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骨髓间充质干细胞移植对大鼠脊髓损伤后血管内皮生长因子基因表达及血管生成的影响

[Effects of bone marrow mesenchymal stem cells transplantation on expression of vascular endothelial growth factor gene and angiogenesis after spinal cord injury in rats].

作者信息

Yu Deshui, Lü Gang, Cao Yang, Li Gang, Zhi Xiaodong, Fan Zhongkai

机构信息

Department of Orthopedics, the First Affiliated Hospital, Liaoning Medical University, Jinzhou Liaoning, 121000, P.R.China.

出版信息

Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2011 Jul;25(7):837-41.

Abstract

OBJECTIVE

To investigate the effect of bone marrow mesenchymal stem cells (BMSCs) transplantation on the motor function recovery, the expression of vascular endothelial growth factor (VEGF) gene, and angiogenesis after spinal cord injury (SCI) in rats, and to explore the treatment mechanism of BMSCs in SCI.

METHODS

BMSCs were isolated and cultured from the marrow of 5 Wistar rats (4 weeks old) and the 3rd-4th passage cells were prepared for the experiment. A total of 87 adult female Wistar rats (weighing 220-250 g) were randomly divided into 3 groups: sham-operated group (group A, n=21), DMEM group (group B, n=33), BMSCs group (group C, n=33). A laminectomy was only performed at T8-10 levels in group A. The SCI models were established by modified Nystrom's compression method in groups B and C, and BMSCs and DMEM were injected in groups B and C respectively at 30 minutes after SCI. Basso-Beattie-Bresnahan (BBB) score was used for the motor function recovery at 3, 7, 14, and 28 days, RT-PCR for the VEGF mRNA at 1, 3, and 5 days, and immunohistochemical staining for angiogenesis at 3, 7, 14, and 28 days.

RESULTS

In groups B and C, the hindlimb locomotor function was improved at different degrees with time, showing significant difference in BBB score between groups B, C and group A (P < 0.05). At 28 days, the BBB score in group C was significantly higher than that in group B (P < 0.05) and there was no significant difference between groups B and C (P > 0.05) at 3, 7, and 14 days after transplantation. The numbers of microvessels in the ventral horns of gray matter around SCI in groups B and C were significantly lower than that in group C (P < 0.05) at 3 days, but there was no significant difference at 7, 14, and 28 days after transplantation (P > 0.05). There was no significant difference in the number of microvessels between group C and group B (P > 0.05) at 3 and 7 days, but the number of microvessels in group C was significantly higher than that in group B (P < 0.05) at 14 and 28 days after transplantation. However, there was no significant difference in the number of microvessels in the white matter around SCI in 3 groups at different time points after transplantation (P > 0.05). The RT-PCR results showed that VEGF mRNA expressed at a low level in group A. Compared with group A, the expression level of VEGF mRNA in groups B and C increased at 1 day and reached the peak at 3 days, then decreased at 5 days after transplantation; and the expression of VEGF mRNA was significantly higher in groups B and C than in group A (P < 0.05), and in group C than in group B (P < 0.05) at 1, 3, and 5 days.

CONCLUSION

BMSCs may promote the motor function recovery by up-regulating VEGF mRNA expression and increasing angiogenesis in the spinal cord after SCI in rats.

摘要

目的

探讨骨髓间充质干细胞(BMSCs)移植对大鼠脊髓损伤(SCI)后运动功能恢复、血管内皮生长因子(VEGF)基因表达及血管生成的影响,以探索BMSCs治疗SCI的机制。

方法

从5只4周龄Wistar大鼠的骨髓中分离培养BMSCs,取第3-4代细胞用于实验。将87只成年雌性Wistar大鼠(体重220-250 g)随机分为3组:假手术组(A组,n=21)、DMEM组(B组,n=33)、BMSCs组(C组,n=33)。A组仅在T8-10节段行椎板切除术。B组和C组采用改良的Nystrom压迫法建立SCI模型,B组和C组分别在SCI后30分钟注射BMSCs和DMEM。于术后3、7、14和28天采用Basso-Beattie-Bresnahan(BBB)评分评估运动功能恢复情况,于术后1、3和5天采用RT-PCR检测VEGF mRNA表达,于术后3、7、14和28天采用免疫组织化学染色检测血管生成情况。

结果

B组和C组大鼠后肢运动功能随时间不同程度改善,B组、C组与A组BBB评分比较差异有统计学意义(P<0.05)。术后28天,C组BBB评分显著高于B组(P<0.05),移植后3、7和14天B组与C组比较差异无统计学意义(P>0.05)。术后3天,B组和C组脊髓损伤周围灰质前角微血管数量显著低于A组(P<0.05),移植后7、14和28天差异无统计学意义(P>0.05)。移植后3和7天,C组与B组微血管数量比较差异无统计学意义(P>0.05),但移植后14和28天,C组微血管数量显著高于B组(P<0.05)。然而,移植后不同时间点3组脊髓损伤周围白质微血管数量比较差异无统计学意义(P>0.05)。RT-PCR结果显示,A组VEGF mRNA表达水平较低。与A组比较,B组和C组移植后1天VEGF mRNA表达水平升高,3天达峰值,5天下降;移植后1、3和5天,B组和C组VEGF mRNA表达水平显著高于A组(P<0.05),C组高于B组(P<0.05)。

结论

BMSCs可能通过上调VEGF mRNA表达及增加大鼠SCI后脊髓血管生成促进运动功能恢复。

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