大鼠面神经损伤后再生过程中BMP-2，6，7及TC10在面神经运动神经元内的表达及作用实验研究
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摘要
BMPs是一类能在骨骼外异位诱导骨和软骨形成的酸性多肽，其作用极为广泛。目前有研究证实它们在CNS胚胎发育的各阶段作用显著，例如神经系统原肠胚形成的调控，CNS背腹化，及CNS胚胎发育后期神经元存活和分化的调控等。而Rho家族通常被认为是细胞间架结构的主要调控者，最新发现其家族成员TC10对舌下神经受损后的再生具有显著促进作用，但关于BMPs及TC10是否在面神经损伤后的再生过程中具有同样的作用未见报道。故本实验研究旨在探讨面神经损伤后再生过程中内源性BMPs及TC10在面神经运动神经元内的表达变化及作用。
一、面神经损伤后面神经运动神经元内BMP-2，6，7的表达变化及作用实验研究
既往针对BMPs在神经系统中的作用研究主要集中在CNS，而对PNS的作用，只局限于对体外培养的神经细胞作用阶段，未见有关内源性BMPs在外周神经损伤后其对应运动神经元内是否具有表达及变化规律的相关报道。本研究建立大鼠面神经切断吻合损伤模型，通过硫酸镍胺增强显色的免疫组化法，检测了正常和切断吻合伤后面神经运动神经元内BMPs的表达变化。结果首次发现并确定内源性BMP-2，6，7在正常状态下的面神经运动神经元胞浆内及胞体突起有表达。损伤后早期BMP-2，6即分别以下调和上调方式应激损伤，至术后4W时BMP-2恢复到正常表达水平，而BMP-6则持续在高表达水平状态；BMP-7阳性表达在损伤后早期下调，随后上升并超过正常表达水平，至术后4w恢复正常表达水平。提示：内源性BMP-2，6，7对面神经发挥正常生理、生化代谢功能发挥一定作用。而当损伤发生后，BMP-2，6可能做为损伤启动子，分别以下调和上调方式参与面神经损伤后早期修复和再生活动，其中BMP-6还可能是面神经再生的维持者；而BMP-7则可能是面神经再生中期过程的参与和促进因子。BMP-2，6，7可能是另一类潜在的NTFs。
二、面神经损伤后面神经运动神经元内BMPR-Ⅰ及其下游信号转导分子Smad1的表达变化及作用实验研究
BMPR分Ⅰ、Ⅱ两型，只有当二者结合时方可与配基形成高亲和力，其直接的下游信号转导分子包括Smad1，5和Smad4。本实验研究建立大鼠面神
君四军厌义公硕士华应沦欠 二 中文贫要
经切断吻合损伤模型，通过硫酸镍胺增强显色的免疫组化法，检测了正常和
切断吻合伤后内源性BMPR－I及其下游信号转导分子Smadl在面神经核团
内运动神经元上的表达及变化规律。结果首次发现并确定了二者在正常状态
下的运动神经元胞浆及胞体突起有微弱表达。而当面神经发生损伤后，二者
均表现出阳性表达升高的反应；并持续 4W以上。提示：做为 BMPS下游信
号转导通路上的中间成分，BMPR刁和 Sma山在 BMP工，6，7营养正常面神
经元胞体，维持面神经轴突功能、结构方面发挥作用。同时，对BMP上氏7
发挥保护神经元胞体、促进新生轴突生长方面的作用的进行，起到积极的信
号转导作用。
三、面神经损伤后面神经运动神经元内TC10的表达变化及作用实验研究
TC属于R上。家族中的＊。GTP结合蛋白。既往研究显示，某些信号
通路分子，其中包括＊＊F，与受体汀hA结合，可以产生一系列中间蛋白；
GTP结合蛋白位于第三位，它可以激活ras基困从而使控制基因表达转录困
子磷酸｛匕 诱导包括 BMP卫，6，7在内的细胞存活和再生所需的各种基困的表
达。有研究显示舌下神经损伤后其运动神经元内 TC与同家族成员 RhOA、
Rac及 Cdc42相比，具有显著性差异．本实验通过建立大鼠面神经切断吻
合损伤模型，采用原位杂交方法，检测了内源性 TC omRNA在面神经核运
动神经元内的表达变化。结果首次发现K1 OInRNA在正常状态下的运动神
经元胞浆及胞体突起有微弱表达。而当面神经发生损伤后，表达显著上调。
提示：TC对面神经损伤后信号转导及再生过程中的运动神经元胞体及轴
突间架结构的重建具有重要作用。
由于BMPR－I、Smadl及TC10均为信号转导通路上的成分，三者于
面神经损伤后均出现表达升高，4W以内都维持在高表达水平；提示它们之
间可能针对面神经再生具有协同作用。
The Expression of BMP-2,6,7 and TC10 in the Facial Nerve Motoneurons during the Regeneration of Rats' Facial Nerve Injury
The Bone Morphogenetic Proteins (BMPs) family of secreted signaling molecules belongs to the transforming growth factor beta superfamily. The initail discovery of BMPs was made based on the abiliity of purified bone extracts to promote ectopic cartilage and bone formation when implanted under the skin or into the moscle of adult rats. However, it is now evident that the BMP family consists of a large number of genetically related molecules. Conserved in evolution, with key role in different periods of central nervous system. Rho families have been implicated in the regulation of cytoskeleton, until recently, some member of this family could promote the hypoglossus. No reports show whether BMPs and TC10 play important role in facial nerve injury. Therefore, our experiments have investigated the expressions of endogenous BMPs and TC10 in the facial motoneuron and their roles.
