Taxol和Y27632联合应用促进脊髓损伤后新生SD大鼠背根神经节神经元轴突生长的体外实验研究

发布时间：2018-05-22 08:36

本文选题：脊髓损伤 + Taxol　；参考：《西南医科大学》2017年硕士论文

【摘要】：目的:研究模拟在脊髓损伤的微环境中,Taxol与Y27632联合应用对新生SD (Sprague Dawley,SD)大鼠背根神经节神经元的轴突生长的影响。方法:①21只SD大鼠随机分为对照组7只,手术组7只(用改良Allen法制作雌性成年SD大鼠T9平面完全性截瘫模型),假手术对照组7只。制模后7天后取出T8～T10段脊髓,并经过分离、过滤形成组织匀液制备脊髓萃取液。②分离、培养以及鉴定新生SD大鼠的背根神经节神经元(dorsal root ganglion neurons, DRGNs)。③分组培养、观察脊髓萃取液对DRG神经元轴突生长,分别为:A组DRGNs+50μlPBS,B组DRGNs+50μl假手术组脊髓萃取液,C组DRGNs+50μl损伤脊髓萃取液,D组DRGNs+50μl对照组脊髓萃取液。培养2d后,各组于倒置显微镜下测量轴突生长的平均长度,然后运用免疫荧光染色技术,测量轴突远端微管荧光平均密度。实验2: DRGNs、脊髓萃取液分别和共同与Y27632,Taxol培养、DRG神经元轴突生长。共分为五组,A组为DRGNs+50μlPBS,B组为DRGNs+50μl脊髓萃取液(假手术组);C组为DRGNs+50μl完全性截瘫大鼠脊髓萃取液+3nM Taxol,D组为DRGNs+50μl完全性截瘫大鼠脊髓萃取液+30μM Y27632; E组,DRGNs+50μl完全性截瘫大鼠脊髓萃取液+3nM Taxol+30μMY27632。培养1d,2d后于倒置显微镜下测量轴突生长的平均长度,然后运用免疫荧光染色技术,测量轴突远端微管荧光平均密度。结果:①实验1:共同培养2d后,各组轴突生长平均长度:A组为 142.1 ±5.7μm,B组为 144.8±3.1μm,C组为47.2±3.5μm,D组为136.9±4.2μm。单因素方差分析示:C组和A、B、D组之间,P0.01,有显著的统计学差异;轴突远端平均微管荧光密度,A组为175.1±2.9 AFU/μm,B组为 191.6±3.1 AFU/μm,C组为64.5± 1.5 AFU/μm,D组为 174.8±3.1AFU/μm。C组和A、B、D组相比较,P0.01,有明显的统计学差异。②实验2:共同培养1d后,各组轴突平均长度:A组为136.5±4.6μm,B组为47.6±3.9μm,C组为 137.9±5.2μm, D组为 158.6±4.6μm,E组为 181.2±5.2μm,单因素方差分析示: B组和A、C、D及E组相对比,P0.01,有显明的统计学上的差异。2d后,轴突平均长度,A组为142.9±1.4μm,B组为44.5±2.2μm,C组为152.6±5.3μm,D组为168.1±3.1μm,E组为194.6±3.2μm。单因素方差分析示:B组与A、C、D及E之间,P0.01,有明显的统计学差异。C组和D、E组相比较,P0. 05,有明显的统计学差异。E组和A、B、C、D组相比较,P0.01,有明显的统计学差异;A组与C组之间,P0. 05,两者无统计学差异。轴突远端平均微管荧光密度,A组为181.8±3.4AFU/μm,B组为61.6±2.7AFU/μm,C组为205.2±2.1AFU/μM, D 组为 401.2±3.5 AFU/μM,E组为470.2±3.6AFU/μm。B组和A、C、D、E组相比较,P0. 01,有明显的统计学差异。E组和A、B、C、D组相比较,P0. 01,有明显的统计学差异;C组与E组之间,P0. 05,两者无统计学差异。结论:①DRGNs置于完全性截瘫大鼠脊髓萃取液中培养,其轴突的再生和延长受到抑制,经过前期实验结果,证实完全性截瘫大鼠脊髓萃取液能模拟脊髓损伤的微环境,抑制新生SD大鼠DRGNs轴突的生长;②单独使用适合剂量的Taxol与Y27632能积极地增进DRGNs轴突生长,由于生长锥形成。联合应用Taxol与Y27632对新生大鼠DRGNs轴突生长促进作用效果比单一应用更为有效,形成多个分支、甚至神经网。
[Abstract]:Objective: To investigate the effects of the combined application of Taxol and Y27632 on the axon growth of dorsal root ganglion neurons in neonatal SD (Sprague Dawley, SD) rats. Methods: (1) 21 rats were randomly divided into 7 rats in the control group and 7 in the operation group (using the modified Allen method to make the female adult SD rat T9 plane complete paraplegia model). 7 rats in the sham operation control group were taken out of the T8 ~ T10 segment after 7 days, and the spinal extract was prepared by filtration to form tissue homogenate. (2) isolation, culture and identification of the dorsal root ganglion neurons (dorsal root ganglion neurons, DRGNs) in newborn SD rats. (3) group culture to observe the axon growth of the spinal cord extraction solution to the DRG neuron. A group DRGNs+50 mu lPBS, B group DRGNs+50 Mu l sham group spinal extract fluid, C group DRGNs+50 u l injured spinal extract fluid, D group DRGNs+50 micron l control group spinal extract fluid. After culture, each group measured the average length of the axon growth under the inverted microscope, and then used immunofluorescence staining technique to measure the average density of the distal axon microtubule fluorescence density Degree. 