课题背景：课题受广东省自然科学基金项目资助，目前进展顺利，已取得的研究成果包括：①成功从基底前脑培养获得具有自我更新能力和多向分化潜能的神经干细胞，移植后可对基底前脑神经元产生明显的补充和保护作用。②成功制备了神经生长因子缓释微球，微球所载的神经生长因子在体外或脑内均能够持续释放4周以上。③神经生长因子缓释微球移植入基底前脑，能够保护该区的胆碱能神经元免受隔-海马伞引起的逆行性溃变。

应用要点：老年性痴呆典型的病变部位是基底前脑，特别是神经元的退行性变，除了胆碱能神经元溃变外，GABA能神经元也发生明显的溃变。实验针对某一典型疾病的特定病变部位，来获得用于细胞移植治疗的细胞来源，此外采用经典的老年痴呆模型来观察神经干细胞移植对基底前脑小白蛋白阳性神经元的影响。

偏倚或不足：①实验所用神经干细胞是从原代取材，经传代培养获得，纯度很可能低于神经干细胞系。②若能够做到单细胞克隆，试验结果的可控性会更高一些。③应进一步从分子水平观察神经干细胞移植对小白蛋白阳性神经元的影响。④小白蛋白是一种钙结合蛋白，为GABA能神经元的一个亚型，若能联合其他GABA特征性标志蛋白实验结果将更有说服力。


摘要
目的：基底前脑是老年性痴呆患者脑皮质下神经元丢失最严重的区域，实验拟验证经神经干细胞移植治疗后老年痴呆鼠基底前脑小白蛋白阳性神经元的变化以及对其空间学习记忆能力方面的影响。
方法：实验于2005-12/2006-07在广州医学院解剖学教研室完成。①动物：清洁级SD雄性大鼠28只，随机数字表法分为3组：正常对照组8只、模型对照组8只、细胞移植组12只。另取10只新生SD鼠用于神经干细胞的分离培养。实验过程中对动物的处置符合动物伦理学标准。②实验方法：模型对照组、细胞移植组大鼠切断左侧穹隆海马伞，建立老年性痴呆动物模型。利用无血清培养技术获得鼠基底前脑神经干细胞，吸取2.5×1010 L－1细胞悬液4 μL，术后即刻细胞移植组损伤侧行细胞移植，坐标为前囟+0.6 mm，外侧+0.6 mm插入，腹侧-5.5 mm。③实验评估：移植后4周，采用Y型迷宫对各组动物进行学习记忆能力检测。麻醉后取脑制备组织切片，ABC法免疫组化染色结合图像分析观察各组大鼠基底前脑小白蛋白阳性神经元的变化。
结果：①对空间学习记忆能力的影响：细胞移植组空间学习能力、记忆能力均显著高于模型对照组（P < 0.05或P < 0.01），基本达到正常水平（P > 0.05）。②对小白蛋白阳性神经元的影响：与正常对照组比较，模型对照组损伤侧内侧隔核和斜角带核的小白蛋白阳性神经元数目分别减少62.5%和30.4%；细胞移植组降低幅度明显小于模型对照组（P < 0.01）。与正常对照组比较，模型对照组损伤侧内侧隔核、斜角带核小白蛋白阳性神经元的面积、周长、灰度均显著降低（P < 0.05或0.01）；细胞移植组上述指标较模型对照组均显著增加（P < 0.05或0.01）。③相关性分析：小白蛋白阳性神经元数目与大鼠在Y型迷宫中的学习次数呈显著负相关（r =-0.76~-0.79，P < 0.01），与记忆能力呈显著正相关（r = 0.78~0.84，P < 0.01）。
结论：神经干细胞移植对老年性痴呆模型鼠基底前脑退变的小白蛋白阳性神经元具有补充和保护作用，并能够明显改善其学习记忆能力，二者呈正性相关。
关键词：神经干细胞；老年性痴呆；小白蛋白阳性神经元；学习；记忆；大鼠

谷海刚，龙大宏，李晓滨，张贵平，苏涛，李佳楣，冷水龙. 神经干细胞移植对老年痴呆模型鼠基底前脑小白蛋白阳性神经元和学习记忆能力的影响[J].中国组织工程研究与临床康复，2008，12(12):2235-2239
[www.zglckf.com/zglckf/ejournal/upfiles/08-12/12k-2235(ps).pdf]

Effect of neural stem cells transplantation on parvalbumin-positive neurons of the basal forebrain and abilities of learning and memory in a rat model of senile dementia

Abstract

AIM：The loss of neurons is the severest in the basal forebrain of senile dementia patients. This study was aimed to verify the effects of neural stem cells (NSC) transplantation on the parvalbumin (PV)-positive neurons in the basal forebrain and the abilities of leaning and memory in the rat model of senile dementia.

METHODS: This study was done in the Department of Anatomy, Guangzhou Medical College from December 2005 to July 2006.①Animals: SPF class adult male SD rats (n=28) were randomly divided into three groups: normal control group (n =8), model control group (n =8), and NSC group (n =12). Furthermore, new-born SD rats (n =10) were used to isolate and culture NSC. All the manipulations were in accordance with the animal ethnic standard.②Methods: The rats of model group and NSC group were made by left fimbria-fornix transaction. NSCs were obtained from the basal forebrain by using the serum-free culturing. And 4 μL NSC suspension (2.5×1010 L-1) was stereotaxically implanted into the injured lesion (coordinates: anterior-posterior, +0.6 mm; lateral, +0.6 mm; dorsal-ventral, -5.5 mm from bregma) instantly after fimbria-fornix transaction.③Evaluation: Four weeks postoperation, the learning and memory abilities were measured by Y-maze test. Then, the brains were taken out after anesthesia and cut on a microtome. The number of PV-positive neurons in the basal forebrain was analyzed by using ABC immunohistochemical staining method and image analysis.

RESULTS: ①Effects on learning and memory abilities: The abilities in NSC group were significantly higher than that in model control group (P < 0.05 or P < 0.01), and almost reached the normal level (P > 0.05).②Effects on PV-positive neurons: Compared with normal control group, PV-positive neurons in the medial septum (MS) and the vertical limb of the diagonal band of Broca (VDB) in model control group decreased by 62.5% and 30.4%, respectively. The decrease extent in NSC group was distinctly smaller than that in model control group (P < 0.01). The area, circumference, and gray value of PV-positive neurons in MS and VDB of model control group were significantly reduced compared with normal control group (P < 0.05 or P < 0.01). However, all the characteristic parameters in NSC group were significantly increased than those in model control group (P < 0.05 or P < 0.01).③Correlative analysis: The number of PV-positive neurons in MS and VDB was negatively correlated with the times of learning of rats (r=-0.76 to -0.79, P < 0.01) and positively correlated with the ability of memory (r=0.78 to 0.84, P < 0.01), which were measured by Y-maze test.

CONCLUSION: NSC transplantation can supply and protect PV-positive degenerative neurons in the basal forebrain and is effective on improving the abilities of learning and memory in the rat model of senile dementia. The relation of them is positive correlative.

Gu HG, Long DH, Li XB, Zhang GP, Su T, Li JM, Leng SL. Effect of neural stem cells transplantation on parvalbumin-positive neurons of the basal forebrain and abilities of learning and memory in a rat model of senile dementia.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(12):2235-2239(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-12/12k-2235(ps).pdf]