1. The experiment study about the changings and roles ofBMP-2,6,7 after facial nerve axtomy.
Previous studies were focused in the effects of BMPs on central nervous system. But to peripheral nerve, there were only localized in neuron culture Relevant information about the changing of BMP-2,6,7 after facial nerve transection were't found. We set up the facial nerve crush injury model in rat to examine these indexes. The result verified that the expression of BMP-2 decrease after injury. Postoperative 2 week, BMP-2 resumes to normal. BMP-6,7 increase gradually after axtomy, and reach maximum at 7 days post injury; then, BMP-6 resumed to normal level on postoperative 4 week. But BMP-7 was still expressed abundantly in the facial motoneurons. These results reveal that some member of BMPs family such as BMP-2,6,7 might be facial nerve potential neurotrophic factors through the change of expression to trigger facial nerve regeneration. The BMP-6 implicates in maintenance of nerve regeneration.
2. The experiment study about the changings and roles of BMPR- I and after downstream signal molecule Smad 1 facial nerve axtomy.
BMPs exert their biological effects through two types of transmembrane
receptors BMPR- I and BMPR- II ,which possess intrinsic serine/threonine kinase activity, and through downstream molecule Smadl transfer signal into the cytoplasm. We identified the expressions of BMPR- I and Smadl in facial nerve axtomy. The results show the upregulation of BMPR-1 and Smadl after injury. The peak value was sustained to 4w group. The result specified that BMPR- I and Smadl have implicated in the processes of the effects of BMP-2,6,7 on nerve regeneration.
3. The effect and expression of small GTP binding protein TClOmRNA on the facial motoneuron regeneration
TC10 belongs to the family of small GTP-binding proteins. Its functions and actions are similar to RhoA. Racl and Cdc42. The alternation between GTP-bound and GDP-bound form is thought to act as bimodal switches, and regulate many intracellular sighalling pathways. Early studies with small GTP-binding proteins play major roles in regulating remodelling of the actin cytoskeleton induced by exteacellular signals. We investigate the change of GTP binding protein TC10 in the facial motoneuron regeneration. The TC10 cDNA probe was derived from facial nucleus by RT-PCR technology. The different expressions of TC10 mRNA are observed by in situ hybridization on normal and regenertional facial motoneurons. In the normal group, the expression of TCI0 is low. The amount of the TC10 mRNA increases gradually from 1-day group following facial nerve injury. At 1-month group, it reaches maximum. TC10 may act an important role in signal transduce and cytoskeleton remodeling after nerve axotomy. Moreover, it could promote the nerve axon growth.
