大鼠骨髓间充质干细胞在脉络膜新生血管微环境中分化的实验研究
摘要
目的研究体外培养的大鼠骨髓间充质干细胞(Mesenchymal StemCells,MSCs)视网膜下移植后在脉络膜新生血管(Chroidal Neovascularization,CNV)微环境中的分化及其对CNV渗漏情况的影响。
方法体外培养挪威棕色大鼠(Brown Norway,BN)MSCs并经流式细胞仪鉴定CD90(+)CD45(-)后,用Brdu进行标记。用氪绿激光视网膜光凝的方法建立25只大鼠双眼CNV模型,并经眼底荧光血管造影(FluoresceinceAngiography,FA)证实CNV生成。将25只大鼠随机分为5组,每组5只。每只大鼠右眼为实验眼,经玻璃体腔注入视网膜下腔MSCs,左眼视网膜下腔注射等量PBS做为对照。术后1,2,3,4,5周分别做FA检查后处死一组动物,摘除眼球行冰冻病理切片后分别用小鼠抗人BrdU单抗、FITC标记羊抗小鼠二抗和小鼠抗大鼠CD31单抗、小鼠抗大鼠Rhodopsin单抗、小鼠抗大鼠全角蛋白(Pan-Cytokeratin,PCK)单抗、Cy3标记羊抗小鼠二抗双重荧光标记。观察术后1,2,3,4,5周MSCs在CNV微环境中的分化,计算视网膜下腔注射MSCs/PBS前、后FA面积并比较CNV渗漏情况的变化。
结果视网膜下移植术后第1周即可见MSCs位于CNV表面,但CD31标记阴性,第2周至第5周可见位于CNV内的MSCsCD31标记阳性,第5周可见移行于RPE层的MSCs rhodopsin和PCK标记阳性。FA分析结果初步表明,实验眼视网膜下移植MSCs后CNV的渗漏面积较对照眼有减少的趋势。
结论MSCs移植入CNV模型大鼠视网膜下腔后第2周至第5周可以向内皮细胞方向分化,第5周可以向光感受器细胞方向和RPE方向分化。视网膜下移植MSCs后有可能使CNV渗漏情况得到一定的改善。
Objective To access the differentiation of subretinal-transplanted rat mesenchymal stem cells (MSCs) in the microenvironment of choroidal neovascularization (CNV) and changes of CNV exudation after the transplantation .
Methods In-vitro cultured Brown Norway (BN) rats MSCs were identified CD90(+) CD45(-)by flow cytometry and labeled with Brdu. 25 CNV rats models were established by Krypton laser photocoagulation and subsequently confirmed by fluorescein angiography(FA). 25 rats were randomly distributed into 5 groups, 5 rats in every group. The right eyes were injected with MSCs into subretinal space, while the left eyes were injected with the same amount of PBS. The rats were killed and globes enucleated 1,2,3,4 or 5 weeks after the surgery respectively. Frozen sections were stained with Brdu primary monoclonal antibody, FITC- labeled secondary monoclonal antibody and CD31 , rhodopsin or pan- cytokeratin(PCK) primary monoclonal antibody respectively combined with Cy3-labeled secondary monoclonal antibody . The sections were observed under fluorescent microscope. Calculate and compare the exudation areas change pre-and post-transplantatively by FA.
Results MSCs distributed on the surface of CNV in the 1~(st) week after surgery with CD31 negative.However, from the 2~(nd) to 5~(th) week, CD31 positive MSCs were found in CNV. Rhodopsin positive and PCKpositive MSCs were found near CNV in the 5~(th) week. FA analysis demonstrated a mild decrease tendency of CNV exudation after the transplantation of MSCs.
Conclusions The results demonstrates that MSCs have the potential to differentiate into endothelium from 2th to 5~(th) weeks, into photoreceptor cells and RPE in 5~(th) weeks after subretinal transplantation in the microenvironment of NCV.CNV exudation may be mildly improved after subretinal transplantation of MSCs.
