【作者】 屈一鸣；

【导师】 冯大雄；

【作者基本信息】 泸州医学院 ， 外科学， 2011， 硕士

【摘要】 目的：(1)观察脂质体介导Rho-ROCKII特异抑制性小分子多肽转染大鼠背根节神经元(DRGNs)可行性及其转染效率；(2)观察Rho-ROCKⅡ特异性抑制的小分子多肽对大鼠背根节神经轴突再生和延长的影响。(3)对比Rho-ROCKⅡ特异性抑制剂Y27632与Rho-ROCKⅡ特异抑制性小分子多肽对大鼠背根节神经轴突再生和延长的影响。方法：(1)随机选取符合条件的健康雌性SD成年大鼠10只,按WD法制成SD大鼠T9平面完全性截瘫的脊髓损伤动物模型,完成造模7d后取T8-10节段脊髓制备脊髓提取液。(2)新生SD大鼠(＜5d)DRGNs分离、培养、鉴定、纯化、扩增。(3)观察最高转染率条件下,不同脂质体/多肽浓度对DRGNs神经轴突长度、TubulinβⅢ阳性表达以及轴突远端平均荧光密度的影响。(4)瘫痪脊髓提取液+DRGNs分别与脂质体/多肽和Y26732共同培养,观察对DRGNs神经轴突长度、TubulinβⅢ阳性表达以及轴突远端平均荧光密度的影响。结果：(1)转染后2天,不同脂质体/多肽浓度下,每组测量60个轴突长度均数为：A组(1ug多肽+2.5μl脂质体)为197±14um,B组(2ug多肽+5μl脂质体)为301±17um,C组(4ug多肽+10μl脂质体)为316±25um,D组(8ug多肽+20μl脂质体)为367±29um,E组(10ug多肽+20 u 1脂质体)为265±16um,F组(12ug多肽+30 u l脂质体)为291±21um。差异有统计学意义,D组最长,两两分析D组与其它组均有统计学差异(P<0.05)；各组TubulinβⅢ免疫荧光强度均值：A-F组分别为197.8±20.1,231.8±19.1,249.8±22.8,289.1±17.1,269.8±21.6,271.6±16.2AFU/um；其中D组(使用8ug多肽+20μl脂质体)轴突远端平均荧光密度高于其它各组,但各组间无统计学意义(P>0.05),各组神经轴突干均有细长生长锥形成,神经轴突延伸,神经轴突干TubulinβⅢ浓聚,生长锥增粗。(2)瘫痪脊髓提取液+DRGNs分别与脂质体/多肽和Y26732共同培养,观察各组测量60个轴突长度均数：A组(DRGNs+PBS)为391±20um,B组(DRGNs+完全性截瘫大鼠脊髓提取液)为107±21um,C组(DRGNs+完全性截瘫大鼠脊髓提取液+脂质体)为121±23um,D组(DRGNs+完全性截瘫大鼠脊髓提取液+Y26732)为307±16um, E组(DRGNs+完全性截瘫大鼠脊髓提取液+脂质体+抑制性小分子多肽)为367±19um,经方差分析,各组差异具有显著性统计学意义(P<0.0001),两两比较,B、C组与A、D、E组之间P<0.05,均有明显统计学意义；B、C两组之间P>0.05,无统计学意义。D、E组之间P<0.05,有统计学意义。轴突远端平均荧光密度分别是：A组为183.4±3.7 AFU/um,B组为56.1±4.2AFU/um,C组为62.7±5.9AFU/um,D组为278.1±6.2AFU/um,E组为313.4±3.6 AFU/um。经方差分析,各组差异具有显著性统计学意义(P<0.0001),两两比较,A、D、E组与B、C组差别均有显著统计学意义(P<0.0001),而B、C组之间无明显统计学差异(P>0.05),E组与A、D组差别有统计学意义(P<0.05)。其荧光密度高,TubulinβⅢ阳性表达强与其他各组。结论：(1)脂质体介导的Rho-ROCKⅡ特异抑制性多肽转染大鼠背根节神经(DRGNs)可促进轴突生长,形成多个轴突或明显轴突分支,使用8ug抑制性多肽20μl脂质体浓度时轴突再生作用更明显。(2)适当浓度的Rho-ROCKⅡ特异抑制性多肽/脂质体转染新生大鼠背根节神经元DRGNs和Y27632均可促进轴突延长,脂质体介导的抑制性多肽转染DRGNs比Y27632干预作用更明显。更多还原

