骨髓源性神经干细胞诱导分化及移植治疗颅脑损伤

Differentiation of bone marrow-derived neural stem cells and their transplantation for treatment of traumatic brain injury

目的:观察体外诱导成人骨髓基质细胞向骨髓源性神经干细胞的分化情况,探讨应用骨髓源性神经干细胞移植治疗脑损伤的可行性。方法:实验于2005-01/10在珠江医院神经外科实验室完成。①选取清洁级SD大鼠6只,随机数字表法分为脑损伤组、假手术组,3只/组。以3名健康成人自愿捐献的骨髓作为实验移植骨髓源性神经干细胞的来源。“CYTOKINE·神经干细胞培养基”由珠江医院神经外科实验室调配,专利申请号:ZL02134314.4。②抽吸骨髓,密度梯度离心法获取骨髓基质细胞,以“CYTOKINE·神经干细胞培养基”+体积分数0.1的胎牛血清诱导骨髓基质细胞向神经细胞分化,倒置相差显微镜追踪观察细胞的形态变化。免疫荧光细胞化学鉴定Nestin、神经元特异性烯醇化酶、胶质酸性纤维蛋白的表达情况。③脑损伤组大鼠以自由落体撞击法建立脑损伤模型,假手术组只作开颅手术,未进行自由落体撞击。造模24h后,两组大鼠尾静脉移植3×106以Dio标记的诱导7d的细胞,荧光显微镜检测移植细胞的存活和迁移。结果:①细胞形态变化观察:原代培养的骨髓基质细胞增殖迅速,诱导分化7～10d可发现典型的细胞团。②抗原的表达情况:成功诱导人骨髓基质细胞向神经细胞方向分化,并表达神经前体细胞特异性标志Nestin、胶质酸性纤维蛋白、神经元特异性烯醇化酶。③移植细胞的存活和迁移检测:人骨髓源性神经干细胞能够在大鼠脑内存活,并向脑损伤区定向迁移,主要分布于损伤灶周围。脑损伤区对侧及假手术组则少见移植细胞的迁移分布。结论:人骨髓基质细胞在合适的条件下能被诱导分化为骨髓源性神经干细胞,可存活并迁移至大鼠脑内并参与脑损伤的修复。

AIM： To observe the isolation and differentiation in vitro of adult human bone marrow stromal cells （BMSCs） into bone marrow-derived neural stem cells （NSCs）, and investigate the feasibility of NSC transplantation for treatment of traumatic brain injury. METHODS： The experiment was finished in the Institute of Neuromedicine, Zhujiang Hospital from January to October in 2005. （1）Six SD rats were randomly divided into brain injury group and sham group, equal in each group. Bone marrow-derived NSCs were harvested from .3 healthy human volunteers. ＂Cytokine-NSCs medium＂ were prepared by the Institute of Neuromedicine, Zhujiang Hospital （Patent No.ZL02134314.4）.（2）Human BMSCs were harvested by means of density gradient centrifugation. ＂Cytokine .NSCs medium＂ supplemented with 10% fetal bovine serum were used to induce BMSCs differentiate along neural pathway. The morphology of cultured BMSCs was observed using inverted microscope. Fluorescent immunocytochemistry was employed to identify the expressions of Nestin, neurone specific enolase （NSE）, and glial fibrillary acidic protein （GFAP）.（3）Models of brain injury were made according to free-falling dart impact method. In the control group, only craniotomy was managed without free-falling dart. And then 3×10^8 cells induced for 7 days ware prelabeled with Dio and intravenously injected into a rat brain trauma model. Fluorescent microscopy was performed to assay the survivance and migration of the graft. RESULTS： （1）The morphology evolvement of cells： The primary BMSCs proliferated rapidly. Typical spherical cell clusters ware formed 7-10 days after induction.（2）Expression of antigen： BMSCs were successfully induced to differentiate along neural pathway and express specific markers of neural precursor cells, including Nestin, NSE and GFAP.（3）Survivance and migration of cells： The human bone marrow-derived NSCs survived and migrated into the parenchyma of the injured brain, which ware not found in the counter lateral or sham group. CONCLUSION： Human BMSCs can differentiate into NSCs under proper experimental conditions, and human bone marrow-derived NSCs can survive in rat brain and take part in the recovery of traumatic brain injury.