一、面神经损伤后面神经运动神经元内BMP-2，6，7的表达变化及作用实验研究
既往针对BMPs在神经系统中的作用研究主要集中在CNS，而对PNS的作用，只局限于对体外培养的神经细胞作用阶段，未见有关内源性BMPs在外周神经损伤后其对应运动神经元内是否具有表达及变化规律的相关报道。本研究建立大鼠面神经切断吻合损伤模型，通过硫酸镍胺增强显色的免疫组化法，检测了正常和切断吻合伤后面神经运动神经元内BMPs的表达变化。结果首次发现并确定内源性BMP-2，6，7在正常状态下的面神经运动神经元胞浆内及胞体突起有表达。损伤后早期BMP-2，6即分别以下调和上调方式应激损伤，至术后4W时BMP-2恢复到正常表达水平，而BMP-6则持续在高表达水平状态；BMP-7阳性表达在损伤后早期下调，随后上升并超过正常表达水平，至术后4w恢复正常表达水平。提示：内源性BMP-2，6，7对面神经发挥正常生理、生化代谢功能发挥一定作用。而当损伤发生后，BMP-2，6可能做为损伤启动子，分别以下调和上调方式参与面神经损伤后早期修复和再生活动，其中BMP-6还可能是面神经再生的维持者；而BMP-7则可能是面神经再生中期过程的参与和促进因子。BMP-2，6，7可能是另一类潜在的NTFs。
二、面神经损伤后面神经运动神经元内BMPR-Ⅰ及其下游信号转导分子Smad1的表达变化及作用实验研究
BMPR分Ⅰ、Ⅱ两型，只有当二者结合时方可与配基形成高亲和力，其直接的下游信号转导分子包括Smad1，5和Smad4。本实验研究建立大鼠面神
君四军厌义公硕士华应沦欠 二 中文贫要
经切断吻合损伤模型，通过硫酸镍胺增强显色的免疫组化法，检测了正常和
切断吻合伤后内源性BMPR－I及其下游信号转导分子Smadl在面神经核团
内运动神经元上的表达及变化规律。结果首次发现并确定了二者在正常状态
下的运动神经元胞浆及胞体突起有微弱表达。而当面神经发生损伤后，二者
均表现出阳性表达升高的反应；并持续 4W以上。提示：做为 BMPS下游信
号转导通路上的中间成分，BMPR刁和 Sma山在 BMP工，6，7营养正常面神
经元胞体，维持面神经轴突功能、结构方面发挥作用。同时，对BMP上氏7
发挥保护神经元胞体、促进新生轴突生长方面的作用的进行，起到积极的信
号转导作用。
三、面神经损伤后面神经运动神经元内TC10的表达变化及作用实验研究
TC属于R上。家族中的＊。GTP结合蛋白。既往研究显示，某些信号
通路分子，其中包括＊＊F，与受体汀hA结合，可以产生一系列中间蛋白；
GTP结合蛋白位于第三位，它可以激活ras基困从而使控制基因表达转录困
子磷酸｛匕 诱导包括 BMP卫，6，7在内的细胞存活和再生所需的各种基困的表
达。有研究显示舌下神经损伤后其运动神经元内 TC与同家族成员 RhOA、
Rac及 Cdc42相比，具有显著性差异．本实验通过建立大鼠面神经切断吻
合损伤模型，采用原位杂交方法，检测了内源性 TC omRNA在面神经核运
动神经元内的表达变化。结果首次发现K1 OInRNA在正常状态下的运动神
经元胞浆及胞体突起有微弱表达。而当面神经发生损伤后，表达显著上调。
提示：TC对面神经损伤后信号转导及再生过程中的运动神经元胞体及轴
突间架结构的重建具有重要作用。
由于BMPR－I、Smadl及TC10均为信号转导通路上的成分，三者于
面神经损伤后均出现表达升高，4W以内都维持在高表达水平；提示它们之
间可能针对面神经再生具有协同作用。
The Expression of BMP-2,6,7 and TC10 in the Facial Nerve Motoneurons during the Regeneration of Rats' Facial Nerve Injury
The Bone Morphogenetic Proteins (BMPs) family of secreted signaling molecules belongs to the transforming growth factor beta superfamily. The initail discovery of BMPs was made based on the abiliity of purified bone extracts to promote ectopic cartilage and bone formation when implanted under the skin or into the moscle of adult rats. However, it is now evident that the BMP family consists of a large number of genetically related molecules. Conserved in evolution, with key role in different periods of central nervous system. Rho families have been implicated in the regulation of cytoskeleton, until recently, some member of this family could promote the hypoglossus. No reports show whether BMPs and TC10 play important role in facial nerve injury. Therefore, our experiments have investigated the expressions of endogenous BMPs and TC10 in the facial motoneuron and their roles.
1. The experiment study about the changings and roles ofBMP-2,6,7 after facial nerve axtomy.
