【作者】 刘健；

【导师】 杨小玉；

【作者基本信息】 吉林大学 ， 外科学， 2012， 博士

【摘要】 重建神经环路是脊髓损伤修复的关键，然而由于受损伤局部内、外环境的抑制性因素影响，离断或严重损毁的轴突再生能力十分有限。研究发现：残存神经传导束可以通过自身可塑性改变发挥重建神经环路进而代偿肢体功能的作用。本论文以大鼠皮质脊髓束（Corticospinal tract ;CST）为靶向研究目标，探讨单侧CST损伤后功能训练对未损伤侧CST投射通路可塑性改变的影响及其保护中枢神经元活性的作用机制。旨在为功能训练促进脊髓损伤修复提供科学证据。本论文研究设计分为假手术组（SHAM）、术后功能训练组(Pyt)和术后未功能训练组(Pyu)，利用锥体束切断术建立单侧CST损伤模型，通过“食物小球抓取实验”（Single pellet reaching task）、“水平阶梯爬行实验”（Horizontal ladderwalking）对大鼠进行术后肢体功能训练，利用神经顺行示踪技术及行为学检查证实未损伤侧CST可塑性改变与患肢功能恢复的关系；通过免疫印迹技术分析GAP-43、Bcl-2、Bax以及Cleaved caspase-3蛋白表达变化；利用原位末端标记技术观察术后各时限大鼠脊髓内神经元细胞活性，借此进一步研究功能训练对大鼠CST可塑性改变的作用以及其对促进中枢神经损伤修复的潜在机制。结果显示：Pyt组大鼠脊髓损伤后前肢和前爪的功能（5.683±0.367）恢复明显较Pyu组（3.466±0.35）大鼠提高P＜0.05，而且长入失神经支配区的轴突数量（I：0.014476±0.000633；II：0.005726±0.000317）明显增多P＜0.05。脊髓损伤3W后大鼠的功能恢复和轴突生长的数量进入平台期。免疫印迹实验结果显示：Pyt组大鼠脊髓内GAP-43和Bcl-2蛋白（GAP-43：0.509±0.022；Bcl-2:0.506±0.023）明显较Pyu组（GAP-43：0.328±0.023;Bcl-2: 0.305±0.021）表达量增高P＜0.05；Pyt组Cleaved caspase-3蛋白(0.192±0.018)较Pyu组(0.269±0.014)表达降低P＜0.05；Pyt组Bax蛋白的表达量与Pyu组大鼠的表达量无明显差异P＞0.05。TUNEL法检测神经元细胞活性发现：Pyt组大鼠脊髓内凋亡细胞的数量(18.669±2.096)较Pyu组(26.163±2.118)明显降低P＜0.05。研究表明：通过损伤可以启动CST自身可塑性改变，而通过特殊形式的肢体训练可以促使CST的这种潜在能力得以更好的发挥，其作用机制涉及上调生长相关蛋白43（Growth-associated protein-43；GAP-43）的表达，使代偿性生长的轴突有能力延伸至失神经支配区与目标神经元细胞连接重建神经环路。同时，功能训练还可以通过上调大鼠脊髓内抗凋亡蛋白Bcl-2表达、抑制促凋亡蛋白Cleaved caspase-3表达，从而减少由损伤或失神经支配而导致的患侧脊髓灰质内神经元细胞的凋亡数量，确保与轴突连接前神经元细胞的活性。结果还表明大鼠CST损伤后的3W内是促进大鼠中枢神经可塑性改变的关键时间窗，利用这个时间窗可以为后续研究皮质脊髓束轴突侧枝发芽以及神经可塑性的新机制提供实验依据。更多还原

