【作者】 肖卫东；

【导师】 陈安民；

【作者基本信息】 华中科技大学 ， 外科学， 2007， 博士

【摘要】 第一部分神经生长导向因子Slit2在成年大鼠急性脊髓损伤后的表达和定位目的:观察轴突生长导向因子Slit2在成年大鼠急性外伤性脊髓损伤(traumatic spinal cord injury,SCI)后不同时间段表达量的变化和分布情况,探讨Slit2对成年大鼠SCI后轴突再生性修复中导向性延伸的可能机制和意义。方法:取成年SD大鼠72只,体重200～250克,雌雄不限,采用Allen’s打击法制作SCI模型,分别于损伤后第2、4、7、14d剖取损伤脊髓组织,应用半定量RT-PCR方法分析Slit2 mRNA表达水平变化,免疫组织化学法观察Slit2蛋白在损伤脊髓区的分布情况,采用免疫荧光染色结合激光共聚焦扫描观察Slit2的表达和星形胶质细胞增生之间的关系。结果:成年大鼠SCI后随着星形胶质细胞的增生,Slit2 mRNA在脊髓损伤区的表达呈明显升高趋势(P<0.05)并于脊髓损伤后第7天达到峰值,光密度相对比值达到0.725±0.005,之后则逐步下降; Slit2蛋白阳性信号先后出现在急性SCI区灰质区星形胶质细胞胞质中及神经元胞膜上,以前、后角及中央管周围较为聚集;免疫荧光组织化学结合激光共聚焦扫描观察则发现随着星形胶质细胞的增生,损伤部位灰质内出现星形胶质细胞标记物GFAP和Slit2在中线区域的共聚焦表达现象,提示Slit2由中线星形胶质细胞分泌并被运输到上述部位发挥作用。结论:成年大鼠SCI后随着星形胶质细胞的增生,脊髓损伤区内有Slit2表达一过性上调现象出现并在损伤脊髓灰质内广泛分布,可能对SCI后的轴索再生性修复过程中生长锥导向性延伸起到关键性的推斥性引导作用。第二部分神经生长导向因子Netrin-1和Slit2在成年大鼠脊髓损伤后的共表达目的:观察成年SD大鼠急性脊髓损伤后不同时间段Netrin-1和Slit2的共表达及分布情况,探讨再生轴突所处微环境中吸引性和排斥性的导向线索相互整合并引导生长锥正确延伸的机制。方法:成年SD大鼠40只随机分为SCI2,4,7,14d组和对照组共5组,每组8只,SCI组制作Allen’s脊髓打击模型按时序取材,免疫荧光激光共聚焦扫描Netrin-1和Slit2蛋白在SCI后损伤脊髓局部的表达及定位情况。结果:SCI后2d,大鼠脊髓挫伤部位即出现轴突生长吸引性因子Netrin-1和排斥性因子Slit2的表达同步上调现象;而后二者表达迅速增强并于SCI后7d达到峰值,在前脚、中央管和后脚三个区域Netrin-1及Slit2荧光强度分别达到83.66±4.15/75.12±6.54、78.27±4.47/72.67±4.65和86.96±2.35/64.47±5.21,且Netrin-1和Slit2在轴突胞膜上有共同表达现象,使代表Netrin-1的绿色荧光和代表Slit2的红色荧光在轴突胞膜上产生融合而发出橙色荧光;SCI后同一时点损伤脊髓各部位Netrin荧光强度无明显差异,表达较均匀(P>0.05),而Slit2前角和中央管荧光强度无差异(P>0.05),但均强于后角(P<0.05);峰值过后二者又缓步下调,但Slit2下调幅度明显较Netrin-1大,SCI后14d时Slit2荧光强度脊髓三个视野中分别下调至42.33±5.64、43.77±4.35和33.43±4.65,而Netrin-1荧光强度则仅轻度下调,分别为79.29±3.68、70.77±3.64和74.28±4.35。结论:成年大鼠SCI后脊髓中枢存在吸引性导向因子Netrin-1和排斥因子Slit2的同步表达升高以及在轴突胞膜上共同表达现象,提示二者均能和轴突胞膜上各自特异性受体结合,这可能是SCI后星形胶质细胞反应性增生的一种表现。SCI后Netrin-1的表达比较均匀而Slit2的表达在受损灰质不同部位不同时间有所差异,这种吸引性和排斥性导向信号在再生脊髓前、后脚和中央管区域的轴突胞膜上以及不同时间段的整合变化可能对再生轴突的生长锥延伸方向起到关键性引导作用。第三部分成年大鼠急性脊髓损伤后轴突再生性修复过程中Rho GTPases的失衡性表达对轴突生长锥延伸的抑制作用目的:观察成年大鼠SCI(Spinal cord injury)后脊髓损伤区域中轴突内小G蛋白Rho族GTP酶中三个主要的蛋白质分子Rac1、Cdc42和RhoA的表达变化及Rho-Rho激酶(Rho associated kinase,ROK)下游作用底物肌球蛋白轻链(Myosin light chain ,MLC)的过度磷酸化变化情况,探讨成年大鼠SCI后轴突再生过程中生长锥易于萎陷的机制。方法:成年SD大鼠36只随机分为SCI4,7,14,21d组,对照组和假手术组共6组,每组6只,SCI组制作Allen’s脊髓打击模型并按时序取材, Western印迹检测Rac1、Cdc42和Rho A的表达变化以及MLC磷酸化程度变化, GST Pull down assay检测RhoA活化程度变化。结果:成年大鼠SCI后,脊髓挫伤部位Rac1和Cdc42蛋白的表达逐步升高(P<0.05),并在第7天达到峰值,之后二者则呈现下降趋势,至SCI后三周时表达基本回归初始水平(P>0.05);与之形成对比的是RhoA总蛋白的表达则无明显变化(P>0.05),但是Rho的下游信号通道,RhoA-ROK通路的作用底物肌球蛋白轻链MLC的磷酸化水平却逐步升高(P<0.05);另一方面,Pull down assay-RhoA活性测定则显示RhoA的活性形式GTP-RhoA在SCI后的表达呈逐步增高趋势(P<0.05),而且这种升高在SCI后三周时并无衰减。结论:成年大鼠SCI后3周时,在脊髓损伤区域的神经元轴突内出现了三个主要的Rho族GTP酶:Rac1、Cdc42和RhoA的表达失衡现象,从Rac1和Cdc42的表达占优势转变为GTP-RhoA的表达直线上升并失去控制,这种情况的出现可能是损伤区域的外周微环境中诸多吸引性和排斥性信号表达相互整合,相互制约机制出现失衡、失控的直接后果; RhoA活性持续异常的升高可以导致肌球蛋白轻链(myosin light chain,MLC)磷酸化程度的持续异常升高即过渡磷酸化,而这种MLC的过渡磷酸化可以导致再生轴突细胞骨架钙敏化(Ca2+ sensitization),即对钙离子的敏感性增高,刺激肌动-肌球蛋白的收缩性,进而使成年SD大鼠损伤脊髓的再生轴突生长锥发生萎陷和神经突起回缩。