Previous studies were focused in the effects of BMPs on central nervous system. But to peripheral nerve, there were only localized in neuron culture Relevant information about the changing of BMP-2,6,7 after facial nerve transection were't found. We set up the facial nerve crush injury model in rat to examine these indexes. The result verified that the expression of BMP-2 decrease after injury. Postoperative 2 week, BMP-2 resumes to normal. BMP-6,7 increase gradually after axtomy, and reach maximum at 7 days post injury; then, BMP-6 resumed to normal level on postoperative 4 week. But BMP-7 was still expressed abundantly in the facial motoneurons. These results reveal that some member of BMPs family such as BMP-2,6,7 might be facial nerve potential neurotrophic factors through the change of expression to trigger facial nerve regeneration. The BMP-6 implicates in maintenance of nerve regeneration.
2. The experiment study about the changings and roles of BMPR- I and after downstream signal molecule Smad 1 facial nerve axtomy.
BMPs exert their biological effects through two types of transmembrane
receptors BMPR- I and BMPR- II ,which possess intrinsic serine/threonine kinase activity, and through downstream molecule Smadl transfer signal into the cytoplasm. We identified the expressions of BMPR- I and Smadl in facial nerve axtomy. The results show the upregulation of BMPR-1 and Smadl after injury. The peak value was sustained to 4w group. The result specified that BMPR- I and Smadl have implicated in the processes of the effects of BMP-2,6,7 on nerve regeneration.
3. The effect and expression of small GTP binding protein TClOmRNA on the facial motoneuron regeneration
TC10 belongs to the family of small GTP-binding proteins. Its functions and actions are similar to RhoA. Racl and Cdc42. The alternation between GTP-bound and GDP-bound form is thought to act as bimodal switches, and regulate many intracellular sighalling pathways. Early studies with small GTP-binding proteins play major roles in regulating remodelling of the actin cytoskeleton induced by exteacellular signals. We investigate the change of GTP binding protein TC10 in the facial motoneuron regeneration. The TC10 cDNA probe was derived from facial nucleus by RT-PCR technology. The different expressions of TC10 mRNA are observed by in situ hybridization on normal and regenertional facial motoneurons. In the normal group, the expression of TCI0 is low. The amount of the TC10 mRNA increases gradually from 1-day group following facial nerve injury. At 1-month group, it reaches maximum. TC10 may act an important role in signal transduce and cytoskeleton remodeling after nerve axotomy. Moreover, it could promote the nerve axon growth.
引文
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