方法体外培养挪威棕色大鼠(Brown Norway,BN)MSCs并经流式细胞仪鉴定CD90(+)CD45(-)后,用Brdu进行标记。用氪绿激光视网膜光凝的方法建立25只大鼠双眼CNV模型,并经眼底荧光血管造影(FluoresceinceAngiography,FA)证实CNV生成。将25只大鼠随机分为5组,每组5只。每只大鼠右眼为实验眼,经玻璃体腔注入视网膜下腔MSCs,左眼视网膜下腔注射等量PBS做为对照。术后1,2,3,4,5周分别做FA检查后处死一组动物,摘除眼球行冰冻病理切片后分别用小鼠抗人BrdU单抗、FITC标记羊抗小鼠二抗和小鼠抗大鼠CD31单抗、小鼠抗大鼠Rhodopsin单抗、小鼠抗大鼠全角蛋白(Pan-Cytokeratin,PCK)单抗、Cy3标记羊抗小鼠二抗双重荧光标记。观察术后1,2,3,4,5周MSCs在CNV微环境中的分化,计算视网膜下腔注射MSCs/PBS前、后FA面积并比较CNV渗漏情况的变化。
结果视网膜下移植术后第1周即可见MSCs位于CNV表面,但CD31标记阴性,第2周至第5周可见位于CNV内的MSCsCD31标记阳性,第5周可见移行于RPE层的MSCs rhodopsin和PCK标记阳性。FA分析结果初步表明,实验眼视网膜下移植MSCs后CNV的渗漏面积较对照眼有减少的趋势。
结论MSCs移植入CNV模型大鼠视网膜下腔后第2周至第5周可以向内皮细胞方向分化,第5周可以向光感受器细胞方向和RPE方向分化。视网膜下移植MSCs后有可能使CNV渗漏情况得到一定的改善。
Objective To access the differentiation of subretinal-transplanted rat mesenchymal stem cells (MSCs) in the microenvironment of choroidal neovascularization (CNV) and changes of CNV exudation after the transplantation .
Methods In-vitro cultured Brown Norway (BN) rats MSCs were identified CD90(+) CD45(-)by flow cytometry and labeled with Brdu. 25 CNV rats models were established by Krypton laser photocoagulation and subsequently confirmed by fluorescein angiography(FA). 25 rats were randomly distributed into 5 groups, 5 rats in every group. The right eyes were injected with MSCs into subretinal space, while the left eyes were injected with the same amount of PBS. The rats were killed and globes enucleated 1,2,3,4 or 5 weeks after the surgery respectively. Frozen sections were stained with Brdu primary monoclonal antibody, FITC- labeled secondary monoclonal antibody and CD31 , rhodopsin or pan- cytokeratin(PCK) primary monoclonal antibody respectively combined with Cy3-labeled secondary monoclonal antibody . The sections were observed under fluorescent microscope. Calculate and compare the exudation areas change pre-and post-transplantatively by FA.
Results MSCs distributed on the surface of CNV in the 1~(st) week after surgery with CD31 negative.However, from the 2~(nd) to 5~(th) week, CD31 positive MSCs were found in CNV. Rhodopsin positive and PCKpositive MSCs were found near CNV in the 5~(th) week. FA analysis demonstrated a mild decrease tendency of CNV exudation after the transplantation of MSCs.
Conclusions The results demonstrates that MSCs have the potential to differentiate into endothelium from 2th to 5~(th) weeks, into photoreceptor cells and RPE in 5~(th) weeks after subretinal transplantation in the microenvironment of NCV.CNV exudation may be mildly improved after subretinal transplantation of MSCs.
引文
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[2] Sugino IK, Wang H, Zarbin MA. Age-related macular degeneration and retinal pigment epithelium wound healing.Mol Neurobiol. 2003 Oct;28(2): 177-94.
[3] Soubrane G, Souied E, Haddad W, Razavi S, Roquet W, Coscas G Alternative therapies for choroidal neovessels resulting from age-related macular degeneration.J Fr Ophtalmol. 2003 Oct;26(8):876-8.)
[4] Friedenstein AJ, Petrakova KV, Kurolesova AI, Grolova GP. Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues.Transplantation.1968;6:230-247.
[5] Friedenstein AJ, Deriglasova UF, Kulagina NN, et al. Precursors for fibroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay method. Exp Hematol. 1974;2:83-92.