2: DRGNs, spinal extract fluid and CO and Y27632, Taxol culture, and DRG neuron axon growth, divided into five groups, A group DRGNs+50 mu lPBS, B group DRGNs+50 Mu l spinal extract fluid (sham operation group), and C group was the spinal extract of complete paraplegia rats Liquid +30 mu M Y27632; E group, DRGNs+50 Mu l complete paraplegic rat spinal extract +3nM Taxol+30 micron MY27632. 1D, 2D after the inverted microscope to measure the average length of the axon growth, and then using immunofluorescence staining technique to measure the average fluorescence average density of the distal axon microtubule. Average length: A group was 142.1 + 5.7 mu m, B group was 144.8 + 3.1 mu m, C group was 47.2 + 3.5 m, D group was 136.9 + 4.2 u M. single factor analysis of variance: C group and A, B, D groups, there were significant statistical differences; the average microtubule fluorescence density of the distal axon was 175.1 + 2.9 The average length of axon in each group was 136.5 + 4.6 mu m, B group was 47.6 + 3.9 mu m, 137.9 + 5.2 micron, 158.6 + 4.6 micron, and 181.2 + 5.2 micron, and single factor analysis of variance analysis showed that after 2: co culture of 1D, the average axon length of each group was compared with that of A, B and D group. 0.01, after statistically significant difference.2d, the average length of axon, A group was 142.9 + 1.4 mu m, B group was 44.5 + 2.2 mu m, C group was 152.6 + 5.3 mu m, D group was 168.1 + 3.1 m, E group was 194.6 + 3.2 M. single factor variance analysis. The difference between the.E group and the group of A, B, C, and D, there were significant statistical differences. There was no statistical difference between the A group and the C group. There was no statistical difference between the group A and the C group. The average microtubule fluorescence density of the distal axon was 181.8 + 3.4AFU/ micron, the group was 61.6 + 205.2 mu, and the group was 401.2 + 3.5. E group compared, P0. 01, there was significant statistical difference between group.E and A, B, C, D group compared, P0. 01, there were significant statistical differences; C group and E group, P0. 05, there was no statistical difference. The spinal extract solution of complete paraplegic rats can simulate the microenvironment of spinal cord injury and inhibit the growth of DRGNs axon in newborn SD rats. (2) the use of suitable dose of Taxol and Y27632 can actively promote the growth of DRGNs axon and form the growth cone. The effect of combined application of Taxol and Y27632 on the growth of DRGNs axon of neonatal rats is more than single. Applications are more effective, forming multiple branches or even neural networks.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.2

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