靳三针疗法对窒息脑瘫幼鼠神经干细胞增殖与分化的实验研究  

Effect of Jin's Three-needle Manipulation on Proliferation and Differentiation of Neural Stem Cells in Immature Rats with Asphyxia-induced Cerebral Palsy

作　　者：刘乡[1] 柴铁劬[2] 孙娟[2] 张瑞[2] 孟令杰[2] 赵丹[2] 周健洪[2] 陈东风[2] 

机构地区：[1]广东省第二中医院,广东广州510095 [2]广州中医药大学,广东广州510405

出　　处：《广州中医药大学学报》2010年第3期231-235,315,316,共7页Journal of Guangzhou University of Traditional Chinese Medicine

基　　金：广东省自然科学基金项目(编号:04010032)

摘　　要：【目的】观察靳三针疗法对脑瘫大鼠神经干细胞(NSCs)增殖与分化的影响。【方法】选用SD新生大鼠,随机分为假手术组、模型组、靳三针疗法治疗组(治疗组,针刺百会、颞Ⅰ针、曲池、内关、足三里、涌泉),模型组与治疗组均采用结扎单侧颈总动脉法复制脑瘫大鼠模型,其中治疗组于手术后24 h开始针刺,共14 d。各组于造模后15 d处死大鼠制备脑组织提取液。另取健康SD新生大鼠,取大脑海马部位,分离、扩增NSCs,采用免疫细胞化学法检测NSCs特征性标记Nestin与B rdu表达,以鉴定培养的细胞及体外分化能力。培养细胞分为正常对照组,低、高浓度组(分别加入100、300μL/mL的治疗组脑组织提取液),培养1、3、5 d后,采用免疫细胞化学法和免疫荧光法检测各组神经元样细胞和星形胶质样细胞的特征性标志物神经丝蛋白(NF)和胶质纤维酸性蛋白(GFAP)的表达,观察各组NSCs分化情况。采用流式细胞术检测各组NF、GFAP阳性细胞数,观察脑组织提取液促进NF、GFAP表达的时效和量效关系。【结果】扩增后的细胞球Nestin与B rdu均呈阳性,证实体外培养的细胞为NSCs,并处于分裂增殖旺盛期。细胞培养1、3、5 d后,NF、GFAP染色均呈阳性,且正常对照组阳性细胞少,低、高浓度组明显增多,呈浓度依赖性,表明NSCs已分化为神经元细胞和星形胶质细胞。同一时间段内低、高浓度组NF、GFAP阳性细胞数均较正常对照组显著增高(P<0.05或P<0.01),高浓度组在培养1、3、5 d后,NF、GFAP阳性细胞数也显著增高(P<0.05或P<0.01),表明治疗组脑组织提取液可促进NF、GFAP表达,且呈一定的量效和时效关系。【结论】靳三针疗法治疗脑瘫可能是通过促进NSCs的增殖和定向分化,从而达到修复脑损伤的目的。Objective To observe the influence of Jin＇s three-needle manipulation（JTM） on neural stem cells（NSCs）in immature rats with asphyxia-induced cerebral palsy and to investigate its possible therapeutic mechanism.Methods Rats were randomized into sham-operation group,model group,and JTM group（acupuncturing on acupoints of Baihui,No.1 of temporal tri-points,Quchi,Neiguan,Zusanli and Yongquan）.Cerebral palsy model was established by ligation of unilateral common carotid artery.Acupuncture was performed 24h after the operation.After treatment for 14 days,cerebral extract was prepared.NSCs were isolated from the hippocampus of neonatal SD rats and then were amplified.Immunocytochemical assay was used for the detection of expression of Nestin and Brdu,which were the specific markers of NSCs and can be used to identify NSCs and its in-vitro differentiation.NSCs were randomized into normal control group,and low-and high-dose cerebral extract groups（with 100 and 300 μL/mL cerebral extract added respectively）.After culturing for 1,3 and 5 day（s）,the expressions of neurofilament protein（NF）and gelatinous fiber acid protein（GFAP）,which were the specific markers of astrocytes and neuron-like cells,were detected by immunocytochemical assay and immunofluorescence assay to examine the differentiation of NSCs.The number of cells with NF and GFAP positive was detected with flow cytometry,and the time-dose effect of cerebral extract on promoting NF and GFAP expression was observed.Results The positive expression of Nestin and Brdu in the amplified cells indicated that the in-vitro cultured cells were NSCs and were in division growth.After culturing for 1,3 and 5 day（s）,the expression of NF and GFAP were positive,indicating that NSCs were differentiated into astrocytes and neuron-like cells.The number of cells with NF and GFAP positive was higher in cerebral extract groups than that in the normal control group,and was the highest in high-dose cerebral extract group（P0.05 or P0.01）,indicating that

关 键 词：靳三针疗法/治疗应用 脑性瘫痪/针灸疗法 神经干细胞/病理学 细胞培养 

分 类 号：R246.6[医药卫生—针灸推拿学]