中国组织工程研究

中国组织工程研究 ›› 2016, Vol. 20 ›› Issue (1): 95-101.doi: 10.3969/j.issn.2095-4344.2016.01.017

• 干细胞移植 stem cell transplantation • 上一篇    下一篇

盐酸川芎嗪联合骨髓间充质干细胞移植对脊髓损伤模型大鼠的神经保护

吴晓明1，高文山2，王 静1，蔡红云2   

  1. 1河北大学，河北省保定市  071000；2河北大学附属医院，河北省保定市  071000
  • 收稿日期:2015-11-27 出版日期:2016-01-01 发布日期:2016-01-01
  • 通讯作者: 高文山，河北大学附属医院，河北省保定市 071000
  • 作者简介:吴晓明，男，1990年生，河北省保定市人，汉族，河北大学在读硕士。
  • 基金资助:
    河北大学医学学科专项基金建设项目(2012B2001)

Neuroprotection of ligustrazine hydrochloride combined with bone marrow mesenchymal stem cell transplantation in rats with spinal cord injury

Wu Xiao-ming1, Gao Wen-shan2, Wang Jing1, Cai Hong-yun2   

  1. 1Hebei University, Shijiazhuang 071000, Hebei Province, China; 2Affiliated Hospital of Hebei University, Shijiazhuang 071000, Hebei Province, China
  • Received:2015-11-27 Online:2016-01-01 Published:2016-01-01
  • Contact: Gao Wen-shan, Affiliated Hospital of Hebei University, Shijiazhuang 071000, Hebei Province, China
  • About author:Wu Xiao-ming, Studying for master’s degree, Hebei University, Shijiazhuang 071000, Hebei Province, China
  • Supported by:

    the Medical Construction Fund of Hebei University, No. 2012B2001

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摘要：

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文题释义：

川芎嗪：是从伞形科藁本属植物川芎中分离提纯的生物碱单体,是中药川芎的主要有效成分，是活血行气祛瘀的一种中药活性生物碱。
骨髓间充质干细胞分离方法： ①全骨髓法：根据干细胞在低血清培养基中有贴壁优势特性，定期换液除去不贴壁细胞，从而达到纯化及扩增骨髓间充质干细胞的目的。②密度梯度离心法：根据骨髓中各细胞成分密度的不同，分离单核细胞进行贴壁培养。③免疫法：如流式细胞仪法、免疫磁珠法等对其进行分离纯化。但经过流式或磁珠分选后的细胞出现了增殖缓慢等一些问题，加之成本较高和技术的难度，在某种程度上限制了这些方法的广泛应用。

 

背景：盐酸川芎嗪具有促进神经修复的作用，可以应用于神经系统损伤的治疗。

目的：观察盐酸川芎嗪与骨髓间充质干细胞移植联合应用大鼠脊髓损伤后的干预对电生理及后肢功能变化的影响。
方法：采用Allen打击法建立T9脊髓全横断损伤模型大鼠，建模后随机分为3组，对照组仅尾静脉注射培养液；骨髓间充质干细胞移植组于尾静脉注射等体积的骨髓间充质干细胞；联合组尾静脉注射骨髓间充质干细胞 +尾静脉滴注盐酸川芎嗪，持续4 h。造模后1，2，4，6，8周后进行BBB评分、改良Tarlov 评分检测大鼠后肢运动功能恢复情况；于造模后72 h采用RT-PCR法检测各组大鼠脊髓损伤区周围Bcl-2、碱性成纤维细胞生长因子的表达变化；于造模后4周行体感诱发电位和运动诱发电位进行神经电生理恢复程度测定；造模后8周行辣根过氧化物酶示踪评估大鼠脊髓神经纤维的再生状况，通过PKH-26标记观察移植细胞的存活、迁移。
结果与结论：造模后1，2，4，6，8周，联合组大鼠后肢BBB评分、改良Tarlov 评分联合组明显优于骨髓间充质干细胞移植组，骨髓间充质干细胞移植组明显优于对照组(P < 0.05)，造模后72 h，PCR法检测结果表明联合组中大鼠脊髓损伤区周围Bcl-2、碱性成纤维细胞生长因子的表达显著高于骨髓间充质干细胞移植组及对照组(P < 0.05)。造模后4周，各组体感和运动诱发电位的潜伏期：联合组<骨髓间充质干细胞移植组<对照组(P < 0.05)。体感和运动诱发电位波幅宽度依次为联合组>骨髓间充质干细胞移植组>对照组(P < 0.05)。造模后8周，骨髓间充质干细胞移植组及联合组有辣根过氧化物酶标记的神经锥体细胞明显的穿越痕迹；联合组中PKH-26标记的阳性细胞数与辣根过氧化物酶阳性细胞数量最多，骨髓间充质干细胞移植组次之，对照组相对最少(P < 0.05)。研究表明，盐酸川芎嗪联合骨髓间充质干细胞移植可促进脊髓损伤大鼠神经细胞再生，促进Bcl-2、碱性成纤维细胞生长因子基因的表达，改善肢体运动功能。 