【Abstract】 [Objective]In this study,we established the rat unilateral CST injury model.through observedthe morphology change of uninjuryed CST collateral sprout and axon elongation tocompare the recovery of rat foreleg function in different time point.Analyse the effectof functional training to rat CST plasticity.Detection the GAP-43,Bcl-2,Bax andcaspase-3 protein expression in rat cervical enlargement.Analyse the mechanism ofthe effect of function training to rat plasticity after SCI,provided the experimentalevidence for repair for central nervous system injury.[Methods]In the present study, pyramidotomy model that resulted in rats with unilateralCST injury was used.Used“Single pellet reaching task”and“Horizontal ladder walking”grade themotor function in different time point.Used BDA trace rats CST,and observed the morphology change of CST .Countthe quantity of axon in denervation .Used immunohistochemistry and western blot method detection theBcl-2,Bax,GAP-43 and caspase-3 protein expression in uninjury spinal cord indifferent time point.Used the TUNEL method label apoptotic cells in denervation.[Result]On the basis of rat CST anatomy,used the pyramidotomy injure the unilateralCST to establish a small sector CST injury model which is more reliable andconvenience to research the central nervous system injury.In this experiment we found that within three weeks after injury, Pyt ratsforelimb and forepaw recovered obviously,which is a gradually increase trend .But therecover of Pyt rats is weak than SHAM.That suggests the function of injury rats is notrecover completely.Pyu rats have certain degree recover,but the level is worse than Pyt rats.we found that though the rats are trained after postoperative three weeks ,butthe fuunction is not further recovery,and the level is same to the three weeks timepoint.It suggests that it is a key time window within postoperative three weeks toimprove rats function.We found that the quantity of axon increase obviously within postoperative threeweeks.After three weeks ,the quantity of axon is stable.The functional Pyt ratsquantity of axon is more than Pyu rats ,P＜0.05.Density scanner analysis revealed that spinal cord of SHAM group rats contain alittle GAP-43 protein expression .There are positive expression in Pyt rats denervatespinal cord after pyramidotomy .The expression of GAP-43 protein peaked at twoweeks ,and dropped gradually.The expression of Pyt rats GAP-43 protein afterpyramidotomy four weeks are close to the SHAM group ,and the expression of Pytrats GAP-43 protein increased obviously than Pyu group rats after pyramidotomy oneweek ,two weeks and three weeks. GAP-43 positive product positioning mainlydistributed in the nuclear, the product is pale brown.There are some positive cellexpression in ventral horn of SHAM group rats spinal cord. There are positive cellexpression in Pyt rats denervate spinal cord after pyramidotomy one week.Theexpression of GAP-43 protein positive cell peaked at two weeks ,and droppedgradually.The expression of Pyt rats GAP-43 protein positive cell after pyramidotomyfour weeks are close to the SHAM group. Interestingly, the expression of Pyt ratsGAP-43 protein positive cell increased obviously than Pyu group rats afterpyramidotomy one week ,two weeks and three weeks P＜0.05.Density scanner analysis revealed that spinal cord of SHAM group rats contain alittle Bcl-2 protein expression .There are positive expression in Pyt rats denervatespinal cord after pyramidotomy one week.The expression of Bcl-2 protein droppedgradually.The expression of function training rats Bcl-2 protein after pyramidotomyfour weeks are close to the SHAM group ,and the expression of Pyt rats Bcl-2 proteinincreased obviously than Pyu group rats after pyramidotomy one week ,two weeksand three weeks.Density scanner analysis revealed that spinal cord of SHAM group rats contain alittle Bax protein expression .There are positive expression in Pyt rats denervate spinalcord after pyramidotomy one week.The expression of Bax protein droppedgradually.The expression of function training rats Bax protein after pyramidotomy four weeks are close to the SHAM group ,and the expression of Pyt rats Bax protein isclose to the Pyu group rats after pyramidotomy one week ,two weeks ,three weeks andfour weeks.Density scanner analysis revealed that spinal cord of SHAM group rats contain alittle Cleaved caspase-3 protein expression .There are positive expression in Pyt ratsdenervate spinal cord after pyramidotomy one week.The expression of Cleavedcaspase-3 protein dropped gradually.The expression of Pyt rats Cleaved caspase-3protein after pyramidotomy four weeks are close to the expression of Pyu rats afterpyramidotomy four weeks,and the expression of Pyt rats Cleaved caspase-3 proteinincreased obviously than Pyu group rats after pyramidotomy one week ,two weeksand three weeks.TUNEL labled positive cell positioning distributed in the nucleus,its boundary isclear. Experiment results show that there are only a little apoptotic cells in SHAMgroup rats spinal cord. There are positive apoptotic cell obviously expression in Pytrats after pyramidotomy one week.The Pyt group rats expression of positive cellreduced obviously than Pyu group rats, and dropped gradually.The expression of Pytrats positive cell after pyramidotomy four weeks are close to the Pyu group rats afterpyramidotomy four weeks P＞0.05.[Conclusion]1. Through the morphological observation of the lesion site of pyramidal tractand behavioral assessment of the forelimb and forepaw, it is accurate and reliable toestablish the model of CST injury of rats, by selectively abscinding the pyramidaltract.2. Pyt rats after spinal cord injury, especially some targeted strengtheningpractice, helps the change of central nervous plasticity. Meantime, we also find thatwithin 3 weeks after spinal cord injury is the key time window to improve andamplify the change of neural function plasticity, which may provide the experimentalevidence for the detailed mechanism of cortical spinal cord axon collateralgermination, extending process and neural plasticity in the future study.3. GAP-43 is found to up-regulate expression after spinal cord injury, whoseexpression quantity can be further enhanced by functional training, which willstrengthen the growth and extension of the axon, providing the experimental evidencefor the thesis that functional training may improve the change of the central nervous plasticity.4. Antiapoptotic protein Bcl-2 is found to up-regulated expression after ratcortical spinal cord injury with the help of functional training, which will inhibit theexpression of Cleaved caspase-3, a promoting apoptosis protein. At the same time,apoptosis neurons in the side of lesion from rats of Pyt group, comparing with the Pyugroup, markedly decrease in quantity, which may be one of the mechanismsunderlying that functional training is protective to the central nervous neurons afterSCI.更多还原