基因修饰神经干细胞移植对大鼠脊髓损伤后神经营养素养-3表达及神经细胞凋亡的影响  

Effect of genetically modified neural stem cells transplantation on neurotrophin-3 expression and neuronal apoptosis after spinal cord injury in rats

作　　者：孔令胜[1] 姚维成[2] 栗世方[2] 贺昭忠[2] 成磊[2] 

机构地区：[1]济宁医学院附属医院,山东济宁272029 [2]青岛大学附属医院,山东青岛266003

出　　处：《济宁医学院学报》2014年第6期393-398,402,共7页Journal of Jining Medical University

基　　金：山东省自然科学基金资助项目(编号:ZR2010HM040)

摘　　要：目的探讨基因修饰神经干细胞(NSCs)移植大鼠损伤脊髓后对神经营养素-3(NT-3)表达及神经细胞凋亡的影响。方法 SD大鼠80只随机分为假手术组(Normal组)、单纯损伤组(SCI组)、NSCs移植组(NSC组)、NT-3基因修饰NSCs移植组(NT-NSC组)。采用电控脊髓损伤打击装置致大鼠SCI模型,25g/cm(10g×2.5cm)力致伤T13脊髓。5-溴脱氧尿嘧啶核苷(BrdU)法标记处于对数生长期的NSCs,SCI后即刻进行NSCs及基因修饰NSCs移植,分1、3、7、14、28d5个时间点运用免疫组织化学的方法检测NT-3、BrdU的表达,采用原位末端脱氧核苷转移酶介导的脱氧尿苷三磷酸生物素缺口末端标记技术(TUNEL法)标记凋亡细胞,改良Rivlin法(斜板试验)观察大鼠后肢运动功能的恢复情况。结果 NT-NSC组与NSC组在脊髓损伤区域内均可检测到Brdu阳性细胞,移植后7、14、28dBrdU阳性NSCs数与NSC组相比明显增多,差异有统计学意义(t=9.439、8.918、4.185,P均<0.05)。NT-NSC组NT-3表达高峰延迟,在14d达到最高值,其平均OD值与其他实验组各时间点相比均增高,差异有统计学意义(P<0.05);移植后7、14、28d凋亡细胞数比NSC组明显减少(P<0.05),斜板试验评分比NSC组明显提高(P<0.05)。结论 NT-3基因修饰NSCs移植较单纯NSCs移植更容易在脊髓损伤区域存活,并能通过增强NT-3的表达来抑制神经细胞的凋亡,从而促进大鼠损伤脊髓的功能恢复。Objective To explore the effect of genetically modified neural stem cells transplantation on neuro‐trophin‐3 expression and neuronal apoptosis after spinal cord injury in rats .Methods 80 SD rats were divided into four groups randomly ：Normal group ,SCI group ,NSC group ,NT‐NSC group .SCI model of Sprague Dawley rats was induced with electrocircuit control spinal cord injuring device method by a 10g × 2 .5 cm impact on the posterior T13 spinal cord .The NSCs were labeled with BrdU .NSCs and NT‐3 genetically modified NSCs transplantation were performed immediately after SCI .5 time points of 1d ,3d ,7d ,14d ,and 28d were observed .The expression of NT‐3 and BrdU were detected by immuno‐histochemical staini （immunohistochemisty and immunofluorescence ） .The im‐proved Rivlin method（namely ,slope test） was used to assess the motor function following spinal cord injury in rats .Results NSCs in NT‐NSC group and NSC group can be detected in the spinal cord after transplantation .After transplantation for 7d ,14d ,28d ,the BrdU positive cells increased obviously than that of NSC group （ t= 9 .439 , 8.918 ,4 .185 ,P〈0 .05 ） .In NT‐NSC group ,the expression peak of NT‐3 was postponed and reached the peak at 14thd ,and their mean OD was prominently higher than that of the other experimental groups （ P〈 0 .05 ） .After transplantation for 7d ,14d ,28d ,TUNEL‐positive cells（immunohistochemistry and immunofluorescence ） were sig‐nificantly decreased that in NT‐NSC group than in NSC group （ P〈 0 .05 ） ,and inclined plane test were obviously＆amp;nbsp;higher than those of NSC group （ P〈0 .05 ） .Conclusion NT‐3 genetically modified NSCs can survive more easily than pure NSCs in the injured site of spinal cord ,and inhibits neuronal apoptosis by enhancing NT‐3 expression af‐ter SCI ,which promote remarkably functional recovery after SCI .

关 键 词：基因修饰神经干细胞 脊髓损伤 神经营养素-3 细胞凋亡 

分 类 号：R651.2[医药卫生—外科学]