[6] Beresford JN.Osteogenic stem cells and the stromal system of bone and marrow.Clin Orthop 1989;270-80
[7] Owen M. Marrow stromal stem cells.J Cell Sci Suppl 1988; 10:63-76
[8] Ferrari,Get al.Muscle regeneration by bone marrow-derived myogenic progenitors.Science 1998; 279:1528-30
[9] Bruder SP, fink DJ, CaplanAI.Mesenchymal stem cells in bone development,bone repair,and skeletal regeneration therapy.J Cell Biochem.1994;56(3):283-94
[10] Minoru Tomita,Yasushi Adachi,Haruhiko Yamada et al.Bone marrow-derived stem cells can differentiate into retinal cells in injured rat retina.Stem cell 2002;20:279-83
[11] Donald O,Jan K,Stefano C et al.Bone marrow cells regenerate infracted myocardium.Nature 2001;410(5):701-5
[12] Orlic D, Kajstura J, Chimenti S et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001;410:701-705.
[13] Lagasse E, Connors H, Al-Dhalimy M et al. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med 2000;6:1229-1234.
[14] Shi Q, Rafii S, Wu MH-D et al. Evidence for circulating bone marrow-derived endothelial cells. Blood 1998;92:362-367.
[15] Krause DS,Thelse ND,Collector MI et al.Multi-orgn,multi-lineage engraftment by a single bone marrow-derived stem cell.Cell 2001;105:369-77
[16] Woodbury D, Schwarz EJ,Prockop DJ,Black IB.Adult rat and human bone marrow stromal cells differentiate into neurons.J Neurosci Res 2000;61:364-70
[17] Makino S,Fukuda K,Miyoshi S et al.Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest 1999; 103:697-705.
[18] R.Deans.Mesehchymal stem cellsxell and gene therapy applications.Eur.Cytokne Netw.2000;11(2):323-4
[19] Friedenstein A, Chailakhyan R , Gerasimov U. Bone marrow osteogenic stem cells : in vitro cultivation and transplantation in diffusion chambers. Cell Tissue Kinet, 1987,20 : 263-272
[20] Theise,N.D.et al.Liver from bone marrow in humans.Hepatology 2000;32:11 -6
[21] Atsushi Otani, Karen Kinder, Karla Ewalt, et al. Bone marrow-derived stem cells target retinal astrocytes and can promote or inhibit retinal angiogesis. Nature Medicine.2002;8(9): 1004-1010.
[22] Tomita S, Li RK, Weisel RD, et al. Autologous transplantation of bone marrow cells improves damaged heart function. Circulation. 1999;100(suppl II ):247-256.
[23]Anthony Kicic, Wei-Yong Shen, Ann S.Wilson, et al. Differentiation of Marrow Stromal Cells into Photoreceptors in the Rat Eye. The Journal of Neuroscience. 2003;23(21):7742-7749.
[24] Morrison SJ,Shah NM,Anderson DJ.Regulatory mechanisms in stem cell biology.Cell 1997;88:287-98
[25] Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284:143-147.
[26] Shibano K,Watanabe J, Iwamoto M,et al .culture of stromal cells derived from medullary cavity of human long bone in the presence of 1,25-dihydroxyvitamin D3,recombinant human bone morphogenetic protein-2,or ipriflavone[J] .Bone. 1998;22(3):251 -8
[27] Pereira RF, Halford KW, O'Hara MD, et al. Cultured adherent cells from lung in irradiated mice. Proc Natl Acad Sci.1995;92:4857-4861.
[28] Van Vlasselaer P, Falla N, Snoeck H. Characterization and purification of osteogenic cells from murine bone marrow by two-color cell sorting using anti-sca-1 monoclonal antibody and wheat germ agglutinin. Blood.1994;84 (3):753-763.
[29] Encina NR, Billotte WG, Hofmann MC. Immunomagnetic isolation of osteoprogenitors from human bone marrow stroma. Lab Invest. 1999;79(4):449-457.
[30] Theise,N.D.et al.Liver from bone marrow in humans.Hepatology 2000;32:l 1-6
[31] Dobi ET, Puliafito CA, Destro M,A new model of experimental choroidal neovascularization in the rat. Arch Ophthalmol. 1989; 107:264-9
[32] David N.Zacks,Eric Ezra,Yosbiko Terada et al. Verteporfin photodynamic therapy in the rat model of choroidal neovasculari -zation:angiographic and histologic characterization. Invest Ophthal -mol Vis Sci. 2002 Jul;43(7):2384-91
[33] Mori K, Gehlbach P, Ando A, McVey D, Wei L, Campochiaro PA. Regression of ocular neovascularization in response to increased expression of pigment epithelium-derived factor. Invest Ophthalmol Vis Sci. 2002 Jul;43(7):2428-34.