关键词: 干细胞, 移植, 盐酸川芎嗪, 脊髓损伤, 再生, 骨髓间充质干细胞, 移植, 大鼠, 电生理, Bcl-2, 碱性成纤维细胞生长因子, 功能

Abstract:

BACKGROUND: Ligustrazine hydrochloride which promotes nerve repair can be applied to the treatment of nervous system injury.
OBJECTIVE: To investigate the effect of ligustrazine hydrochloride combined with bone marrow mesenchymal stem cells transplantation on electrophysiological property and hindlimb function of rats with spinal cord injury.
METHODS: T9 spinal cord transection injury models were made in rats using Allen’s method, and then rat models were randomized into three groups: rats in control group received tail vein injection of culture solution; rats in cell transplantation group underwent bone marrow mesenchymal stem cell transplantation via the tail vein; rats in combined group were subjected to the tail vein injection of ligustrazine hydrochloride and bone marrow mesenchymal stem cells that lasted for 4 hours. At 1, 2, 4, 6, 8 weeks after modeling, Basso, Beattie and Bresnahan scores and modified Tarlov scores were used to detect the motor function of rats. At 72 hours after modeling, RT-PCR method was used to detect the expression of Bcl-2 and basic fibroblast growth factor around the injured region. At 4 weeks after modeling, somatosensory and motor evoked potentials were measured for evaluation of neurophysiological recovery. At 8 weeks after modeling, horseradish peroxidase tracer was used to assess the regeneration of rat spinal cord nerve fibers; PKH-26 labeling was used to observe the survival and migration of transplanted cells.
RESULTS AND CONCLUSION: At 1, 2, 4, 6, 8 weeks after modeling, Basso, Beattie and Bresnahan scores and modified Tarlov scores were significantly higher in the combined group than the cell transplantation followed by the control group (P < 0.05). At 72 hours after modeling, the expression of Bcl-2 and basic fibroblast growth factor around the injured region was significantly higher in the combined group than the cell transplantation group and control group (P < 0.05). At 4 weeks after modeling, the latencies of somatosensory and motor evoked potentials were ranked as follows: combined group < cell transplantation group < control group (P < 0.05); the amplitudes of somatosensory and motor evoked potentials were ranked as follows: combined group > cell transplantation group > control group (P < 0.05). At 8 weeks after modeling, horseradish peroxidase-labeled pyramidal cells in the cell transplantation group and combined group showed apparent crossing signs; the number of PKH-26-positive cells and horseradish peroxidase-positive cells was the most in the combined group followed by the cell transplantation group, and was the least in the control group (P < 0.05). These findings indicate that ligustrazine hydrochloride combined with bone marrow mesenchymal stem cells transplantation can facilitate nerve cell regeneration, promote the expression of Bcl-2 and basic fibroblast growth factor, and improve motor function in rats after spinal cord injury. 

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