【Abstract】 Objective:(1) To explore the transfection efficiency of that the inhibitory polypeptide of Rho-ROCKII was introduced into dorsal root ganglia neurons(DRGNs) in vitro with Lipofectamine 2000 and the optimized factors affecting the transfection efficiency of liposome. (2) To observe the effect on DRGNs axonal growth and extension with application of Inhibitory polypeptide of Rho-ROCKII in vitro. (3) To Compare the effect on DRGNs axonal growth and extension of rats treated with Y27632 and the micromolecule polypeptide. Methods:(1) 10 adult female Sprague-Dawley (SD) rats were randomly selected into a groups and were subjected to weight-drop impact causing complete paraplegia, The T8-10 spinal cord extracts (SCE) were harvested in complete paraplegia group at 7th day after spinal cord injury in rats. (2) All thoracic-lumbar DRGNs of neogenic SD rats (<5d) were harvested, and then DRNGs were harvested, indentified, Cultured, purified and amplified. (3) Based on the most optimized transfection condition (the ratio of liposome and polypeptide is 2.5:1), The axonal length, the mean fluorescence density of positive expression of TubulinβⅢat axonal distal end of DRGNs were observed at different concerations of liposome/inhibitory polypeptide respectively. (4)The axonal length, the mean fluorescence density of positive expression of TubulinβⅢat axonal distal end of DRGNs were observed in vitro after co-culture with complete paralysis SCE+ Y26732 or liposome/ inhibitory polypeptide at 2 days respectively. Result:(1)The mean lengths of neuronal axons of neogenesis DRGNs was 197±14um in group A, 301±17um in group B,316±25um in group C,367±29um in group D,265±16um in group E and291±21um in group F respectively at 2 days after co-culture. The axonal length in group D was significantly longer compared to group A, B, C and E respectively.there was no difference among group A,B, C and E. The mean fluorescence densities at axonal distal end were 197.8±20.1 AFU/um in group A,231.8±19.1AFU/um in group B,249.8±22.8 AFU/um in group C,289.1±17.1 AFU/um in group D,269.8±21.6AFU/um in group E and 271.6.8±16.2 AFU/um in group F respecviely at 2 days after co-culture.The mean fluorescence densities in group D was significantly stronger than it in group A,B, C and E and there is no significant difference among group A,B, C and E. (2) The mean lengths of neuronal axons of neogenesis DRGNs was391±20um in group A (DRGNs+PBS),107±21 um in group B (DRGNs+ complete paralysis SCE),121±23um in group C (DRGNs+complete paralysis SCE+liposome),307±16um in group D (DRGNs+complete paralysis SCE+Y26732) and 367±19um in group E (DRGNs+complete paralysis SCE+liposome+inhibitory polypeptide)respectively at 2 days after co-culture. The axonal length in group B and group C were shorter than it in group A,D,and E, and group E was longer than group D, there was no difference between the groups B and C. The mean fluorescence densities at axonal distal end were 183.4±3.7AFU/um in group A,56.1±4.2AFU/um in group B, 62.7±5.9 AFU/um in group C,278.1±6.2AFU/um in group D and 313.4±3.6AFU/um in group E in group F respecviely at 2 days after co-culture. The mean fluorescence densities at axonal distal end in groupA,D,E were stronger than groupB, C and group E was stronger than group A,D, there was no difference between the groups B and C. Conclusion:(1) The inhibitory polypeptide of Rho-ROCKⅡtransfectd by Lipofectamine 2000 can promote axon extend, induce regeneration of axon branches. The highest efficiency was achieved in the condition of 8ug polypeptide and 20μl liposome. (2) The effect of promoting axon extend of the inhibitory polypeptide is significantly stronger than Y27632.更多还原