双重荧光标记验证静脉移植碱性成纤维细胞生长因子基因修饰骨髓间充质干细胞在脑缺血模型大鼠脑内的存活

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中国组织工程研究与l临床康复 争J2眷第 J 2008—12—16出版 Journal ofClinical Rehabilitative Tissue Engineering Research December 6,2008 Vo1.12,No.51 鼬fc 

Survival and differentiation of basic fibroblast growth 

factor gene modified bone marrow mesenchymal stem 

cells following vein transplantatiOn in cerebral ischemia 

model rats va l idated by dou ble i m m U nOfl uorescence 

staining冰☆ 

Yang Jie,Zhang Fu-feng,Gu Wen-ping,Xiao Bo,Tang Bei-sha,Yang Qi_dong 

Abstract BACKGROUND:Basic fibroblast growth factor(bFGF)can accelerate the bone marrow mesenchymal stem cells(BMSCs) proliferation and differentiation into nerve cells.which is considered as a mitogen of glial cells. oBJECTIVE:To investigate the survival and differentiation of bFGF gene modified BMSCs transplanted on rat models of cerebral ischemia by double immunofluorescence staining,and to study the differentiation trend of BMSCs into neuron—like cells and glial cells. DESIGN.TⅡ E AND SETTING:The randomized control animal experiment was completed in the central laboratory of Experimental Animal Center of Central South University from July 2005 to March 2006. MATERIAL:Fifty Sprague.Dawley rats were divided into 4 groups at random:sham operation group( =10),cerebra1 ischemia—reperfusion injury group( =l0),BMscs group(,z=l5)and bFGF modified BMscs group(n=15). METHODS:Except sham operation group.rats in the other three groups were prepared for local cerebral ischemia—reperfusion models.Then BMSCs or bFGF modified BMSCs were intravenously transplanted into cerebral ischemic rats.and the same volume of DMEM were injected in the cerebral ischemia—reperfusion injury group. MAIN OUTCOME MEASUltES:Survival rate and differentiation of grafted cells were observed by 5一bromo一2一deoxyuridine(BrdU)一NeuN,and BrdU—glial fibrillary acidic protein(GFAP)double immunofluorescence staining; the neurological scores and infarction volumes in each group. RESULTS:At 7 days after implantation.the number of BrdU,NeuN—positive cells and BrdU—GFAP—positive cells in the bFGF modified BMSCs group was higher than those on the BMSCs group(P<0.05),but there were no significant differences in the co expression cells by double immunofluorescence staining between the two groups(P>0.05).At 7 days following reperfusion.neural function of cerebralischemia rats were improved and infarction volume was reduced in both BMSCs group and the bFGF modified BMSCs group.and bFGF modified BMSCs group was superior to BMSCs group. CoNCLUSIoN:BMSCs modified by bf、GF can survive in cerebral ischemic region and diferentiate into neuron and glial cells,which are more proper for repairing nerves. 

INTR0DUCTl0N 

A large amount of studies have reported that bone marrow mesenchymal stem cells(BMSCs1 can differentiate into neurons and are prone to conduct exogenous gene transfection and expression.BMSCs transplantation provides a potential prospect for the treatment of nervous system disease.At present, BMSCs show a lower differentiation rate into nerve cells and an unclear mechanism underlying the cell growth in transplanted area when BMSCs are used to repair the ischemic iniury.Therefore,it is an urgent matter to solve the orientated differentiation of BMSCs into nerve cells and increase the neuroprotection effect of BMSCs.Basic fibroblast growth factor(bFGF)plays a neuroprotective role on the cerebral ischemic iniury and can promote the BMSCs proliferation and diferentiation into nerve cells【IJ.However,bFGF degrades in vivo and requires many times of injection when in combination with BMSCs.Therefore,BMSCs modified by bFGF were intravenously transplanted into the experimental ischemic rats in this study. which aims to investigate the differentiation of BMSCs and the therapeutic e骶ct of bFGF—modified 

ISSN 1673.8225 CN 2l—l539fR CoDEN:ZLXHAH BMSCs.Furthermore,these evidences may provide a basis for the treatment of cerebral ischemic disease. 

MATERlALS AND METH0DS 

Design Randomized control animal experiment. 

Time and setting Experiment was performed in the central laboratory of Experimental Animal Center of Central South University from July 2005 to March 2006. 

Materials Fifty male Sprague—Dawley(SD)rats,cleaning grade and weighing 250—300 g,were offered by the Experimental Animal Center of Xiangya Hospital, Central South University(China,license number: SYXK2005—0005).All experimental protocols were in accordancewiththeGuidefortheCareandUseof Laboratory Animals approved by Ethics Committee t2]. Fifty rats were divided into 4 groups at random: sham operation group(n=10),cerebral ischemia—reperfusion injury group =10),BMSCs group(n=15)and bFGF modified BMSCs group Department of Neurology,Xiangya Hospital OfCentral South University, Changsha 410008, Hunan Province, China Yang Jie☆.Doctor, Associate professor, Department of Neurology,Xiangya Hospital 0f Central South University, Changsha 410008, HunBn Province, China yangjie6523@ 163.corn Supported by:the Namral Science Foundation ofHnnan Province, No.03JJY3057* Received:2008_02.01 Accepted:2008--08—-19 (08—50-2-813/(3) Yang J,ZhangFF,Gu WPXiaoB.Tang BS,Yang OD.Survival and diferentiation of basic fibroblast growth factor gene modified bone marrow mesenchymal stem cells following vein transplantation in cerebml ischemia model rats validated by double immunofluorescence staining.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(51): 10015—10019(China) [11Up://www.crter.cn http://en.zglckf.com】