CAJ | 학술논문

背景:骨髓基质细胞具有体内、外迁移特性,但目前关于其迁移的具体机制还不十分清楚.目的:探讨基质细胞来源因子1及其受体CXCR4存骨髓基质细胞体内、外迁移中的作用.设计、时间及地点:细胞学体内实验,于2007-03/06在新加坡国立大学解剖系完成.材料:清洁级Wistar新生大鼠1只,用于骨髓基质细胞培养.清洁级成年Wistar大鼠40只,随机分为损伤模型组、假手术组,20只/组.方法:骨髓基质细胞体外趋化实验在48孔Boyden小室上进行,取25 μL基质细胞来源因子1,分别以5,50,500 μg/L质量浓度加到趋化板的下层,以8 μm孔径的聚碳酸酯膜覆盖;并设立空白对照,单纯添加DMEM条件培养基.以含生血清白蛋白的DMEM条件培养基调整细胞浓度至1.5×109L-1,50 μL细胞悬液加到Boyden小窜上层,37℃、CO2培养箱培养10 h.损伤模型组大鼠制各脊髓全横断损伤,假手术组大鼠只打开椎板.脊髓全横断后1 h,将5-(6-)羟基荧光素双乙酸琥珀酰亚胺酯荧光标记的骨髓基质细胞悬液1.0 mL(1×109L-1)经颈内静脉注入体内.主要观察指标:免疫荧光组化染色观察骨髓基质细胞趋化因子受体CXCR4的表达,基质细胞来源因子1体外对骨髓基质细胞的趋化迁移作用,Real-time PCR定量检测脊髓损伤区基质细胞来源因子1 RNA的表达,荧光显微镜下观察骨髓基质细胞向脊髓损伤区的体内迁移.结果:纯化的骨髓基质细胞呈CXCR4阳性.与空白对照比较,5,50,500 μg/L基质细胞来源因子1均可明显促进骨髓基质细胞的体外趋化迁移(P<0.06),且质量浓度为50 μg/L时趋化作用达峰值.与假手术组比较,损伤模型组造模后7 d基质细胞来源因子1 RNA的表达明显升高(P<0.05),14 d时恢复至正常水平.细胞注射2周后与假手术组比较,损伤模型组损伤区迁移细胞数明显增加(P<0.05).结论:基质细胞来源因子1体内、外可趋化骨髓基质细胞迁移,基质细胞来源因子1及其受体CXCR4参与骨髓基质细胞向脊髓全横断损伤区的迁移. 揹景:骨髓基質細胞具有體內、外遷移特性,但目前關于其遷移的具體機製還不十分清楚.目的:探討基質細胞來源因子1及其受體CXCR4存骨髓基質細胞體內、外遷移中的作用.設計、時間及地點:細胞學體內實驗,于2007-03/06在新加坡國立大學解剖繫完成.材料:清潔級Wistar新生大鼠1隻,用于骨髓基質細胞培養.清潔級成年Wistar大鼠40隻,隨機分為損傷模型組、假手術組,20隻/組.方法:骨髓基質細胞體外趨化實驗在48孔Boyden小室上進行,取25 μL基質細胞來源因子1,分彆以5,50,500 μg/L質量濃度加到趨化闆的下層,以8 μm孔徑的聚碳痠酯膜覆蓋;併設立空白對照,單純添加DMEM條件培養基.以含生血清白蛋白的DMEM條件培養基調整細胞濃度至1.5×109L-1,50 μL細胞懸液加到Boyden小竄上層,37℃、CO2培養箱培養10 h.損傷模型組大鼠製各脊髓全橫斷損傷,假手術組大鼠隻打開椎闆.脊髓全橫斷後1 h,將5-(6-)羥基熒光素雙乙痠琥珀酰亞胺酯熒光標記的骨髓基質細胞懸液1.0 mL(1×109L-1)經頸內靜脈註入體內.主要觀察指標:免疫熒光組化染色觀察骨髓基質細胞趨化因子受體CXCR4的錶達,基質細胞來源因子1體外對骨髓基質細胞的趨化遷移作用,Real-time PCR定量檢測脊髓損傷區基質細胞來源因子1 RNA的錶達,熒光顯微鏡下觀察骨髓基質細胞嚮脊髓損傷區的體內遷移.結果:純化的骨髓基質細胞呈CXCR4暘性.與空白對照比較,5,50,500 μg/L基質細胞來源因子1均可明顯促進骨髓基質細胞的體外趨化遷移(P<0.06),且質量濃度為50 μg/L時趨化作用達峰值.與假手術組比較,損傷模型組造模後7 d基質細胞來源因子1 RNA的錶達明顯升高(P<0.05),14 d時恢複至正常水平.細胞註射2週後與假手術組比較,損傷模型組損傷區遷移細胞數明顯增加(P<0.05).結論:基質細胞來源因子1體內、外可趨化骨髓基質細胞遷移,基質細胞來源因子1及其受體CXCR4參與骨髓基質細胞嚮脊髓全橫斷損傷區的遷移.
