TDZD-8对新生大鼠背根节神经元轴突再生影响的体外实验研究

In vitro effect of TDZD-8 on DRG neurons axonal regeneration in rats

中文关键词:  脊髓损伤  背根节  轴突再生  TDZD-8  葡萄糖原合成酶-3β抑制剂

英文关键词:Spina cord injury  Dorsal root ganglias  Axonal regeneration  TDZD-8  Glycogen synthase kinase 3β inhibitor

中文摘要:

  【摘要】　目的：探讨葡萄糖原合成酶-3β（glycogen synthase kinase 3β，GSK-3β）的抑制剂TDZD-8对新生大鼠背根节（dorsal root ganglion，DRG）神经元轴突生长和延长的影响。方法：取新生（<5d）SD大鼠胸腰段背根节进行原代培养、提纯和鉴定。用WD法制成健康成年雌性SD大鼠T9平面完全性截瘫的脊髓损伤动物模型，造模7d后取T8～T10节段脊髓制作脊髓提取液。将不同浓度TDZD-8与成年大鼠脊髓提取液和新生大鼠DRG神经元分组培养：A组，DRG神经元+磷酸缓冲液（phosphate-bufered saline，PBS）；B组，DRG神经元+正常脊髓提取液；C组，DRG神经元+假手术脊髓提取液；D组，DRG神经元+全瘫脊髓提取液；E组，DRG神经元+全瘫脊髓提取液+不同浓度TDZD-8。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组、B组和C组之间平均神经轴突长度及轴突远端Tubulin βⅢ表达强度比较，无统计学意义，D组明显减小，与A、B和C组分别比较，有统计学意义（P＜0.01）。0.5～3μM TDZD-8处理组平均轴突长度及Tubulin βⅢ荧光表达强度均明显高于A、B、C组，与D组比较，更为显著，差异均具有统计学意义（P＜0.01）。5～25μM TDZD-8处理组平均轴突长度与A、B、C组分别比较，明显缩短且有统计学意义（P＜0.01），与D组比较无统计学差异（P>0.05）；轴突远端Tubulin βⅢ表达比A、B、C、D组及0.5～3μM TDZD-8处理组明显增强，有统计学意义（P＜0.01）。结论：完全瘫痪脊髓提取液明显抑制DRG神经元轴突生长，轴突回缩。低浓度TDZD-8能促进轴突生长，形成多个轴突或轴突分支，而高浓度TDZD-8明显抑制轴突生长，导致轴突回缩。

英文摘要:

  【Abstract】 Objective：To explore the in vitro effect of TDZD-8（inhibitor of GSK-3β） on DRG neurons axonal regeneration in rats.Method：All thoracic-lumbar DRGs of newborn SD rats（<5d） were harvested under the stereopsis microstat，and then DRG neurons were cultured，purified and indentified.15 adult female SD rats were randomly divided into three groups and 5 were subjected to weight-drop impact inducing complete paraplegia，5 were in sham group and 5 in normal group respectively.The T8-T10 spinal cord extracts（SCEs） were harvested from 3 different groups respectively at the 7th day after operation.5 groups were assigned as follows，group A was DRG neurons + PBS，group B was DRG neurons + normal SCE，group C was DRG neurons + sham operation SCE，group D was DRG neurons + complete paraplegia SCE and group E was DRG neurons + complete paralysis SCE + TDZD-8.The average axonal length and positive expression of Tubulin βⅢ at distal end of neuronal axons were observed after 2 days co-culture.Result：There was no difference in average axonal length among group A，B and C respectively，while the axonal length in group D was significant shorter than that in group A，B and C respectively.The mean Tubulin βⅢ fluorescence density of axonal distal end in group D was significant weaker than that in group A，B and C respectively and no difference was noted between group A，B and C.In 0.5-3μM TDZD-8 treatment groups，there were not only longer in average axonal length and higher in expression of Tubulin βⅢ at axonal distal end than in those in group A，B and C respectively，but significant longer than that in group D.In 5-25μM TDZD-8 treatment groups，the average axonal length was shorter than that in group A，B and C，while the expression of Tubulin βⅢ at axonal distal end was significant higher than that in other groups respectively（P＜0.01）.Conclusion：The complete paraplegia SCEs inhibit DRG neurons axonal growth obviously，which induce axonal retraction and growth cone collapse.Low dose of TDZD-8 can promote axon growth while high dose can inhibit axon growth obviously.TDZD-8 can enhance the expression of Tubulin βⅢ at neuronal axon shaft and growth cone，especially in high dose.