Y27632对脊髓损伤微环境下新生大鼠背根节神经元轴突再生的影响

In vitro effect of Y27632 on dorsal root ganglion neurons axonal regeneration in neogenetic rats with spinal cord injury

中文关键词:  脊髓损伤  背根节  轴突再生  Y27632  Rho蛋白激酶Ⅱ  大鼠

英文关键词:Spinal cord injury  Dorsal root ganglia  Axonal regeneration  Y27632  Rho-associated coiled-coil-containing protein kinase Ⅱ  Rat

中文摘要:

  【摘要】 目的：探讨Rho蛋白激酶Ⅱ（Rho-associated coiled-coil-containing protein kinase Ⅱ，ROCK Ⅱ）特异性抑制剂Y-27632对脊髓损伤微环境下新生大鼠背根节（dorsal root ganglion，DRG）神经元轴突再生和延长的影响。方法：取新生（<5d）SD大鼠胸腰段DRG神经元进行原代培养、提纯和鉴定。将健康雌性SD成年大鼠15只随机分为脊髓损伤组、假手术组和正常组，每组5只；用WD法制成T9平面脊髓损伤动物模型，造模7d后取T8~T10节段脊髓制作脊髓提取液。将新生大鼠DRG神经元分为5组进行培养：A组，DRG神经元+PBS；B组，DRG神经元+正常组脊髓提取液；C组，DRG神经元+假手术组脊髓提取液；D组，DRG神经元+损伤脊髓提取液；E组，DRG神经元+损伤脊髓提取液+不同浓度（5、10、20、30、40、50μmol/L）Y27632。培养2d后观察并比较各组新生大鼠DRG神经元轴突平均长度和微管（Tubulin βⅢ）荧光表达强度。结果：A、B和C组平均神经轴突长度及轴突远端tubulin βⅢ表达强度比较无统计学差异（P>0.05）；D组明显减小，与A、B和C组分别比较有统计学差异（P<0.05）。5、10μmol/L Y27632治疗组平均轴突长度以及轴突远端和生长锥tubulin βⅢ表达强度比D组有所增加（P<0.05），10μmol/L Y27632治疗组增加更明显，但小于A、B和C组（P<0.05）。20～50μmol/L Y27632治疗组平均轴突长度以及轴突远端和生长锥tubulin βⅢ表达强度三组间比较无统计学差异（P>0.05）；与5～10μmol/L Y27632治疗组、D组比较明显增加（P<0.05）；与A、B、C组比较平均轴突长度无统计学差异（P>0.05），平均荧光密度明显增加（P<0.05）。结论：损伤脊髓提取液能明显抑制新生大鼠DRG神经元轴突生长，导致轴突回缩。加入5~10μmol/L Y27632能促进轴突生长，20~50μmol/L Y27632更能明显促进轴突生长。

英文摘要:

  【Abstract】 Objective：To explore the in vitro effect of Rho-associated coiled-coil-containing protein kinase Ⅱ（ROCK Ⅱ） inhibitor（Y27632） on dorsal root ganglion（DRG） neurons axonal growth and extension with in neogenetic rats with spinal cord injury.Method：All thoracic-lumbar DRGs harvested from new-born Sprague-Dawley（SD） rats（<5d） were cultured，purified and indentified.15 adult female SD rats were randomly divided into three groups with each group of 5 animals.5 animals were subjected to weight-drop impact inducing T9 spinal cord injury（SCI），5 in sham operation group and 5 in normal group.T8-T10 spinal cord extracts（SCEs）were harvested in each group at 7th day after spinal cord injury.The experiment was divided into 5 groups which termed as followings：DRG neurons + phosphate-bufered saline（PBS）（group A），DRG neurons + normal SCEs（group B），DRG neurons + sham operation SCEs（group C），DRG neurons + SCI SCEs（group D） and DRG neurons + SCI SCEs +（5μmol/L，10μmol/L，20μmol/L，30μmol/L，40μmol/L，50μmol/L）Y27632（group E）.The average axonal length and positive expression of tubulin βⅢ at distal end of neuronal axons were observed after 2 days′ co-culture respectively in intro.Result：There was no difference with regarding to average axonal length among group A，B and C respectively，while the axonal length in group D was significant shorter than in group A，B and C.The mean tubulin βⅢ fluorescence densities of axonal distal end in group D was significant weaker than in group A，B and C no difference existed between group A，B and C.In 5-10μmol/L Y27632 group，there were average longer axonal length and higher expression of tubulin βⅢ at axonal distal end than in group D，but less than in group A，B and C respectively.10μmol/L Y27632 group showed longer length and higher expression than 5μmol/L Y27632 group.In 20-50μmol/L Y27632 groups，average axonal length showed no difference but higher expression of tubulin βⅢ at axonal distal end existed compared with group A，B and C，which was more than 5-10μmol/L Y27632 groups and group D.There no difference among 20-50μmol/L Y27632 groups.Conclusion：The SCI SCEs inhibit DRG neurons axonal growth obviously，5~10μmol/L Y27632 could promote axon growth，while 20~50μmol/L Y27632 could promote axon growth obviously.