【作者】 赵建美；

【导师】 汪健；

【作者基本信息】 苏州大学 ， 儿科学（专业学位）， 2013， 博士

【摘要】 一、 β-1,4半乳糖基转移酶-I和V在坐骨神经损伤后大鼠腓肠肌中的表达及意义目的：观察β-1,4-半乳糖基转移酶I和V(β1,4galactosyltransferase I、V,β-1,4-GalT-I、V)在大鼠坐骨神经夹伤和切断后腓肠肌中的表达变化，探讨其在坐骨神经损伤后腓肠肌萎缩和再生过程中可能存在的生物学作用。方法：制备大鼠坐骨神经夹伤和切断模型；测定坐骨神经功能指数；实时荧光定量PCR检测β-1,4-GalT-I mRNA和β-1,4-GalT-V mRNA在坐骨神经夹伤和切断后不同时间腓肠肌中的表达变化；荧光原位杂交技术观察β-1,4-GalT-ImRNA和β-1,4-GalT-V mRNA的定位情况。结果：(1)坐骨神经功能指数检测发现随着夹伤时间的延长，坐骨神经功能指数逐渐升高，至夹伤4w时基本恢复至至正常水平。而坐骨神经切断后坐骨神经功能指数均为-100。(2)在坐骨神经夹伤模型中，实时荧光定量PCR结果显示β-1,4-GalT-I mRNA、β-1,4-GalT-V mRNA在正常大鼠腓肠肌中表达量较高。坐骨神经夹伤后6h开始逐渐下降，至伤后2w时达到最低水平，第4w时才恢复至相对正常水平。原位杂交结果表明：在正常情况及坐骨神经夹伤后4w时β-1,4-GalT-ImRNA、β-1,4-GalT-V mRNA定位于大量肌细胞及肌卫星细胞中。(3)在坐骨神经切断模型中，实时荧光定量PCR结果显示，β-1,4-GalT-I mRNA、β-1,4-GalT-V mRNA在伤后6h开始逐渐下降，至伤后4w仍维持在低水平。原位杂交结果表明：4w时检测到β-1,4-GalT-I mRNA、β-1,4-GalT-V mRNA定位于少许肌卫星细胞中。结论：β-1,4-GalT-I mRNA、β-1,4-GalT-V mRNA在坐骨神经夹伤和切断后不同时间腓肠肌中的表达水平不同，提示β-1,4-GalT-I mRNA、β-1,4-GalT-V mRNA的表达与坐骨神经损伤类型及损伤时间有关，且β-1,4-GalT-I mRNA、β-1,4-GalT-V mRNA主要表达于肌卫星细胞中，说明β-1,4-GalT-I mRNA、β-1,4-GalT-V mRNA在坐骨神经损伤后腓肠肌再生过程中发挥重要的作用。二、β-1,4-糖苷键在坐骨神经损伤后大鼠腓肠肌中的表达及意义目的：观察β-1,4-糖苷键（Galβ1,4-N-acetylglucosamine, Galβ-1,4-GlcNAc)在大鼠坐骨神经夹伤和切断后腓肠肌中的表达情况，探讨其在坐骨神经损伤后腓肠肌萎缩和再生过程中可能存在的生物学作用。方法：制备大鼠坐骨神经夹伤和切断模型；采用凝集素印迹分析（Lectin blot）和RCA免疫荧光染色(RCA immunofluorescent staining，RCA-I)技术观察Galβ-1,4-GlcNAc在正常及坐骨神经损伤后大鼠腓肠肌中的表达变化及定位情况。结果：(1)凝集素印迹分析表明Galβ-1,4-GlcNAc的表达在坐骨神经夹伤后6h开始逐渐下降，至伤后2w时达到最低水平，第4w时恢复至相对正常水平。与β-1,4-GalT-I和V mRNA的表达趋势相一致。(2)利用特异性结合Galβ-1,4-GlcNAc的RCA-I研究证实：在正常情况及坐骨神经夹伤后4w时Galβ-1,4-GlcNAc定位于大量肌细胞及肌卫星细胞中，而在坐骨神经夹伤后2w以及坐骨神经切断后2w和4w时可检测到Galβ-1,4-GlcNAc定位于少许肌卫星细胞中。结论： Galβ-1,4-GlcNAc在坐骨神经夹伤和切断后不同时间腓肠肌中的表达水平不同，提示Galβ-1,4-GlcNAc表达与坐骨神经损伤类型及损伤时间有关，且Galβ-1,4-GlcNAc主要表达于肌卫星细胞中，说明Galβ-1,4-GlcNAc在坐骨神经损伤后腓肠肌再生过程中发挥着重要的作用。三、Kv4.2与PSD-95在慢性坐骨神经损伤后大鼠脊髓及坐骨神经的表达及意义目的：探讨电压门控型钾通道4.2(Voltage gated potassium channel4.2, Kv4.2)与突触后致密区蛋白95（Post-synaptic density protein-95, PSD-95）在慢性坐骨神经损伤后大鼠脊髓及坐骨神经的表达变化及在脊髓中的共定位情况，观察是否参与了疼痛传导过程。方法：制备大鼠慢性坐骨神经压迫性损伤模型（Chronic constriction injury, CCI），通过逆转录聚合酶链反应（Reverse transcription polymerase chain reaction,RT-PCR)及免疫印迹（Western Blotting）等方法分别测定Kv4.2与PSD-95在坐骨神经及脊髓中的mRNA和蛋白水平的表达变化，采用免疫荧光双标技术探讨Kv4.2和PSD-95与胶质细胞及神经元在脊髓中的共定位情况。结果：(1) Kv4.2mRNA在正常坐骨神经组织中度表达，坐骨神经损伤后1d到5d下降，随后升高，7d达到峰值；蛋白水平未检测出。(2) Kv4.2mRNA在正常脊髓中高度表达，坐骨神经损伤后1d到5d下降，7d后增高，但总体水平较正常水平低；蛋白水平在正常脊髓中度表达，在损伤后3d降到最低点，5d开始缓慢上调，持续到14d仍显著增加。(3) PSD-95mRNA在正常坐骨神经中度表达，损伤后5d显著上调持续到10d，14d有下降趋势，但仍比正常水平高；蛋白水平在损伤后1d到3d时明显降低，而在损伤后5d时明显增加，一直持续到14d。(4) PSD-95mRNA在正常脊髓中高度表达，坐骨神经损伤后缓慢下降，损伤后7d开始上调，10d达到高点；蛋白水平在损伤后显著下调，10d后开始上调，并且变化趋势基本与Kv4.2相似。(5)在腰脊髓切片中，荧光免疫双标发现Kv4.2与PSD-95共定位于神经元，并且在损伤后集中在脊髓背侧角区域，与疼痛相关，而与星型胶质细胞无共定位。结论：Kv4.2、PSD-95在慢性坐骨神经损伤后不同时间坐骨神经及脊髓中的表达水平不同，提示Kv4.2、PSD-95的表达与坐骨神经损伤时间有关，且Kv4.2与PSD-95共定位于神经元，并且在损伤后集中在脊髓背侧角区域，与疼痛相关，而与星型胶质细胞无共定位，说明慢性坐骨神经损伤后，坐骨神经本身及其上游脊髓中的Kv4.2及PSD-95表达水平明显改变，二者共同参与了慢性疼痛过程，并且与感受伤害性刺激神经元兴奋性有关。更多还原

