骨髓间充质干细胞静脉移植对损伤大鼠脊髓VEGF、bFGF表达及血管新生的影响
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摘要
目的:将标记荧光的骨髓间充质干细胞(bone mesenchymal stem cells ,MSCs静脉移植到脊髓损伤(spinal cord injury,SCI)大鼠模型体内,观察MSCs在损伤区的存活及血管内皮生长因子(vascular endothelial growth factor,VEGF)和碱性成纤维生长因子(basic fibroblast growth factor,bFGF)表达和血管新生情况,为MSCs移植治疗SCI及其机理提供实验依据。
方法:1.体外分离培养MSCs,流式细胞仪检测其表面标志。
2.实验动物分组:
成年健康SD雄鼠42只(体重200g-250g),随机平均分成7组,每组6只,即3组为MSCs移植组;3组为对照组;1组为正常组
3.脊髓损伤模型的制备:
用自制改良的Allen装置撞击脊髓T12节段,建立脊髓外伤性截瘫模型。
4.羧基荧光素二醋酸盐琥珀酰亚胺酯( carboxyfluorescein succinimidyl ester,CFSE)体外标记MSCs
5. MSCs移植
MSCs移植组6只:术后3天经尾静脉注射MSCs
对照组6只:注射等量磷酸缓冲盐溶液(phosphate-buffered saline,PBS)
6.取材和检测
6.1心脏灌注、取材、切片
6.2激光共聚焦显微镜观察CFSE标记的MSCs在体内存活情况及分泌生长因子VEGF和bFGF的情况。
6.3逆转录-聚合酶链反应(Reverse Transcription-Polymerase ChainReaction ,RT-PCR)法检测VEGF和bFGFmRNA表达变化情况。
6.4免疫组化检测第VIII因子相关抗原表达情况
6.5采用Basso-Beattie-Bresnahan(BBB)评分和爬网格训练评估大鼠行为学改变。
7.统计学分析
结果: 1.激光共聚焦显微镜显示脊髓损伤后经尾静脉移植的MSCs可达到脊髓损伤处聚集、存活且可以分泌生长因子VEGF和bFGF。
2.RT-PCR结果显示VEGF和bFGFmRNA表达在损伤后比正常增高;移植后1天,MSCs移植组与对照组相比(P>0.05),无统计学意义;移植后3天和14天,MSCs移植组均高于同一时间段的对照组且随时间延长表达持续升高。
3.第VIII因子相关抗原免疫组化结果显示:移植后1天,MSCs移植组与对照组相比(P>0.05),无统计学意义;移植后3天和14天,MSCs移植组微血管密度(microvessel density,MVD)均高于同一时间段的对照组且随时间延长表达持续增多。
4.BBB评分和爬网格训练显示移植MSCs可促进脊髓损伤大鼠后肢运动功能恢复。
结论:静脉移植MSCs能在大鼠脊髓损伤区存活且分泌生长因子VEGF和bFGF,从而促进血管新生。
Objective:To observe the survival and migration of Bone Marrow Mesenchymal Stem Cells labeled and the change on expression of VFGF and bfgf factors at different times after transplantion MSC via caudal vien to treat contused spinal cord injuried adult SD rats model, and to provide experimental evidence for the reparing function and menchanism which MSCs treat spinal cord injury.
Methods:1.Rat bone marrow mesenchymal stem cells(MSCs)from adult rats were cultured in vitro,their specific cellular surface marker were analysed with flow cytometer.
2.Experimental animal and groups:
42 healthy adult srague-dawely(SD)famal rats,mean weigh 200-250g,were randomly divided into 7 groups including normal control group、control group and MSCs transplantation groups,each groups 6 rats.
3.Establish contused spinal cord injuried SD rat model: To adapt the Allen's improved method-20g weight iron bar fall from 5cm high and strike at T12 segment spinal cord to get contused spinal cord injuried rat models.
4. MSC are prelabeled with CFSE in vitro before transplantation.
5. Transplantation of MSCs:
Inject MSCs into the spinal cord injury site after injury 3days in transplant groups,but PBS are injuried in contused control groups.
6. Materials and Estimates
6.1 Heart perfusion、draw the spinal cord from rat、slice
6.2 The survive of CFSE-MSCs which were transplanted into spinal cord injuried rats via vein was observed by using confocal microscopy
6.3 The expression of VEGFand bFGFmRNA was detected in spinal cord by using RT-PCR.
6.4 Immunohistochemistry identification of VIII-Ag expression
6.5 Functional evaluation using the open-field BBB scoring system and climd grid training.
7. statistical analysis
Result: 1. CFSE-MSCs which were transplanted into spinal cord injuried rats via vein could survival in the spinal cord by using confocal microscopy ,show the ability of secreting VEGF,bFGF.
2. RT-PCR revealed that the expression of VEGFand bFGFmRNA was higher in the MSCs transplantation group than that of the control group on the 3th、14th day and the counted number of VEGFand bFGFmRNA increase obviously in the MSCs transplantation group. But MSCs transplantation group compared with control group on 1th day(P>0.05 ) , the expression of VEGFand bFGFmRNA was not statistical significance.
3. Immunohistochemistry revealed that the expression of MVD was higher in the MSCs transplantation group than that of the control group on the 3th、14th day and the counted number of MVD increase obviously in the MSCs transplantation group. But MSCs transplantation group compared with control group on 1th day(P>0.05) , the expression of VEGFand bFGFmRNA was not statistical significance.
