作者：张坡，黄岚，宋明宝，赵晓辉，崔 斌，周音频，尹阳光，朱光旭作者单位：基金项目：国家自然科学基金资助项目（No,30470729) 第三军医大学新桥医院全军心血管研究所，重庆 400037 . B3 S/ [: P; N# C4 k% B 6 A- I: M/ x' ?% w$ `% x" I - V- V: r6 R/ c2 F: ?5 U 0 L3 s; g( @* _* o" v
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【摘要】 目的：探讨大鼠骨髓单个核细胞在体外经条件培养基诱导定向分化成平滑肌祖细胞的可行性。方法：分离4～6周龄的大鼠胫骨、股骨，以4℃预冷的DMEM培养基冲出骨髓，密度梯度离心法分离骨髓单个核细胞，在血小板源生长因子-BB （ PDGF-BB）和碱性成纤维细胞生长因子 （ b-FGF）作用下，培养8～12d形成贴壁的梭形细胞。采用CD34与α-SMA免疫荧光染色进行鉴定，RT-PCR法测定其α-SMA mRNA表达情况。结果： 在PDGF-BB作用下，82%的骨髓单个核细胞α-SMA染色阳性，78%细胞CD34染色阳性，新鲜分离的骨髓单个核细胞不表达α-SMA mRNA，体外培养后表达α-SMA mRNA。结论：体外培养的骨髓单个核细胞能分化为平滑肌祖细胞，可作为研究平滑肌细胞分化和筛选抑制再狭窄药物的工具。 * Y: W$ K7 I- R9 V0 l S
【关键词】骨髓细胞；干细胞；肌细胞，平滑肌9 \9 I* l/ c: Q/ I7 I. k4 G
Bone marrow mononuclear cells of adult rat to differentiate into smooth muscle progenitor cells in vitro/ZHANG Po, HUNAG Lan, SONG Ming|bao, ZHAO Xiao|Hui，CUI Bin, ZHOU Yin|pin, YIN Yang|guang, ZHU Guang|xu//Chinese Journal of Cardiovascular Rehabilitation Medicine,2007，16（2）：1287 S- _2 B! k# i# L6 l
$ c$ n' W7 m* @$ Y7 u5 jAbstract：Objective:To explore the potential of rat bone marrow mononuclear cells (MNCs) to differentiate into vascular smooth muscle progenitor cells.Methods:After the tibias and femurs were dissected from 4~6 week|old rats, the marrow was flushed out with ice|cold DMEM mediumThe MNCs were obtained with density gradient centrifugation and cultured in DMEM medium containing 20鸖, 50ng/ml Platelet|derived growth factor-BB(PDGF-BB), 10ng/ml basic fibroblast growth factor(b-FDF).The cultured cells in vitro were identified with smooth muscle cell|specific α actin (α-SMA) and CD34 by immunofluorescent techniqueRT-PCR technique was used to identify the α-SMA mRNA level in fresh isolated marrow mononuclear cells and SPCs.Results:Smooth muscle|like cells outgrew from the culture of MNCs in presence of PDGF-BB and b-FGF.These cells were positive stain for α-SMA and CD34 on immunofluorescenceα-SMA mRNA was not expressed in fresh isolated MNCs but in the cells cultured for 8-12 days.Conclusion:Vascular smooth muscle cells can outgrow from bone MNCsThis model may be useful in study of smooth muscle cell differentiation and the origin of neo|intimal cells after vascular injury.% F7 K6 a7 [5 t _0 a- e5 |3 g
! h2 F9 ~+ A! p/ R& T9 oAuthor′s address：Cardiovascular Research Institute of PLA, Xinqiao Hospital of the Military Medical University, Chongqing 400037, China ) ?5 e6 `/ n5 B* G5 d# e' s % A& c; }1 q+ q- K+ kKey words：Bone marrow cells; Stem cell; 9 t$ w v9 e( s9 d9 i+ w8 v. b" v% X( P% K5 a
Muscle cell,smooth muscle 成年机体外周血中存在一种CD34阳性，血小板源生长因子-BB（Platelet|derived growth factor, PDGF-BB）受体阳性，与造血干细胞具有多种相同表面抗原的细胞群，在体外培养能够增殖，并逐步分化成为平滑肌细胞，在体内可被血管损伤动员，参与损伤血管修复和病理性新内膜的生成，因此这种细胞被认为是循环平滑肌祖细胞（smooth muscle progenitor cells, SPCs）[1, 2]。正常动物外周血中SPC含量很低［3］，分离培养困难。骨髓作为外周造血干细胞的产生组织，其中是否存在SPCs，在体外是否可以分化成平滑肌细胞，目前尚不清楚，本研究旨在为研究平滑肌细胞分化调控和血管损伤后新生内膜增生发病机制提供新的实验佐证。/ ]6 Y4 K! H3 Q
' D! p" F6 y0 P/ W3 L [3]Cho HJ, Kim TY, Cho HJ, et alThe effect of stem cell mobilization by granulocyte|colony stimulating factor on neointimal hyperplasia and endothelial healing after vascular injury with bare|metal versus Paclitaxel|eluting stents[J].J Am Coll Cardiol, 2006,48(2):366-374.4 Y$ X9 O7 u1 i
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[7]Simper D, Stalboerger PG, Panetta CJ, et alSmooth muscle progenitor cells in human blood[J].Circulation, 2002,106(10):1199-1204. d9 r, o0 Y3 c4 Y6 u1 S* q+ z
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3 O; N4 l! L# o8 x% `) V: @+ V [9]张 坡, 黄 岚, 蒋世忠,等.小鼠骨髓基质细胞的生物学特性及血管新生能力[J].中华心血管病杂志, 2003,31(09): 681./ Y [5 g m ?" H" V' c+ j
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& k$ [8 W: g# ?! r" `* o [10]Zulli A, Buxton BF, Black MJ, et alCD34 Class III positive cells are present in atherosclerotic plaques of the rabbit model of atherosclerosis[J].Histochemistry and Cell Biology, 2005,124(6):517-522. 7 U+ Q* f5 h: @1 A2 i- B" t9 o4 C8 Z) g' j- ?- K4 Z$ A/ P4 S9 U
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[11]Schober A, Hoffmann R, Opree N, et al.Peripheral CD34 cells and the risk of in|stent restenosis in patients with coronary heart disease[J].Am J Cardiol, 2005,96(8):1116-1122.作者: immail 时间: 2015-5-26 16:43