联合应用Y27632和TDZD-8促进大鼠DRG神经元轴突再生

Promotion of axonal regeneration of DRG neurons with combined use of Y27632 and TDZD-8

目的探讨联合应用ROCKⅡ（ Rho - associated coiled - coil - containing protein kinase Ⅱ, ROCK Ⅱ）和糖元合成酶-3β（glycogen synthase kinase，GSK-3B）抑制剂对新生大鼠背根神经节（dorsal root ganglion，DRG）神经元轴突再生和延长的影响。方法（1）取新生sD（Sprague—Dawley，SD）大鼠（〈5d）2只，显微操作下取胸腰段背根节，进行原代培养。（2）健康雌性成年sD大鼠15只，随机数字表法分为：手术瘫痪组（5只）、假手术对照组（5只）和正常组（5只），瘫痪组用WD（weight—drop，WD）法制成SD大鼠T9平面完全性截瘫的脊髓损伤动物模型，假手术组仅做椎板切除，造模7d后取1r8。节段脊髓制作脊髓提取液。（3）将Y27632（ROCKⅡ抑制剂）、TDZD-8（GSK-3β抑制剂）及伤后7d全瘫脊髓提取液与新生大鼠DRG神经元分组培养2d。A组：DRG神经元＋PBS，B组：DRG神经元＋全瘫脊髓提取液，C组：DRG神经元＋全瘫脊髓提取液＋不同浓度的Y26732，D组：DRG神经元＋全瘫脊髓提取液＋不同浓度的TDZD-8，E组：DRG神经元＋全瘫脊髓提取液＋Y26732＋TDZD-8，观察并比较各组新生大鼠DRG神经元轴突平均长度和微管蛋白Bm（TubulinβⅢ）荧光表达强度。结果（1）与A组比较，B组轴突长度明显减小（P〈0．01），轴突远端TubulinβⅢ表达强度明显减弱（P〈0．01）。（2）C组中，5、10μmoL／LY27632治疗组轴突长度以及轴突远端和生长锥Tubulinl3llI表达强度比B组有所增加，但小于A组（P〈0．05）；20～50μmoL／LY27632治疗组轴突长度以及轴突远端和生长锥TubulinβⅢ表达强度较B组增加明显（P〈0．01），其中30μmoL／LY27632治疗组最明显。（3）D组中，0．5—3μmoL／LTDZD-8治疗组轴突长度比A和B组明显增加（P〈0．01），其中1μmoL／LTDZD-8治疗组轴突长度最长；而5—25μmoL／LTDZD-8治疗组轴突长度明显缩短，与B组比较，差异无统计学意义，且明显小于A组（P〈0．01）；TDZD-8各浓度组轴突干和生长锥TubulinlβⅢ表达均明显增强，与A和B组比较差异有统计学意义（P〈0．01）。（4）E组联合应用30μmol／LY27632和1μmoWLTDZD-8，其轴突长度明显长于B组（P〈0．01）和A组（P〈0．01），且轴突的分枝更多；与A组比较，E组轴突干和生长锥TubulinβⅢ表达更强（P〈0．05）。结论完全瘫痪脊髓提取液能明显抑制DRG神经轴突生长，导致生长锥塌陷；高浓度比低浓度的Y27632能明显促进轴突生长，形成细长的生长锥；低浓度比高浓度的TDZD-8能促进轴突生长，形成粗大的生长锥；适当浓度的Y27632和TDZD-8联合应用可促进轴突延长与轴突分枝形成，有利于形成神经环路。

Objective To explore the effect on effect of combined use of Y27632 （ ROCK II inhibitor） and TDZD-8 （GSK-313 inhibitor） on axonal regeneration of dorsal root ganglion （DRG） neurons in neogenetic rats. Methods All the thoracolumbar DRGs of two neogenetic Sprague-Dawley （ SD ） rats （ 〈 5 days） were harvested under the stereopsis microstat, and then the DRG neurons were cultured,purified and indentified. Fifteen adult female SD rats were randomly divided into three groups, ie, complete paraplegia group （5 rats）, sham operation control group （5 rats） and normal group （5 rats） respectively. The Ts_10spinal cord extracts （SCEs） were harvested in the complete paraplegia group, sham operation control group and normal group respectively at day 7 after spinal cord injury. The experiment was divided into group A （DRG neurons ＋ PBS）, group B （DRG neurons ＋ complete paralysis SCE）, group C （ DRG neurons ＋ complete paralysis SCE ＋ different concentration Y27632） , group D （ DRG neurons ＋ complete paralysis SCE ＋ different concentration TDZD-8） and group E （ DRG neurons ＋ complete paralysis SCE ＋ proper concentration Y27632 and TDZD-8）. The average axonal length and expression intensity of Tubulin βⅢ at distal end of neuronal axons were observed after two days of co-culture respectively in intro. Results （ 1 ） The average axonal length and expression intensity of Tubulin βⅢ at axon shaft and growth cone in the group B were significantly shorter and weaker than that in the group A, with statistical difference. （2） In the group C, the average axonal length and expression intensity of Tubulin βⅢ at axon shaft and growth cone in 5-10 μmol/L Y27632 treatment groups were more than that in the group B but lower than that in the group A. The average axonal length and expression intensity of Tubulin βⅢ at axon shaft and growth cone in 20-50 μmol/L Y27632 treatment group were lohger and stronger than that in the group A and the group B, especially the group B. Among different concentration Y27632 treatment groups, there was a longest average axonal length and strongest expression intensity of Tubulin βⅢ in 30 μmil/L treatment group. （3） In the group D, there was a longer average axonal length in 0.5-3 μmol/L TDZD-8 treatment group than that in the group A and the group B, with the longest average axonal length in 1 μmol/L TDZD-8 treatment group. In 5-25 μmol/L TDZD-8 treatment groups, the average axonal length showed no difference compared with the group B but was shorter than that in the group A. In all different concentration TDZD-8 treatment groups, the expression intensity of Tubulin βⅢ at axon shaft and growth cone was significantly stronger than that in the groups A and B. （4） In the group E, although the average axonal length was increased in the group E, there was no statistical difference compared with the group A, 30 μmol/L Y27632 treatment group and 1 μmol/L TDZD-8 treatment group. There was a significantly longer average axonal length in the group E than it in the group B and the expression intensity of Tubulin βⅢ at axon shaft and growth cone was stronger in the group E compared to the group A, 30 μmol/L Y27632 treatment group and 1 μmol/L TDZD-8 treatment group. Conclusion The complete paralysis SCEs obviously inhibits DRG axonal growth, induces axonal retraction and growth cone collapse. High concentration of Y27632 can more obviously promote the axon growth compared with the low concentration, while the low concentration of TDZD-8 can obviously promote the axon growth. Combined use of appropriate concentration of TDZD-8 and Y27632 can promote the axon growth and induce the axons branching, as facilitates the formation of the axon circuit.