第四部分Rho-ROK通路的抑制对体外模拟脊髓缺血及再灌注诱导的神经元损伤中的保护作用目的:观察Rho-ROK通路抑制剂Y-27632对体外培养的神经元细胞模拟SCI后脊髓缺血及再灌注损伤过程中细胞骨架的保护作用,探讨成立哺乳动物SCI后轴突再生过程中Rho/Rho激酶的过渡激活对于生长锥易于萎陷的可能机制。方法:体外培养神经母细胞瘤细胞株N2a细胞,分为对照组、模拟缺血组和干预Ⅰ,Ⅱ组:将N2a神经元细胞置于5%CO2,95%N2的37℃培养箱中培养120min以模拟脊髓SCI后在所处的缺血缺氧环境中的变化和反应,干预Ⅰ组预先加入Rho激酶抑制剂Y-27632对在缺氧环境中培养的N2a神经元作用,干预Ⅱ组则在缺氧培养120min后再加入Y-27632对缺血再灌注中的神经细胞起作用,然后用FITC标记的鬼笔毒环肽染色神经细胞的纤维状肌动蛋白(F-actin)骨架,采用免疫荧光技术观察其重组及变构的变化;同时我们采用MTT细胞增殖实验分别检测用不同浓度Y-27632干预后及干预Ⅰ,Ⅱ组的细胞活力。结果:正常培养的N2a细胞生长良好,轴突、树突形态清晰,胞体比较大,有明显的深色的细胞核,胞浆区较为透亮,免疫荧光下可见纤维状肌动蛋白丝主要分布于细胞周边,成丝带状,应力纤维少;而经过缺氧培养后,可见细胞骨架发生重组变化,应力纤维明显增多,同时出现周边肌动蛋白丝带模糊,轴突回缩现象;然而预先加入Y-27632孵育后再经缺氧培养的神经细胞则无明显的重组变化,轴突回缩不明显,周边肌动蛋白丝带依然清晰,胞浆内应力纤维也较少;而缺氧培养后再加入Y-27632孵育也可明显逆转这一过程,使已经发生轴突回缩的神经细胞胞膜上重新出现轴突并生长;MTT细胞增殖实验则显示Y-27632能显著提高N2a细胞模拟缺血和缺血再灌注24h后的存活率(P<0.05),而且其保护效率和浓度在一定范围内成正比。结论:采用Rho激酶抑制剂Y-27632对在体外培养的神经细胞模拟SCI后的缺血缺氧及再灌注损伤过程进行干预,可以明显抑制缺血缺氧及再灌注损伤所导致的神经元轴突内肌动蛋白细胞骨架的塌陷和回缩,从而可以防止甚至进而逆转生长锥的萎陷及神经突起的回缩,即Y-27632对神经损伤具有保护作用,而且这种保护作用主要是通过对Rho激酶(Rho association kinase,ROK)作用,对Rho-ROK通路进行抑制而实现的。更多还原

【Abstract】 PartⅠ: Slit2 Expression Variety And Distribution In Adult SD Rats During Acute Phase of Spinal Cord InjuryObjective:To observe the Slit-2 expression distribution and expressive variety during SCI of the rats,then investigate the molecular biological reason of the growth cone guidance.Methods :Make the model of SCI with Allen’s method, 72 adult SD rats were randomly divided into SCI 2, 4, 7, 14d groups, sham operation group and normal control group. After the spinal cords of rat were injured, hemi-quantitative Reverse transcription polymerase chain reaction was applied to detect the expressive variety of Slit2 mRNA. On the other hand, Immunocytochemistry was done to detect the cell location of Slit2 protein expression, immunofluorescence was done to investigate the relation of Slit2 protein expression hoist and the reactive astrocytes hyperplasiaResults :There was little detectable Slit2 mRNA expression in normal control group and sham operation group spinal cord tissue samples. However,the mRNA expression of Slit2 had began to be upregulated just 2 days after spinal cord injury(on day 2) of the experimental rats(p<0.05), and it increased on day 2 to day 4 . The expression of Slit2 mRNA reached to a peak level on day 7 after SCI, and the signal intensity for Slit2 mRNA decreased from day 7 to day 14; On the other hand,immunohistochemistry showed that there was little detectable expression of Slit2 protein in normal control group tissue. However, Slit2 was positive in the spinal cord gray astrocytes of all SCI rats, microscope demonstrated many secretive brown-yellow color granules with a high density in cells. The irregular granules with a high density were Slit2, they appeared in the astrocytes of spinal cord gray on day 2 of SCI, the