中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (19): 3465-3470.doi: 10.3969/j.issn.1673-8225.2010.19.010

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

骨髓基质细胞条件液联合神经干细胞移植对帕金森病大鼠行为认知功能的影响：效果优于单纯神经干细胞移植吗？

顾 平1，2，张忠霞1，王彦永1，2，崔冬生1，耿 媛1，王铭维1，2   

  1. 1河北省脑老化与认知神经科学实验室，河北省石家庄市  050031； 
    2河北医科大学第一医院神经内科，河北省石家庄市 050031
  • 出版日期:2010-05-07 发布日期:2010-05-07
  • 通讯作者: 王铭维，博士，主任医师，教授，博士生导师，河北省脑老化与认知神经科学实验室，河北省石家庄市 050031；河北医科大学第一医院神经内科，河北省石家庄市 050031 wmw@jyyy.com.cn
  • 作者简介:顾 平，女，1970年生，江苏省无锡市人，汉族，1993年河北医科大学毕业，博士，主任医师，副教授，硕士生导师，主要从事神经病学方面的研究。 smallrainn@gmail.com
  • 基金资助:

    河北省自然科学基金（C2008000993）

Effects of combined transplantation of bone marrow stromal cell conditioned medium and neural stem cells on the behavior and cognition of Parkinson’s disease rats: Whether the outcomes of their combination are better than neural stem cell transplantation alone?

Gu Ping1,2, Zhang Zhong-xia1, Wang Yan-yong1,2, Cui Dong-sheng1, Geng Yuan1, Wang Ming-wei1,2   

  1. 1Hebei Provincial Laboratory of Brain Aging and Cognitive Neuroscience, Shijiazhuang   050031, Hebei Province, China;
    2Department of Neurology, First Hospital, Hebei Medical University, Shijiazhuang  050031, Hebei Province, China
  • Online:2010-05-07 Published:2010-05-07
  • Contact: Wang Ming-wei, Doctor, Chief physician, Professor, Doctoral supervisor, Hebei Provincial Laboratory of Brain Aging and Cognitive Neuroscience, Shijiazhuang 050031, Hebei Province, China; Department of Neurology, First Hospital, Hebei Medical University, Shijiazhuang 050031, Hebei Province, China wmw@jyyy.com.cn
  • About author:Gu Ping, Doctor, Chief physician, Associate professor, Master’s supervisor, Hebei Provincial Laboratory of Brain Aging and Cognitive Neuroscience, Shijiazhuang 050031, Hebei Province, China; Department of Neurology, First Hospital, Hebei Medical University, Shijiazhuang 050031, Hebei Province, China smallrainn@gmail.com
  • Supported by:

    the Natural Science Foundation of Hebei Province, No. C2008000993*

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摘要：

背景：将骨髓基质细胞和神经干细胞分别单独移植于帕金森病模型鼠脑内已取得良好效果，但尚未见联合移植的报道。
目的：观察骨髓基质细胞条件液与神经干细胞联合移植对帕金森病模型大鼠行为认知功能的影响，并与单纯神经干细胞移植效果进行比较。
方法：用Neurobasal+B27培养3~6代的骨髓基质细胞24 h后，收集并离心上清液，即为骨髓基质细胞条件液。体外分离培养大鼠神经干细胞后，行BrdU标记。55只大鼠随机取8只作为正常组，剩余47只采用立体定向仪单侧黑质致密部和腹侧被盖区注射6-羟基多巴胺建立帕金森病动物模型。32只造模成功，随机分为3组，选取右侧纹状体两个坐标点为移植点，单纯神经干细胞组每点注入神经干细胞悬液5 μL，联合组每点注入神经干细胞悬液+骨髓基质细胞条件液5 μL，模型组不注入任何液体。观察帕金森病大鼠旋转行为的改变，通过水迷宫试验对大鼠认知功能进行评价，免疫组织化学染色检测
黑质区酪氨酸羟化酶蛋白的表达及移植区BrdU的表达。
结果与结论：与模型组比较，移植后1~8周单纯神经干细胞组、联合组大鼠旋转次数均显著减少(P < 0.01)，移植后第4，8周单纯神经干细胞组、联合组大鼠逃避潜伏期均明显缩短(P < 0.05)，在平台象限的游泳时间百分比、游泳距离百分比、穿越平台次数均明显增加(P < 0.05)；后2组间各指标比较均无明显差异(P > 0.05)。移植后第8周，各组大鼠6-羟基多巴胺注射侧黑质区酪氨酸羟化酶阳性神经元和神经纤维表达基本缺失；单纯神经干细胞组、联合组移植区可见一定数量的BrdU阳性细胞表达，多数位于针道附近，部分细胞沿胼胝体迁移。提示骨髓基质细胞条件液与神经干细胞联合移植能改善帕金森病模型大鼠的旋转行为和认知能力，与单纯神经干细胞纹状体移植的效果基本相似。

关键词: 帕金森病, 行为, 认知, 移植, 骨髓基质细胞条件液, 神经干细胞

Abstract:

BACKGROUND: Good results have been achieved by the transplantation of bone marrow stromal cells (BMSCs) and neural stem cells (NSCs) separately into the brain of Parkinson’s disease (PD) model rat, while the transplantation of conditioned medium of BMSCs associated with NSCs has not been reported.

OBJECTIVE: To observe the effects of intracephalic transplantation of NSCs combined with BMSC conditioned medium on the behavior and cognition of PD model rats, and compare with NSC transplantation alone.

METHODS: Following 24 hours of culture using Neurobasal+B27, BMSCs at 3-6 passages were harvested and the supernatant was centrifuged, i.e., BMSC conditioned medium. Following in vitro culture, rat NSCs were labeled by BrdU. Of 55 rats, 8 rats were randomly selected as a normal group. The remaining rats were used to establish PD models by injection of 6-hydroxydopamine (6-OHDA) in the lateral substantia nigra and ventral tegmental area using a stereotactic apparatus. Thirty-two models were successfully established and randomly assigned to three groups. Two coordinate points at the right corpus striatum were selected as a transplantation point. Simple NSC group was injected with NSC suspension (5 μL) at each point. Combination group was infused with the mixture of NSC suspension + BMSC conditioned medium (5 μL). Model group did not receive any liquid. Behavior changes were observed in PD rats. Cognition function was assessed in rats using a Morris water maze test. Tyrosine hydroxylase protein expression in the substantia nigra and BrdU expression in the transplanted region were detected utilizing immunohistochemistry.

RESULTS AND CONCLUSION: Compared with model group, the rotation times of rats in simple NSC group, combination group decreased significantly from 1st to 8th weeks after transplantation (P < 0.01). The escape latency of rats in simple NSC group and combination group was significantly decreased (P < 0.05). The crossing times, percentage of swimming distance and swimming time in the platform quadrant in the Morris water maze test were increased after transplantation (P < 0.05). No significant difference was found in each index between the later two groups (P > 0.05). Expression of tyrosine hydroxylase-immunoreactive cells and nerve fiber expression were deleted in the rat substantia nigra undergoing 6-OHDA in each group at 8 weeks following transplantation. BruU-positive cells were determined in the transplanted regions in the simple NSC and combination groups; most located near to needle channel, and some migrated along the corpus callosum. Results indicated that transplantation of NSCs combined with BMSC conditioned medium can improve rotational behavior and cognitive ability of PD rats, which is similar to corpus striatum transplantation of simple NSCs.

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