大鼠坐骨神经离断吻合后外周血单核细胞对脊髓前角运动神经元的影响

Effect of peripheral blood mononuclear cells on spinal motor neurons after end-to-end anastomosisof transected sciatic nerves in rats

目的观察大鼠坐骨神经离断伤端端吻合后外周血单核细胞（PBMCs）对对应脊髓前角运动神经元的影响，并初步探讨其作用机制。方法30只SD大鼠按随机数字表法分为空白对照组、坐骨神经离断组（模型组）、坐骨神经离断吻合组（缝合组）、坐骨神经离断吻合＋PBMCs组（PBMCs组）和坐骨神经离断吻合＋1640培养基对照组（溶媒对照组），每组6只。除空白对照组外，均于梨状肌下缘0．5cm处锐性离断左侧坐骨神经，缝合组、PBMCs组和溶媒对照组随即进行神经外膜吻合，PBMCs组端端吻合处给予细胞悬浮液0．2ml约1×10^7个PBMCs，溶媒对照组给予0．2ml1640培养基。术后4周，HE染色观察脊髓前角运动神经元形态变化；TUNEL法检测对应脊髓的细胞凋亡情况；Westernblot法检测对应脊髓脑源性神经营养因子（BDNF）和胶质细胞源性神经生长因子（GDNF）蛋白水平。结果HE染色示模型组脊髓前角运动神经元多呈空泡状，核固缩，胞体变形；溶媒对照组、缝合组部分细胞胞体收缩变形，部分神经元可见胞浆溶解，细胞质呈空泡状，部分细胞核固缩；PBMCs组可见部分神经元形态近于正常，细胞核居中，但仍可见部分神经元胞浆溶解，部分细胞质呈空泡状。TUNEL染色示凋亡细胞呈棕黄色，凋亡细胞计数空白对照组为0．44±0．10，模型组为5．78±1．11，缝合组为5．22±0．51，PBMCs组为2．56±0．42，溶媒对照组为3．78±0．19。与空白对照组比较，模型组凋亡细胞明显增多（P〈0．05）；与模型组比较，缝合组未见明显差异，而PBMCs组和溶媒对照组凋亡细胞明显减少（P〈0．05），且PBMCs组较溶媒对照组明显减少（P〈0．05）。脊髓组织中BDNF蛋白表达：空白对照组为1．83±0．72，模型组为1．35±0．46，缝合组为1．29±0．44，PBMCs组为1．87±0．55，溶媒对照组为1．22±0．50；脊髓组织中GDNF蛋白表达：空白对照组为1．47±0．48，模型组为1．17±0．51，缝合组为1．46±0．67，PBMCs组为1．68±0．50，溶媒对照组为1．24±0．48。与空白对照组比较，模型组脊髓中BDNF蛋白水平明显降低（P〈0．05），GDNF蛋白水平有降低趋势（P〉0．05）；与模型组比较，PBMCs组BDNF和GDNF蛋白水平明显增高（P〈0．05），而缝合组和溶媒对照组与模型组比较差异无统计学意义（P〉0．05）。结论大鼠坐骨神经离断可导致对应脊髓前角运动神经元损伤、凋亡，而采用端端吻合后给予PBMCs，可减轻大鼠坐骨神经对应脊髓前角运动神经元凋亡，其机制可能与PBMCs促进脊髓组织中BDNF和GDNF蛋白表达有关。

Objective To investigate the effects of peripheral blood mononuclear cells （PBMCs）on spinal motor neurons after end-to-end anastomosis of transected sciatic nerves in rats and further explore the possible mechanisms concerned. Methods A total of 30 SD rats were randomly divided into five groups ： blank control group, sciatic nerve transaction group （ model group） , nerve anastomosis after sciatic nerve transaction （anastomosis group）, nerve anastomosis with PBMCs treatment after sciatic nerve transaction （PBMCs group）, and nerve anastomosis with solvent control treatment after sciatic nerve transaction （solvent control group）, with six rats per group. Except for the rats in blank control group, transection of the left sciatic nerves conducted 0.5 cm under the piriformis muscle was performed in all animals, immediately followed by end-to-end anastomosis of sciatic nerves in anastomosis, PBMCs and solvent control groups. The rats in PBMCs group were given 0. 2 ml PBMCs （ 1 × 107 PBMCs ） at anastomosis, and the rats in solvent control group were injected with 0.2 ml PRMI-1640. After rats were on regular diet for 4 weeks, morphological examination was performed by HE staining and the counting of apoptotic ceils was evaluated by TdT mediated dUTP nick end labeling （TUNEL） staining. The protein expression of brain derived neurotrophic factor （BDNF） and glial cell derived neurotrophic factor （GDNF） were detected by Western blot. Results In model group, HE staining showed that most of the spinal motor neurons became vacuoles with karyopyknosis and cytomorphosis. In anastomosis group and solvent control group, some spinal motor neurons had contraction, deformation and even plasmolysis, with vacuoles status of cytoplasm, karyopyknosis in some cells. In PBMCs group, some spinal motor neurons were normal with nucleus in the middle while some other spinal motor neurons were seen plasmolysis and vacuoles status of cytoplasm. Through TUNEL, the numbers of apoptotic cells were 0.44 ±0.10 in blank control group, 5.78± 1.11 in model group, 5.22 ±0.51 in anastomosis group, 2.56 ±0. 42 in the PBMCs group, and 3.78 ± 0.19 in solvent control group, respectively. Compared with blank control group, the apoptosis of spinal motor neurons was significantly increased in model group （P 〈 0. 05 ）. Compared with model group, the apoptosis of spinal motor neurons was significantly decreased in treatment groups except anastomosis group （ P 〈 0. 05 ）. Compared with solvent control group, the apoptosis of spinal motor neurons was significantly decreased in PBMCs group （ P 〈 0.05 ）. The BDNF protein levels of the spinal cord tissue were 1.83 ± 0.72 in blank control group, 1.35 ± 0.46 in model group, 1.29± 0.44 in anastomosis group, 1.87 ± 0.55 in PMBCs group, and 1.22 ± 0.50 in solvent control group, respectively. Compared with blank control group, the protein expression of BDNF was decreased in model group （ P 〈 0.05 ）. Compared with model group, the protein levels of BDNF and GDNF were increased in PMBCs group （ P 〈 0. 05 ）. There was no significant difference between anastomosis group and solvent control group in regard of protein levels of BDNF and GDNF （P 〉 0.05 ）. Conclusions The transaction of sciatic nerves may induce injury and apoptosis of the spinal motor neurons. End-to-end anastomosis combined with PMBCs can effectively ameliorate apoptosis of the spinal motor neurons, the mechanism of which may be related to PMBCs that may enhance protein levels of BDNF and GDNF in spinal cord.