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

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

Key words:  brain-derived neurotrophic factor(BDNF)  neural stem cells (NSCs)  traumatic brain injury (TBI)  Ras/Raf/Erk1/2[

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3 μL（1×10⁸/mL）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。

Abstract:

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. [WTHZ]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/mL）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. [WTHZ]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). [WTHZ]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 Trkβ activates 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.