【Abstract】 1. The expression and significance of β-1,4-GalT I, V ingastrocnemius muscles after sciatic nerve injuryObjective: To observe the expression of β-1,4-Galactosyltransferase I and V in ratgastrocnemius muscles after sciatic nerve crush and transection, as well as toexplore their potential biological roles during the atrophy and regeneration ofgastrocnemius muscles.Methods: Sprague-Dawley (SD) rat sciatic nerve crush and transection injury modelswere established. Sciatic nerve funtion index was detected. Gene expression of β-1,4-Galactosyltransferase I and V were detected in gastrocnemius muscles aftersciatic nerve crush and transection by Real-time fluorescence quantitative PCR aswell as in situ hybridization.Results:(1) Sciatic nerve function index assay showed that the higher sciatic nerve functionindex, the longer time, and recovered to the normal level at4weeks after sciaticnerve crush. The sciatic nerve function index keeps with-100after sciatic nervetransection.(2) In the sciatic nerve crush model, real time PCR analysis revealed that β1,4-GalT Iand V mRNAs expressed at a high level in normal gastrocnemius muscles anddecreased gradually from6hours, reached the lowest level at2weeks, thenrestored gradually to relatively normal level at4weeks after sciatic nerve crush.In situ hybridization indicated that β-1,4-GalT I and V mRNAs localized innumerous myocytes and muscle satellite cells under normal conditions and at4weeks after sciatic nerve crush.(3) In the sciatic nerve transection model, β-1,4-GalT I and V mRNAs decreasedgradually from6hours, and remained on a low level at4weeks in gastrocnemiusmuscles after sciatic nerve transection. In situ hybridization indicated that β- 1,4-GalT I and V mRNAs localized in numerous myocytes and muscle satellitecells under normal conditions and in a few muscle satellite cells at4weeks aftersciatic nerve transection.Conclusions: The expression of β-1,4-GalT I and V mRNAs are different in ratgastrocnemius muscles in different time after sciatic nerve crush and transection,which indicate that the expression of β-1,4-GalT I and V mRNAs are associatedwith the type and time of sciatic nerve injury. They localize mainly in musclesatellite cells. The expression of β-1,4-GalT I and V mRNAs are involved in theprocess of denervation and reinnervation, which suggests that β-1,4-GalT I andV mRNAs may play an important role in the muscle regeneration. 2. The expression and significance of Galβ-1,4-GlcNAcgroup in gastrocnemius muscles after sciatic nerve injuryObjective: To observe the expression and significance of Galβ-1,4-GlcNAc group inrat gastrocnemius muscles after sciatic nerve crush and transection, as well as toexplore its potential biological role during the atrophy and regeneration ofgastrocnemius muscles.Methods: Sprague-Dawley (SD) rat sciatic nerve crush and transection injury modelswere established. Lectin blot and Lectin-fluorescent staining with RCA-I, wereperformed to investigate the expression and significance of Galβ-1,4-GlcNAcgroup in gastrocnemius muscles after sciatic nerve injury.Results:(1) Lectin blot analysis showed that the expression level of the Galβ-1,4-GlcNAcgroup decreased from6hours, reached the lowest level at2weeks, and restoredto relatively normal level at4weeks after sciatic nerve crush, which wasconsistent with the expression of β1,4-GalT I and V mRNAs.