Slit2 protein was expressed in the astrocytes and neurocytes cytoplasm on day 4. The expression of Slit2 protein distributed diffusely in the spinal cord gray of SCI on day 7, but the granules density of central canal and anterior motor column was significantly higher than the other region. Our study showed that on day 7, the expression of Slit2 protein of SCI group had a 28.92% higher level than control tissue(x2=0.019,P<0.05 ); The Slit2 immunofluorescence positive signal also appeared at the extensive area of spinal cord gray, located in the astrocyte.Conclusion:This study demonstrates that Slit2 mRNA was expressed in the damaged local and vicinity part of gray matter, Slit2 proteins were excreted from astrocytes and transported to plasma membrane of growth cone of the regenerative axon, it is likely to do something importantly with axon regeneration after SCIPartⅡ: Expression Variety and Distribution of Netrin-1 and Slit2 In Adult SD Rats During Acute Phase of Spinal Cord Injury through immunofluorescence techniques by Confocal Laser MicroscopyObjective:To observe the Netrin and Slit expression distribution and expressive variety during posttraumatic acute phase of SCI and investigate the molecular biological mechanism of the regenerative growth cone guidance.Methods:40 SD rats were randomly divided into 5 groups( SCI2, 4, 7, 14d groups and one normal control group),the model of SCI was made by Allen’s method. The expression and distribution of Netrin-1 and Slit2 were detected through immunofluorescence techniques, the co-expression of then were observed by confocal laser microscopy.Results:significant expressions of Netrin and Slit were showed at injured location at 2nd day post SC(IP<0.05), extensive green fluorescence and manipulus scattered red fluorescence were showed at 3 fields microscopically;on day 4, the expressions of Netrin and Slit were intensified significantly(P<0.05), the former expressed more extensively with greater amplitude in contrast with the latter, the co-expression on the cell membrane of neuron was obvious;on day 7, Netrin persistently high-expressed, Slit peaked its expression, the co-focalization of Netrin and Slit was presented microscopically, but a circle orange fluorescence was emerged from the cell membrane of neuron. Slit expression was significantly intensified at spinal cord posterior horn;both’s expressions were weakened after 2 weeks, but the down-regulation of Slit was more significant compared with Netrin. hinted the expressions of Netrin and Slit post SCI was time-dependent;Netrin fluorescence intensity had no significant variation at same time of spinal cord damage at different regions(P>0.05),but the fluorescence of Slit anterior angle was stronger than that of central canal and