课题背景：骨髓基质细胞在体内外培养均可分化神经干细胞和成熟的神经细胞，能够透过血脑屏障在脑中成活并迁移，促进脑梗死后神经功能的恢复，重建血管，比神经保护剂具有更长的治疗时间窗。实验立足其抗神经细胞凋亡及机制研究。

应用要点：①实验通过对骨髓基质细胞传代培养，BrdU体外标记，表明静脉注射的骨髓基质细胞能够到达缺血脑组织并存活。②应用TUNEL法检测凋亡细胞在骨髓基质细胞移植前后脑组织中的表达，并证实具有抗凋亡作用。

偏倚或不足：缺乏相应时间点的功能学数据，另外未进行双标免疫组化，后续试验将进一步补充。

摘要
目的：骨髓基质细胞能透过血脑屏障在脑中存活并迁移至脑梗死区，而在脑梗死区转化生长因子β1的增加将有助于减轻神经元的损害。
目的：观察静脉移植骨髓基质细胞对大鼠脑缺血再灌注损伤神经元转化生长因子β1的影响。
设计、时间及地点：随机对照设计，于2005-09/2006-03在中国医科大学实验动物中心完成。
材料：清洁级成年Wistar大鼠36只，随机分为模型对照组、盐水组、细胞移植组，12只/组。另取2月龄Wistar大鼠2只用于制备骨髓基质细胞。
方法：贴壁法+胰蛋白酶消化分离培养骨髓基质细胞，传至3~5代用于实验，移植前24 h向培养基中加入BrdU进行体外标记。采用改良线栓法建立左侧大脑中动脉梗死大鼠模型，造模后24 h，细胞移植组于尾静脉输入浓度为3×109 L-1骨髓基质细胞悬液1 mL，盐水组尾静脉注射1 mL生理盐水，模型对照组不给予任何处理。
主要观察指标：免疫组化染色检测BrdU阳性细胞及转化生长因子β1的表达，原位TUNEL法检测神经细胞凋亡情况。
结果：造模后3 d，细胞移植组脑梗死灶周围可见大量BrdU阳性细胞，盐水组及模型对照组未见BrdU阳性细胞；细胞移植组转化生长因子β1的表达明显高于盐水组及模型对照组（P < 0.01）；细胞移植组纹状体梗死区神经细胞凋亡数明显低于盐水组及模型对照组（P < 0.01）。
结论：经静脉移植的骨髓基质细胞可选择性进入脑缺血区，增强脑损伤区转化生长因子β1的表达，从而减少神经细胞的凋亡。
关键词：骨髓基质细胞；转化生长因子β1；细胞凋亡；局灶性脑缺血

陈敬，赵彬.骨髓基质细胞静脉移植对局灶性缺血再灌注损伤脑组织转化生长因子β1的影响[J].中国组织工程研究与临床康复，2008，12(16):3011-3014 [www.zglckf.com/zglckf/ejournal/upfiles/08-16/16k-3011(ps).pdf]

Effects of intravenous transplantation of marrow stromal cells on transforming growth factor beta 1 in brain tissues of rats with focal cerebral ischemia/reperfusion injury

Abstract

BACKGROUND：Marrow stromal cells (MSCs) can cross blood-brain barrier, and survive and traverse into cerebral infarction region. The increase of transforming growth factor beta 1 (TGF-B1) in the cerebral infarction region can reduce neuron lesion.
OBJECTIVE: To investigate the effect of intravenous transplantation of MSCs on TGF-B1 expression in neurons from rats with cerebral ischemia/reperfusion.
DESIGN, TIME AND SETTING: A randomized control experiment was performed at the Experimental Animal Center of China Medical University from September 2005 to March 2006.
MATERIALS: Thirty-six clean adult Wistar rats were equally randomized into a model control group, a saline group and a cell transplantation group. An additional two Wistar rats aged two months were used to prepare MSCs.
METHODS: Adhesion + trypsin digestion was used to isolate and culture MSCs. Third to fifth passages of cells were used in the study. MSC was in vitro labeled with bromodeoxyuridine (BrdU) 24 hours before transplantation. Modified suture occlusion was used to establish rat models of left side middle cerebral artery occlusion (MCAO). Twenty-four hours after MCAO, 1 mL of MSCs (3×109 L-1) were injected into the caudal vein in the cell transplantation group; 1 mL saline was injected into the saline group; Rats in the model control group did not receive any treatment.
MAIN OUTCOME MEASURES: The distribution of BrdU-positive cells and the expression of TGF-B1 were determined by immunohistochemistry. Nerve cell apoptosis was detected by TUNEL.
RESULTS: Three days after MCAO, BrdU-positive cells were mainly distributed around the infarction area in the cell transplantation group. There was no BrdU-positive cells in the model control group and saline group. The expression of TGF-B1 was significantly higher in the cell transplantation group than in the model control and saline groups (P < 0.01). The number of nerve cell apoptosis at the striatum in the cell transplantation group was significantly reduced compared to the model control and saline groups (P < 0.01).
CONCLUSION: Intravenous injected MSCs can traverse into the cerebral infarction region, and promote the expression of TGF-β1 and reduce the nerve cell apoptosis.

Chen J, Zhao B.Effects of intravenous transplantation of marrow stromal cells on transforming growth factor beta 1 in brain tissues of rats with focal cerebral ischemia/reperfusion injury.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(16):3011-3014 [www.zglckf.com/zglckf/ejournal/upfiles/08-16/16k-3011(ps).pdf]