摘要
目的探讨人脑源性神经营养因子（humanbrain-derivedneurotrophicfactor，hBDNF）基因修饰骨髓间充质干细胞（mesenchymalstemcells，MSCs）移植入大鼠体内后在脊髓损伤区的存活情况、hBDNF蛋白的表达情况及其对大鼠脊髓损伤后神经细胞凋亡的影响。方法240只成年雄性SD大鼠按随机数字表法分为假手术组、损伤组、hBDNF-大鼠MSCs（rMSCs）组和空载体-rMSCs组，每组60只。用Allen法建立大鼠脊髓损伤模型。造模后7d，于L4。，间隙蛛网膜下腔向hBDNF-rMSCs组、空载体-rMSCs组和损伤组分别注射等体积的hBDNF基因修饰rMSCs悬液、空载体基因修饰rMSCs悬液和PBS。移植后1，2，3，7和14d分别取损伤脊髓组织，用荧光显微镜观察带有增强型绿色荧光蛋白（enhancedgreenfluorescentprotein，EGFP）基因的rMSCs在体内的存活分布情况，用Westernblot法检测hBDNF蛋白的表达水平，以原位末端标记法（TUNEL）检测神经细胞的凋亡情况。结果hBDNF-rMSCs组和空载体-rMSCs组均检测到EGFP基因表达的绿色荧光信号；hBDNF-rMSCs组表达hBDNF蛋白，其表达水平随时间而变化，移植后2d即可检测到hBDNF蛋白表达，移植后7dhBDNF表达量最高，此后逐渐下降；移植后2，3，7和14d，hBDNF-rMSC组TUNEL阳性细胞数量最少，空载体-rMSCs组次之，损伤组相对较多，差异有统计学意义（P〈0．05）。结论脊髓损伤后hBDNF基因修饰rMSCs经蛛网膜下腔移植能聚集生长在脊髓损伤区域，并表达hBDNF蛋白；hBDNF基因修饰rMSCs能够抑制神经细胞的凋亡。
Objective To discuss the survivorship of rat mesenchymal stem cells （rMSCs） and the expression of human brain-derived neurotrophic factor （hBNDF） protein after transplantation of the hBDNF-modified rMSCs （hBDNF-rMSCs） to the adult rats with spinal cord injury （SCI） and discuss the effect of hBDNF-rMSCs on the apoptosis of rat neural cells. Methods A total of 240 adult male Spra- gue-Dawley rats were randomly divided into sham operation group, SCI group, hBDNF-rMSCs transplanta- tion group and empty vector-rMSCs transplantation group, with 60 rats in each group. The SCI model was established by using the modified Allen technique. At day 7 after modeling, an equal volume of hBDNF- rMSCs suspension, empty vector-rMSCs suspension and phosphate buffered saline （PBS） were injected through the L4.5 subarachnoid space into the hBDNF-rMSCs group, empty vector-rMSCs group and SCI group, respectively. Then, the injured spinal cord tissues were obtained from each group at days 1,2,3, 7 and 14 after transplantation to observe the viability and distribution of rMSCs with enhanced green fluo- rescent protein gene by fluorescent microscope, measure the expression of hBDNF protein by Western blot and detect the apoptosis of neural cells by TdT-mediated dUTP nick end labeling （TUNEL）. Results Both hBDNF-rMSCs and empty vector-rMSCs groups showed green fluorescence expression of rMSCs. The hBDNF protein expression was observed in hBDNF-rMSCs group and changed with time, ie, the expres- sion was detected at day 2 after transplantation, reached the highest level at day 7 and then decreased gradually. Among the hBDNF-rMSCs, empty vector-rMSCs and SCI groups, the number of TUNEL positivecells was the least in hBDNF-rMSCs group, followed by the empty vector-rMSCs group and the number was relatively more in SCI group at days 2,3,7 and 14 after transplantation, with significant differences a- mong groups （ P 〈 0.05 ）. Conclusions Transplantation of the hBDNF-modified rMSCs through sub- arachnoid approach are able to survive and assemble at the injured spinal cord area and express hBDNF protein. The hBDNF-modified rMSCs can inhibit the apoptosis of neural cells after SCI.
出处
《中华创伤杂志》
CAS
CSCD
北大核心
2012年第4期375-380,共6页
Chinese Journal of Trauma
关键词
脊髓损伤
脑源性神经营养因子
骨髓间充质干细胞
基因治疗
凋亡
Spinal cord injuries
Brain-derived neurotrophic factor
Mesenchymal stemcells
Gene therapy
Apoptosis