posterior horn(P<0.05).Conclusion:High level expressions and co-expression on plasma membrane of Netrin and Slit indicate that they may play some important roles in the progression of axon regeneration. PartⅢ: Expression Unbalance of The Rho GTPases: Rac,Cdc42 and Rho In Adult SD Rats During Acute Phase of Spinal Cord Injury and Growth Inhibition of Regenerative AxonObjective:Traumatic spinal cord injury(TSCI) is characterized by a progressive cell loss and a lack of axonal regeneration. In the central nervous system(CNS), the Rho GTPases: Rac, Cdc42 and RhoA are considered the molecule switch of the F-actin cytoskeleton recombination, having affinity with axon retractation.To observe the expression of the Rho-GTPases Rac,Cdc42 , Rho and the phosphorylation levels of myosin light chain(MLC) during posttraumatic acute phase of SCI ,we attempt to investigate the mechanism of the growth cones collapse of regenerative axon.Methods:36 SD adult rats were randomly divided into 6 groups( SCI4, 7, 14, 21d groups ,sham operation group and control group),the model of SCI was made by Allen’s method. The expressive variety of the Rho GTPases: Rac1,Cdc42, RhoA protein and the phosphorylation levels of MLC were detected through Western Blotting technique.On the other hand, GST Pull down assay was used to determine the level of GTP-RhoA.Results:The expressions of Rac1 and Cdc42 were manifestly increased at 4 day post SCI(P<0.05)and peaked at 7 day followed by a rapid decrease, both expressions fundamentally regressed to the initial levels at 3 weeks; On the contrast,RhoA total protein expression had no apparent variations post SCI, which compares unfavorably with Rac1 and Cdc42,RhoA expression levels had merely tenuous elevations compared with sham-operated groups at 4,7,14,21d post SCI, but with no statistical significance(P>0.05).On the other hand, Damaged spinal cord MLC phosphorylated level had a gradually elevated tendency with a time-lapse mode compared with sham-operated groups(P<0.05),nonetheless, no apprarant changes were detected in the expressions of myosin B chain total protein(P>0.05),and GST Pull down assay displayed in vivo regenerating axon GTP-RhoA expression had a gradually elevated tendency with a time-lapse mode post SCI(P<0.05).Conclusion:The expression unbalance of the GTPases Rac,Cdc42 and Rho may be important for the collapse of the growth cones of regenertative axon. we confirmed that the activation of Rho had increased notwithstanding its total protein had no apparent changes to switch on Rho-ROK pathway. Rho GTPases activations are regulated by in vitro cellular targeting signalling, on these grounds, we consider that Rho over activation in vivo regenerating axon post SCI plays a negative role for growth cone