CAJ | 학술논문

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、28d 5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 N T‐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、28d BrdU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。
목적：탐토기인수식신경간세포（NSCs）이식대서손상척수후대신경영양소‐3（NT‐3）표체급신경세포조망적영향。방법 SD대서80지수궤분위가수술조（Normal조）、단순손상조（SCI조）、NSCs이식조（NSC조）、NT‐3기인수식NSCs이식조（NT‐NSC조）。채용전공척수손상타격장치치대서SCI모형，25g／cm （10g ×2.5cm）력치상T13척수。5‐추탈양뇨밀정핵감（BrdU）법표기처우대수생장기적NSCs ，SCI후즉각진행NSCs급기인수식NSCs이식，분1、3、7、14、28d 5개시간점운용면역조직화학적방법검측NT‐3、BrdU적표체，채용원위말단탈양핵감전이매개도적탈양뇨감삼린산생물소결구말단표기기술（TUNEL법）표기조망세포，개량Rivlin법（사판시험）관찰대서후지운동공능적회복정황。결과 N T‐NSC조여NSC조재척수손상구역내균가검측도Brdu양성세포，이식후7、14、28d BrdU양성NSCs수여NSC조상비명현증다，차이유통계학의의（t＝9.439、8.918、4.185，P균＜0.05）。NT‐NSC조NT‐3표체고봉연지，재14d체도최고치，기평균OD치여기타실험조각시간점상비균증고，차이유통계학의의（ P＜0.05）；이식후7、14、28d조망세포수비 NSC조명현감소（ P＜0.05），사판시험평분비NSC조명현제고（P＜0.05）。결론 NT‐3기인수식NSCs이식교단순NSCs이식경용역재척수손상구역존활，병능통과증강NT‐3적표체래억제신경세포적조망，종이촉진대서손상척수적공능회복。
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 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 .