배경:골수기질세포구유체내、외천이특성,단목전관우기천이적구체궤제환불십분청초.목적:탐토기질세포래원인자1급기수체CXCR4존골수기질세포체내、외천이중적작용.설계、시간급지점:세포학체내실험,우2007-03/06재신가파국립대학해부계완성.재료:청길급Wistar신생대서1지,용우골수기질세포배양.청길급성년Wistar대서40지,수궤분위손상모형조、가수술조,20지/조.방법:골수기질세포체외추화실험재48공Boyden소실상진행,취25 μL기질세포래원인자1,분별이5,50,500 μg/L질량농도가도추화판적하층,이8 μm공경적취탄산지막복개;병설립공백대조,단순첨가DMEM조건배양기.이함생혈청백단백적DMEM조건배양기조정세포농도지1.5×109L-1,50 μL세포현액가도Boyden소찬상층,37℃、CO2배양상배양10 h.손상모형조대서제각척수전횡단손상,가수술조대서지타개추판.척수전횡단후1 h,장5-(6-)간기형광소쌍을산호박선아알지형광표기적골수기질세포현액1.0 mL(1×109L-1)경경내정맥주입체내.주요관찰지표:면역형광조화염색관찰골수기질세포추화인자수체CXCR4적표체,기질세포래원인자1체외대골수기질세포적추화천이작용,Real-time PCR정량검측척수손상구기질세포래원인자1 RNA적표체,형광현미경하관찰골수기질세포향척수손상구적체내천이.결과:순화적골수기질세포정CXCR4양성.여공백대조비교,5,50,500 μg/L기질세포래원인자1균가명현촉진골수기질세포적체외추화천이(P<0.06),차질량농도위50 μg/L시추화작용체봉치.여가수술조비교,손상모형조조모후7 d기질세포래원인자1 RNA적표체명현승고(P<0.05),14 d시회복지정상수평.세포주사2주후여가수술조비교,손상모형조손상구천이세포수명현증가(P<0.05).결론:기질세포래원인자1체내、외가추화골수기질세포천이,기질세포래원인자1급기수체CXCR4삼여골수기질세포향척수전횡단손상구적천이.
BACKGROUND: Marrow stromal cells (MSCs) own the characteristic of migration. However, the mechanisms underlying the migration of these cells remain unclear. OBJECTIVE: To explore the roles of stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 in trafficking of MSCs migration. DESIGN, TIME AND SETTING: The in vivo cytology experiment was performed at Department of Anatomy, National University of Singapore from March 2007 to June 2007. MATERIALS: MSCs were isolated and purified from a Wistar neonatal rat. Forty adult female Wistar rats were randomly divided into sham operation and experimental groups, with 20 animals in each group. METHODS: The chemotaxis assay was performed at a 48 well Boyden chamber, and a total of 25 μL SDF-1 was added to the lower layer of chamber, covered with 8 μm polycarbonate membrane filter; SDF-1 cultured in DMEM conditioned medium was served as a blank control group. Cell concentration was regulated to 1.5×109L-1/L. 50 μL and cell suspension was added into the upper layer of chamber, cultured at CO2 incubator with temperature of 37 ℃ for 10 hours. Rats in the experimental group were prepared for transected spinal cord injury models, and in the sham operation'group, only the vertebral plate was opened. 1.0 mL (1×109L-1/L) MSCs suspension labeled with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) was injected through internal jugular vein at 1 hour after completely transected spinal cord. MAIN OUTCOME MEASURES: Expression of chemokine receptor CXCR4 in MSCs, as well as the effect of SDF-1 on the migration of MSCs was observed by immunofluorescence, change of SDF-1 in lesion site of spinal cord was detected by real-time PCR analysis, as well as the in vivo migration of intravenously injected MSCs was detected by fluorescence microscopy. RESULTS: The pudfied MSCs were positive to CXCR4. Compared to the blank control group, SDF-1 with concentrations of 5, 50, and 500 μg/L could accelerated the migration of MSCs (P < 0.05), which reached a peak with concentration of 500 μg/L. The expression of SDF-1 RNA was obvious increased in the experimental group than that of the sham operation group (P < 0.05), and returned to a normal level at 14 days. At 2 weeks after cell injection, the number of MSCs migrated to the lesion site of completely transected spinal cord was significant increased than sham operation group (P < 0.05). CONCLUSION: SDF-1 may contribute to MSCs migration in vitro and in vivo. SDF-1 and its receptor CXCR4 are involved in the migration of injected MSCs to the lesion site of completely transected spinal cord.Ding P, Xue LP, Yang ZY, Wang CQ, Wang JH, Feng ZT, Ling EA.Chemokine stromal cell-derived factor-1 and its receptor CXCR4 mediate migration of marrow stromal ceils into the lesion site of completely transected spinal cord.