elongation, especially as the attractive signaling is attenuated at 3 weeks post SCI, we presume that the episode of disequilibrium was occurred in the attractive and repellent signals of SCI microenvironment at 3 weeks post SCI in rats ,this sort of disequilibrium is likely induced by astrocyte persistent hyperplasy, which directly leading to unbalanced expressions among in vivo regenerating axons Rac,Cdc42 and Rho, resulting in uncontrollable enhanced Rho activation followed by GTP-Rho activating ROK,after ROK phosphorylation Ca2+ non-dependent myosin B chain phosphatase myosin binding with subunit to inactivate it, thus cut down the dephosphorylation function on phosphorylational, causing MLC over-phosphorylation, MLC over-phosphorylation is likely one of the chief causes leading to growth cone ultimate detelectasis, which make albumen cytoskeleton calcium–sensitized, accordingly contributing on the polymerization and depolymerization of actin-globulin, ultimately leading to growth cone detelectasis,axon recovery. PartⅣ: Protection Effect of The Inhibition of Rho Association Kinase During N2a cell Mimic Ischemia and Ischemia Reperfusion Injury in VitroObjective:To observe the reorganization of F-actin of neuron during mimic ischemia and ischemia reperfusion injury in vitro,we attempt to investigate the mechanism of the growth cones collapse of regenerative axon and the protection effect of the inhibition of Rho-ROK.Methods:After N2a cells induced by ischemia and ischemia-reperfusion were treated with different dilute Y-27632, a specific inhibitor of Rho association kinase, cell damage was analyzed by cell proliferation assay(MTT assay); on the other hand , N2a cells were then prepared for routine scanning observation through Immunofluorescence techniques by Confocal Laser Microscopy which stained with Fitc-phalloidin for F-actin visualization.Results:Ischemia induced a striking reorganization of actin cytoskeleton with a weakening of fluorescent intensity of the peripheral filament actin bands and formation of the long and thick stress fibers, lamellipodia and filopodia, but Pretreatment of Y-27632 could reversed the changes of ultrasturcture on the cellular surface, the stress fibers were diminished by pretreatment of it, and the protection of Y-27632 has related with its concentration in determinate bound. MTT assay show that Y-27632 could prolong the survival time of the N2a cells after mimic ischemia-reperfusion for 24h.Conclusion:The activation of Rho had a exceptional hoist after SCI. The activation of Rho association kinase(ROK) is likely to induce the collapse of the growth cone . Suppression of Rho association kinase activity could promote axonal growth on inhibitory spinal cord through ischemia and ischemia-reperfusion.更多还原