4. The result of BBB scoring system and climd grid training indicated that MSCs could improve the recovery of motor funcyion of spinal cord injuried rat model.
Conclusion: MSCs,after being implanted into injured spinal cord by intravenous administration ,shows the ability of secreting VEGF,bFGF,with subsequent angiogenesis.
方法:1.体外分离培养MSCs,流式细胞仪检测其表面标志。
2.实验动物分组:
成年健康SD雄鼠42只(体重200g-250g),随机平均分成7组,每组6只,即3组为MSCs移植组;3组为对照组;1组为正常组
3.脊髓损伤模型的制备:
用自制改良的Allen装置撞击脊髓T12节段,建立脊髓外伤性截瘫模型。
4.羧基荧光素二醋酸盐琥珀酰亚胺酯( carboxyfluorescein succinimidyl ester,CFSE)体外标记MSCs
5. MSCs移植
MSCs移植组6只:术后3天经尾静脉注射MSCs
对照组6只:注射等量磷酸缓冲盐溶液(phosphate-buffered saline,PBS)
6.取材和检测
6.1心脏灌注、取材、切片
6.2激光共聚焦显微镜观察CFSE标记的MSCs在体内存活情况及分泌生长因子VEGF和bFGF的情况。
6.3逆转录-聚合酶链反应(Reverse Transcription-Polymerase ChainReaction ,RT-PCR)法检测VEGF和bFGFmRNA表达变化情况。
6.4免疫组化检测第VIII因子相关抗原表达情况
6.5采用Basso-Beattie-Bresnahan(BBB)评分和爬网格训练评估大鼠行为学改变。
7.统计学分析
结果: 1.激光共聚焦显微镜显示脊髓损伤后经尾静脉移植的MSCs可达到脊髓损伤处聚集、存活且可以分泌生长因子VEGF和bFGF。
2.RT-PCR结果显示VEGF和bFGFmRNA表达在损伤后比正常增高;移植后1天,MSCs移植组与对照组相比(P>0.05),无统计学意义;移植后3天和14天,MSCs移植组均高于同一时间段的对照组且随时间延长表达持续升高。
3.第VIII因子相关抗原免疫组化结果显示:移植后1天,MSCs移植组与对照组相比(P>0.05),无统计学意义;移植后3天和14天,MSCs移植组微血管密度(microvessel density,MVD)均高于同一时间段的对照组且随时间延长表达持续增多。
4.BBB评分和爬网格训练显示移植MSCs可促进脊髓损伤大鼠后肢运动功能恢复。
结论:静脉移植MSCs能在大鼠脊髓损伤区存活且分泌生长因子VEGF和bFGF,从而促进血管新生。
Objective:To observe the survival and migration of Bone Marrow Mesenchymal Stem Cells labeled and the change on expression of VFGF and bfgf factors at different times after transplantion MSC via caudal vien to treat contused spinal cord injuried adult SD rats model, and to provide experimental evidence for the reparing function and menchanism which MSCs treat spinal cord injury.
Methods:1.Rat bone marrow mesenchymal stem cells(MSCs)from adult rats were cultured in vitro,their specific cellular surface marker were analysed with flow cytometer.
2.Experimental animal and groups:
42 healthy adult srague-dawely(SD)famal rats,mean weigh 200-250g,were randomly divided into 7 groups including normal control group、control group and MSCs transplantation groups,each groups 6 rats.
3.Establish contused spinal cord injuried SD rat model: To adapt the Allen's improved method-20g weight iron bar fall from 5cm high and strike at T12 segment spinal cord to get contused spinal cord injuried rat models.
4. MSC are prelabeled with CFSE in vitro before transplantation.
5. Transplantation of MSCs:
Inject MSCs into the spinal cord injury site after injury 3days in transplant groups,but PBS are injuried in contused control groups.
6. Materials and Estimates
6.1 Heart perfusion、draw the spinal cord from rat、slice
6.2 The survive of CFSE-MSCs which were transplanted into spinal cord injuried rats via vein was observed by using confocal microscopy
6.3 The expression of VEGFand bFGFmRNA was detected in spinal cord by using RT-PCR.
6.4 Immunohistochemistry identification of VIII-Ag expression
6.5 Functional evaluation using the open-field BBB scoring system and climd grid training.
7. statistical analysis
Result: 1. CFSE-MSCs which were transplanted into spinal cord injuried rats via vein could survival in the spinal cord by using confocal microscopy ,show the ability of secreting VEGF,bFGF.
2. RT-PCR revealed that the expression of VEGFand bFGFmRNA was higher in the MSCs transplantation group than that of the control group on the 3th、14th day and the counted number of VEGFand bFGFmRNA increase obviously in the MSCs transplantation group. But MSCs transplantation group compared with control group on 1th day(P>0.05 ) , the expression of VEGFand bFGFmRNA was not statistical significance.
3. Immunohistochemistry revealed that the expression of MVD was higher in the MSCs transplantation group than that of the control group on the 3th、14th day and the counted number of MVD increase obviously in the MSCs transplantation group. But MSCs transplantation group compared with control group on 1th day(P>0.05) , the expression of VEGFand bFGFmRNA was not statistical significance.
4. The result of BBB scoring system and climd grid training indicated that MSCs could improve the recovery of motor funcyion of spinal cord injuried rat model.
Conclusion: MSCs,after being implanted into injured spinal cord by intravenous administration ,shows the ability of secreting VEGF,bFGF,with subsequent angiogenesis.
引文
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