(2) RCA-I lectin histochemistry analysis demonstrated that Galβ1–4GlcNAc grouplocalized in numerous membranes of myocytes and muscle satellite cells innormal and at4weeks after sciatic nerve crush, and in a few muscle satellitecells at2and4weeks after sciatic nerve transection.Conclusions: The expression level of the Galβ-1,4-GlcNAc group is different in ratgastrocnemius muscles in different time after sciatic nerve crush andtransection, which indicates that the expression of Galβ-1,4-GlcNAc group isassociated with the type and time of sciatic nerve injury. Galβ-1,4-GlcNAcgroup localizes in muscle satellite cells. The expression of Galβ-1,4-GlcNAcgroup is involved in the process of denervation and reinnervation, whichsuggests that Galβ-1,4-GlcNAc group may play an important role in the muscleregeneration. 3. The expression and significance of Kv4.2and PSD-95insciatic nerve and spinal cord during CCIObjective: To investigate the expression of the voltage gated potassium channel4.2（Kv4.2）and post-synapic density protein (PSD-95) in sciatic nerve and spinalcord during the process of chronic constriction injury(CCI) of the rat sciaticnerve. Then, observe the colocalization of Kv4.2and PSD-95in spinal cordduring this process.Methods: Sprague-Dawley (SD) rat chronic constriction injury(CCI) models wereestablished. Reverse transcription PCR and Western Blotting were used toinvestigate the changes of Kv4.2and PSD-95at mRNA and the protein levelsrespectively. The colocalization of Kv4.2with PSD-95was investigated byimmunofluorescence double staining.Results:(1) The expression of Kv4.2mRNA in sciatic nerve was moderate, decreased from1d to5d after injury and peaked at7d; And protein was not detected.(2) The expression of Kv4.2mRNA in spinal cord was high, decreased from1d to5d after injury and increased at7d, but the overall level was low; The proteinlevel was moderate in spinal cords, decreased to the lowest point at3d afterinjury, increased slowly at5d, increased significantly at14d.(3) The expression of PSD-95mRNA in sciatic nerve was moderate, increased from5d to10d, decreased at14d, but was still higher than normal; The protein leveldecreased from1d to3d after injury, increased significantly at5d, up to14d.(4) The expression of PSD-95mRNA in spinal cord was high, decreased after injury,increased from7d, reached a high point at10d; The protein level decreasedafter injury, increased significantly at10d, which was similar to Kv4.2.(5) In the lumbar spinal cord slices, immunofluorescence double staining resultsshowed that PSD-95colocalized with Kv4.2.They both colocalized with neuronin spinal dorsal horn, but not with astrocytes, which were involved in paintransmission.Conclusion: The expressions of Kv4.2and PSD-95mRNAs are different in ratsciatic nerve and spinal cord in different time after chronic constriction injury, which indicates that the expressions of Kv4.2and PSD-95mRNAs areassociated with the time of chronic constriction injury and expression sites.Kv4.2and PSD-95both colocalized with neuron in spinal dorsal horn, but notwith astrocytes, which were involved in pain transmission. All these suggest thatthe expressions of Kv4.2and PSD-95in the sciatic nerve itself and its upstreamcentral nervous system spinal cord change significantly; they may participate inthe process of chronic pain and relate to the neuronal excitability.更多还原