[34] Renno RZ, Youssri AI, Michaud N.Expression of pigment epithelium-derived factor in experimental choroidal neovascularizati Invest Ophthalmol Vis Sci. 2002 May;43(5): 1574-80
[35]Morrison SJ,Shah NM,Anderson DJ.Regulatory mechanisms in stem cell biology.Cell 1997;88:287-98
[36] Galli R, Borello U, Grittia, et al. Skeletal myogenic potential of human and mouse neural stem cells. [J]. Nat Neurosci. 2000;3: 986-991.
[37] Bjornson CR, Rietze TL, Reynolds BA, et al. Turning brain into blood: a hematopoietic fate adopted by adult neural stem cells in vivo. Science. 1999;283:534-537.
[38] Ling ZD, Potter ED, Lipton JW, et al. Differentiation of mesencephalic progenitor cells into dopaminergic neuros by cytokines. Exp Neurol. 1998;149:411-423.
[39] Takahashi M, Palmer TD, Takahashi J, Gage FH. Widespread integration and survival of adult-derived neural progenitor cells in the developing optic retina. Mol Cell Neurosci.1998;12:340-348.
[40] Young MJ, Ray J, Whiteley SJ, et al. Neuronal differentiation and morphological integration of hippocampal progenitor cells transplanted to the retina of immature and mature dystrophic rats. Mol Cell Neurosci. 2000; 16:197-205.
[41] Tropepe V,Coles BL,Chiasson BJ et al.Retinal stem cells in the adult mammalian eye.Science 287:2032-2036
[42]Okamoto N, Tobe T, Hackett SF, et al. Transgenic mice with increased expression of vascular endothelial growth factor in the retina: a new model of intraretinal and subretinal neovascularization. Am J Pathol.1997;151:281-91
[43]Mackay AM, Beck SC, Murphy JM, et al. Chondrogente differentiation of cultured human mesenchymal stem cells from marrow. Tissue Eng.1998;4(4):415-428.
[44] Goshima J, Goldberg VM, Caplan AI. The osteogenic potential of culture-expanded rat marrow mesenchymal cells assayed in vivo in calcium phosphate ceramic blocks. Clin Orthop Rel Res.1991 ;262:298-311.
[45] Paul H. Bone Fomation in vivo: Comparison of osteogenesis by transplanted mouse and human marrow stromal fibroblsts. Trnasplantation.1997;63:1059.
[46] Wakitani S, Saito T, Caplan AI. Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine. Muscle Nerve. 1995; 18:417-428.
[47] Sanchez Romos J, Song S, Cardozo Pelaez F, et al. Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp Neurol. 2000; 164:247-256.
[48] Zhao LR, Duan WM, Reyes M, et al. Human bone marrow stem cells exhibit neural phenotypes and a meliorate neurological deficits after grafting into the ischemic brain of rats. Exp Neurol, 2002; 109(3):337-346.
[49] Aliello LP, Pierce EA,Foley ED,et al.Suppression of retinal neovas -cularization in vivo by inhibition of vascular endothelial growth factor(VEGF) using soluble VEGF-receptor chimeric proteins.Proc Natl Acad Sci USA.1995;92:10457-61
[50] Robinson GS,Pierce EA,Rook SL, et al.Oligodeoxynucleotides inhibit retinal neovascularization in a murine model of proliferative retinopathy.Proc Natl Acad Sci USA 1996;93:4851-6
[51] Luna J,Tobe T,Mousa SA,Reilly TM,et al .Campochiaro PA. Anta- gonists of integrin alpha-v beta-s inhibit retinal neovascularization in a murine model.Lab Invest. 1996;75:563-73
[52] Seo M-S,Kwak N,Ozaki H,et al.Dramatic inhibition of retinal and choroidal neovascularization by oral administration of a kinase inhibitor.Am J Pathol. 1999; 154:1743-53
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