摘要
目的:观察携带小鼠脑源性神经营养因子cDNA表达片段的重组腺病毒载体AxCA-BDNF,转染大鼠坐骨神经后基因表达的情况。方法:实验于2000-03/2002-04在第三军医大学大坪医院野战外科研究所、北京军区总医院神经外科实验室完成。实验材料:体质量200g左右的Wistar大鼠90只,雌雄不限。实验方法:①制备坐骨神经损伤模型:用1%戊巴比妥钠(40mg/kg)腹腔注射麻醉大鼠。在大鼠右侧股后部作纵行切口,无菌条件下显露坐骨神经,自股中部切除10mm坐骨神经,然后随机分为3组,每组30只,按不同方式处理:A组:坐骨神经缺损+硅胶管+AxCA-BDNF原液8μL;B组:坐骨神经缺损+硅胶管+脑源性神经营养因子溶液8μL;C组:坐骨神经缺损+硅胶管+空白病毒稀释液8μL。②灌注固定和组织切片制备:分别于术后3d、7d、14d、1个月、2个月、4个月共6个时相点,每组各取5只大鼠进行灌注。另外取5只正常Wistar大鼠作为正常对照。应用原位杂交和免疫组化等手段从脑源性神经营养因子mRNA和蛋白水平,定性和半定量分析测定坐骨神经损伤后脑源性神经营养因子基因表达。结果:90只大鼠均进入结果分析。A组3d、7d、14d、1个月时近、远端神经干和脊髓(L3～6)中脑源性神经营养因子mRNA水平均远远高于B、C组(近端神经干:A组:90.70±3.32,108.32±3.95,110.51±2.13,60.39±3.24;B组:15.46±1.89,20.50±2.31,94.37±2.45,48.32±2.37;C组:14.49±2.03,22.42±2.24,95.28±3.66,46.47±2.11;远端神经干:A组:96.24±4.58,113.10±5.83,116.28±5.22,66.91±3.87;B组:21.37±3.39,28.56±2.67,105.62±4.31,52.54±4.15;C组:20.75±3.56,27.33±2.52,104.45±4.98,53.40±3.76;脊髓:A组:100.43±4.17,109.69±6.06,103.47±5.47,60.84±4.84;B组:50.51±4.32,37.36±3.15,35.05±3.82,35.82±3.35;C组:51.03±3.91,38.98±3.75,39.36±3.34,34.64±2.84,P<0.05,P<0.01),脑源性神经营养因子水平也高于C组。结论:通过腺病毒介导转染的脑源性神经营养因子基因在大鼠坐骨神经内得到了有效表达,并通过轴突逆行转运到了相应的脊髓神经元。
AIM： To observe the expression of the brain-derived neurotrophic factor （BDNF） gene after the microinjection of adenovirus-mediated gene transfer of BDNF （AxCA-BDNF） to the sciatic nerve for peripheral nerve regeneration. METHODS： The experiment was conducted in the Research Institute of Surgery, Daping Hospital, Third Military Medical University of Chinese PLA and Department of Neurosurgery, General Hospital of Beijing Military Command Area of Chinese PLA from March 2000 to April 2002. ①Ninety Wistar rats of 200 g were selected to prepare the sciatic nerve injury models. A longitudinal incision of right posterior thigh was made in 1% somnopentyl （40 mg/kg） anesthetized rats to expose the sciatic nerve, and 10 mm sciatic nerve was cut from the middle part of thigh. Then the animals were randomly divided into three groups with 30 rats in each group： Group A was sciatic nerve defects ± silicone tube graft ± 8 μL AxCA-BDNF solution; Group B was sciatic nerve defects ± silicone tube graft ± 8 μL BDNF; Group C was sciatic nerve defects ± silicone tube graft ± 8 μL virus buffer. ②Five rats of each group were selected and infused with the previous solution 3, 7, and 14 days, 1, 2, and 4 months after operation. Additional 5 normal rats served as control. BDNF mRNA and protein levels were detected with the in situ hybridization and immunocytochemistry to identity quantitatively and semi-quantitatively BDNF gene expression after sciatic nerve injury. RESULTS： Ninety rats were involved in the result analysis. At 3, 7, and 14 days and 1 month, the BDNF mRNA levels in the proximal and distal nerve stem and L3-6 segment spinal cord of the Group A were significantly higher than those of the Groups B and C （proximal nerve stem： Group A： 90.70±3.32, 108.32±3.95, 110.51±2.13, 60.39±3.24; Group B： 15.46±1.89, 20.50±2.31, 94.37±2.45, 48.32±2.37; Group C： 14.49±2.03, 22.42±2.24, 95.28±3.66, 46.47±.2.11; distal nerve stem： Group A： 96.24±4.58, 113.10±5.83, 116.28±5.22, 66.91±3.87; Group B： 21.37±3.39, 28.56_±.2.67, 105.62±4.31, 52.54±4.15; Group C： 20.75±3.56, 27.33±2.52, 104.45±4.98, 53.40±3.76; spinal cord： Group A： 100.43-±4.17, 109.69±6.06, 103.47±5.47, 60.84± 4.84; Group B： 50.51±4.32, 37.36±3.15, 35.05±3.82, 35.82±3.35; Group C： 51.03±3.91, 38.98±3.75, 39.36±3.34, 34.64±2.84, P 〈 0.05, P 〈 0.01）, and the BDNF levels of Group A were higher than those of Group C. CONCLUSION： The adenovirus-mediated 8DNF gene is expressed in the sciatic nerve of rats, and transferred to the spinal cord motoneurons through retrograde axonal transport.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第23期4553-4557,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家重点基础研究发展规划专项基金资助项目部分资助(G1999054206)~~
