BDNF基因转染神经干细胞移植脑损伤大鼠移植细胞存活率和Trkβ、Erk1/2、Ras及Trx的表达  

Neural stem cells overexpressing brain-derived neurotrophic factor improve cell survival and elevate the expression of Trkβ,Erk1/2,Ras and Trx following transplantation in a rat model of traumatic brain injury

作　　者：尹良伟[1] 王禹兹 陈涛[2] 张舒珊 初海鹰[2] 孔力[2] 马海英[2] YIN Liang-wei WANG Yu-zi CHEN Tao ZHANG Shu-shan CHU Hai-ying KONG Li MA Hai-ying(Comprehensive Wards 6, Dalian Central Hospital, Dalian 116033, China Department of Histology and Embryology, Dalian Medical University, Dalian 116044, China)

机构地区：[1]大连市中心医院综合六病房,辽宁大连116033 [2]大连医科大学组织胚胎学教研室,辽宁大连116044

出　　处：《大连医科大学学报》2016年第5期422-427,共6页Journal of Dalian Medical University

基　　金：国家自然科学基金项目(31300812)

摘　　要：目的探讨脑源性神经营养因子基因修饰神经干细胞(BDNF/NSCs)移植大鼠创伤性脑损伤(TBI)模型后,对移植细胞存活数量的影响及其机制。方法采用Feeney法制备TBI模型,于造模后72 h,随机分为两组:BDNF/NSCs移植组及NSCs移植组,将3μL(1×108/m L)BDNF/NSCs或NSCs直接移植于脑损伤区。分别于移植后1、2、3和4周取脑组织,冻存或制备冰冻切片。在荧光显微镜下计数移植细胞存活数量,并采用免疫组织化学技术检测p-Erk1/2、Ras蛋白的表达;通过RT-PCR技术检测脑损伤区及周围组织中Trx、Trkβ基因的表达。结果 BDNF/NSCs组和NSCs组细胞存活数随移植时间延长而减少,在移植后的不同时间点,BDNF/NSCs组移植细胞存活率均明显高于NSCs组(P<0.05);BDNF/NSCs组在移植后不同时间点移植细胞的p-Erk1/2、Ras蛋白表达强度均明显高于NSCs组(P<0.05);与NSCs组比较,BDNF/NSCs组损伤灶及周围脑组织中Trx、Trkβ基因的表达水平均明显增强(P<0.05)。结论 BDNF基因转染NSCs移植大鼠TBI后,能够明显提高移植细胞的存活率,上调Trkβ、p-Erk1/2、Ras及Trx的表达。BDNF与其受体Trkβ结合后,可能通过Ras/Raf/Erk途径激活其下游的Trx基因从而激活细胞的抗氧化作用,进而减少移植细胞的氧化损伤,提高移植细胞在脑组织损伤区的存活率。Objective To investigate the survival of engrafted cells after transplantation of NSCs modified by BDNF gene（ BDNF / NSCs） into rat models of traumatic brain injury（ TBI） and underlying mechnisms. Methods TBI models built as described by Feeney were divided into two groups after 72 hours,BDNF / NSCs- transplanted group and NCSs- transplanted group,both directly transplanted with 3 μL（ 1 × 108/ m L） BDNF / NSCs or NSCs,respectively.Brain samples were freezed or made into freezed slices at 1,2,3,and 4 weeks after transplantation. The survival of engrafted cells was accounted with fluroescent microscope. Immunohistochemical staining and RT- PCR were conducted to evaluted the expression of Erk and Ras protein and the expression of the Trx and Trkβ gene. Results The survival numbers in both BDNF / NSCs- transplanted group and NSCs- transplanted group were decreased with the passage of time. However,the survival in BDNF / NSCs- transplantated group was significantly more than that in NSCs- transplantate group during the experimental period（ P〈0. 05）. Immunohistochemical staining showed the expressions of p-Erk1 /2 and Ras proteins in BDNF / NSCs- transplanted group were higher than those in NCSs- transplanted group（ P〈0. 05）,and RT- PCR showed the expressions of Trx and Trkβ genes around the injured areas in BDNF / NSCs- transplanted group were increased significantly compared to those in NCSs- transplanted group（ P〈0. 05）. Conclusions BDNF gene transfection improves the survival of engrafted NSCs in rat models of TBI,and upregulates the levels of Trkβ,p- Erk1 /2,Ras and Trx. The underlying mechanisms may be that BDNF gene combining with its receptor Trkβactivates its downstream Trx gene through the anti- oxidate pathway Ras / Raf / Erk1 /2,subsequently reduces the oxidative damage of engrafted cells and improves the survival rate in TBI.

关 键 词：脑源性神经营养因子 神经干细胞 创伤性脑损伤 Ras/Raf/Erk 

分 类 号：R651.15[医药卫生—外科学]