hBDNF基因修饰骨髓间充质干细胞移植对大鼠脊髓损伤后神经细胞凋亡的影响  

Apoptosis of neural cells in response to hBDNF-modified rMSCs transplantation after spinal cord injury in rats

作　　者：刘凯[1] 刘文革 王振宇 姚志鹏 

机构地区：[1]福建医科大学协和临床学院,福州350001 [2]福州市鼓楼区协和医院骨科

出　　处：《中华创伤杂志》2012年第4期375-380,共6页Chinese Journal of Trauma

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

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 

分 类 号：R651.2[医药卫生—外科学]