiple mesodermal phenotype. Am Surg, 1999,65:22-26．
56． Nakahara H, Dennis JE. Bruder SP. In vitro differentiation of bone and hypertrophic cartilage from periosteal-derived cell. ExpCell Re, 1991, 195: 492-503．
57． Nuttall ME, Patton AJ, Patton AJ, et al. Human trabecular bone cells are able to expressboth osteoblastic and adipocytic phenotype:implications for osteopenic disorders. J Bone Miner Res, 1998, 13 : 371-382．
58． Ghilzon R, McCulloch CA, Zohar R. Stromal mesenchymal progenitor cells in process citation. Leuk Lymphoma, 1999, 32: 211-222．
59． Kicic A, Shen W, Rakoczy PE. The potential of marrow stromal cells in stem cell therapy. Eye, 2001, 15: 695-707．
60． Justesen J, Stenderup K, Kassem MS. [Mesenchymal stem cells. Potential use in cell and gene therapy of bone loss caused by aging and osteoporosis]. Ugeskr Laeger, 2001, 163: 5491-5495．
61． Hoerstrup SP, Kadner A, Melnitchouk S, et al. Tissue engineering of functional trileaflet heart valves from human marrow stromal cells. Circulation, 2002, 106: 1143-1150．
62． Jose J.M., Alejandro E.. Paillette C. Mesenchymal stem cells. Exp Bio Med, 2001, 226: 507-520．
63． Le Blanc K. [Mesenchymal stem cells. Basic science and future clinical use]. Lakartidningen, 2002, 99: 1318-21, 1324．
64． Peterson BE, Bowen WC, Partrene KD, et al. Bone Marrow as a potential source of hepatic oval cells. Science, 2000, 284: 1168-1170．
65． Sambook J. Molecular Cloning, a laboratary manual. 2nd edition. 1989．
66． Grant C A, Ponnazhagan S, Wang XS. Evaluation of recombinant adeno-associated virus as a gene transfer vector for the retina. Curr Eye Res, 1997, 16: 956-.
67． Wu ZJ, Wu XB, Cao H, et al. A new high-efficiency method for generating recombinant adeno-associated virus. SCIENCE IN CHINA ( Series C ), 2001, 31: 423-430．
68． Zacks DN, Samson CM, Loewenstein J, et al. Electroretinograms as an indicator of disease activity in birdshot retinochoroidopathy. Graefes Arch Clin Exp Ophthalmol, 2002, 240: 601-607．
69． Garway-Heath DF, Holder GE, Fitzke FW, et al. Relationship between electrophysiological, psychophysical, and anatomical measurements in glaucoma. Invest Ophthalmol Vis Sci, 2002, 43: 2213-2220．
70． Qiao C, Wang B, Zhu X, et al. A novel gene expression control system and its use in stable, high-titer 293 cell-based adeno-associated virus packaging cell lines. J Virol, 2002, 76: 13015-13027．
71． Yuasa K, Sakamoto M, Miyagoe-Suzuki Y, et al. Adeno-associated virus veclor-medialed gene transfer into dystrophin-deficient skeletal muscles evokes enhanced immune response againsl the transgene product. Gene Ther, 2002, 9: 1576-1588．
72． Tsai TH, Chen SL, Xiao X, et al. Gene therapy for trealment of cerebral ischemia using defective recombinanl adeno-associated virus vectors. Methods, 2002, 28: 253-258．
73． Wu WC, Lai CC, Chen SL, et al. Gene therapy for delached relina by adeno-associated virus vector expressing glial cell line-derived neurotrophic factor. Invest Ophthalmol Vis Sci, 2002, 43: 3480-3488．
74． Pelled G, G T, Asian H, el al. Mesenchymal stem cells for bone gene therapy and tissue engineering. Curr Pharm Des, 2002, 8: 1917-1928．
75． Van Damme A, Vanden Driessche T, Collen D, et al. Bone marrow stromal cells as targets for gene therapy. Curr Gene Ther, 2002, 2: 195-209．
76． Pei X. Stem cell engineering: the new generalion of cellular therapeutics. Int J Hemalol, 2002, 76: 155-156．
77． Hochedlinger K, Jaenisch R. Monoclonal mice generated by nuclear Iransfer from malure B and T donor cells. Nature, 2002, 415: 1035-1038．
78． Koc ON, Lazarus HM. Mesenchymal stem cells: heading into the clinic. Bone Marrow Transplant, 2001, 27: 235-239．
79． Wu P, Ye Y, Svendsen CN. Transduction of human neural progenitor cells using recombinanl adeno-associaled viral vectors. Gene Ther, 2002, 9: 245-255．
80． Owens RA. Second generalion adeno-associated virus lype 2-based gene Iherapy systems with the potential for preferential integration into AAVS1． Curr Gene Ther, 2002, 2: 145-159．
81． Lian JB, Stein GS, Stein JL, et al. Marrow transplanlalion and targeted gene therapy to the skeleton. Clin Orthop, 2000, 379 (Suppl): S146-155．
82． Wolf HK, Buslei R, Schmidt-Kaslner R, et al. NeuN: a useful neuronal marker for diagnostic histopathology. J Histochem Cytochem, 1996, 44: 1167-1171．
83． Nolte C, Malyash M, Pivneva T, el al. GFAP promoler-conlrolled EGFP-expressing transgenic mice: a tool to visualize astrocyles and aslrogliosis in living brain lissue. Glia, 2001, 33: 72-86．
84． Wolf HK, Buslei R, Schmidl-Kaslner R, el al. NeuN: a useful neuronal marker for diagnoslic hislopalhology. J Histochem Cytochem, 1996, 44: 1167-1171．
85． Pleines UE, Morganti-Kossmann MC, Rancan M, el al. S-100 beta reflecls the extenl of injury and oulcome, whereas neuronal specific enolase is a belter indicator of neuroinflammation in patienls with severe Iraumatic brain injury. J Neurotrauma, 2001, 18: 491-498．
86． Satoh F, Umemura S, Yasuda M, et al. Neuroendocrine Marker expression in thyroid epithelial lumors. Endocr Parhol, 2001, 12: 291-299．
87． Yuge K, Nakajima M, Uemura Y, et al. Immunohistochemical features of the human retina and retinoblastoma. Virchows Arch, 1995, 426: 571-575．
88． Eng LF, Vanderhaeghen JJ, Bignami A, et al. An acidic protein isolated from fibrous astrocytes. brain research, 1971, 28: 351-354．
89． Lazarides E. Intermediate filaments as mechanical integrators of cellular space. Nature, 1980,28:249-256．
90． Pei RJ, Liu YH, Su B, et al. Do the CK18 related proteins change in general in epithelial cancers? Res Commun Mol Pathol Pharmacol, 2000, 108: 253-260．
91． Kurzen H, Esposito L, Langbein L, et al. Cytokeratins as markers of follicular differentiation: an immunohistochemical study of trichoblastoma and basal cell carainoma. Am J Dermatopathol, 2001, 23: 501-509．