p62在大鼠慢性脊髓损伤中的作用机制研究
摘要
研究目的：随着科技进步和人类平均年龄的增加，中老年脊柱退变性疾病发病率越来越高，慢性脊髓损伤尤为常见，严重影响生活质量。脊柱退行性改变导致慢性机械性脊髓损伤，造成运动和感觉功能的隐匿性和持续性进展。慢性脊髓损伤严重影响患者的生活质量，给家庭和社会带来了沉重的负担。慢性脊髓损伤机制尚未阐明，慢性脊髓损伤是由于多种因素所导致的，发病机制不明，虽然有许多针对该领域的研究，但都未获得明确的病因。近年来，凋亡反应在脊髓损伤中的研究越来越多，参与慢性脊髓损伤的凋亡通路很多，包括经典的Fas信号通路途径。近年来，有研究者发现，p62与NFkB介导的凋亡通路密切相关，但未见针对慢性脊髓损伤的相关研究，p62可能在慢性脊髓损伤中发挥重要作用。因此，本研究通过建立慢性脊髓损伤动物模型，初步探索p62在慢性脊髓损伤中的作用。
研究方法:采用后路慢性压迫装置造成SD大鼠T8的脊髓慢性损伤。将36只SD大鼠分为对照组，1周、4周、8周、12周和16周模型组。首先，通过自制的后路慢性脊髓压迫装置，固定在大鼠的T7至T9水平，通过间断的螺钉拧入，连续6周每周拧入1个螺距0.3mm，造成脊髓慢性压迫，6周后停止拧入，持续观察。对出现术后并发症或神经功能急性损伤大鼠采取废弃处理，及时补充每组的模型动物数量。慢性压迫螺钉每周拧入一个螺距0.3mm，连续拧入6周，后10周采取静态观察，不再拧入。每4周进行X线检测螺钉压迫程度，通过HE染色观察大体形态变化，采用PCR和Western Blot检测p62、NFkB、LC3、caspase3在造模后不同时间点的表达量，通过免疫组化观察上述指标在不同时间点的表达变化情况，通过TUNEL检测术后不同时间点的凋亡进展。活体动物神经功能评分方面，采用BBB评分进行神经功能评价，观察术后不同时间点的大鼠神经功能变化情况。
结果：造模后复查侧位片提示慢性压迫装置在位，造成椎管内压迫。大鼠的运动和感觉功能障碍出现在造模后1周和8周时，神经功能评分降低(18.33±0.82；14.83±0.75；12.67±0.82)，与对照组相比有统计学意义(P=0.000；P=0.000；P=0.000)，提示慢性压迫造成了神经功能障碍。至造模后12周及16周神经功能BBB评分略有升高(14.83±0.75；16.0±0.82，P=0.000；P=0.000)，但与对照组相比，均有统计学意义(P<0.05)。组织学方面，造模后1周时，HE染色切片观察未见明显神经元变性以及白质的髓鞘改变；4周时出现轻度的神经元变形以及白质的脱髓鞘改变，出现少量空泡样变性；造模后8周时出现神经元固缩变性，白质空泡样改变，脱髓鞘改变。造模后12周时，出现较为明显的神经元变性，空泡样变以及空泡样变；造模后16周时，神经元广泛变性，空泡样变加重，脱髓鞘改变更加明显。TUNEL凋亡检测发现从造模后1周开始出现阳性细胞，持续表达至造模后16周，逐渐出现核固缩碎裂等典型的凋亡特征。PCR结果提示，造模后p62的表达持续增加，造模后至12周时，p62的表达持续增加，至造模后16周时，表达相对值较12周略有下降；LC3、NFkB以及caspase3结果均提示造模后1周至16周，上述基因的表达量均持续增加；WB结果提示，p62的表达量至造模后12周时表达量持续增加，12周至16周时增加不明显，LC3、NFkB以及casepase3的表达量在造模后各时间点均显著增加。
结论:大鼠的后路慢性压迫装置能够较稳定的模拟慢性脊髓损伤疾病，可重复性好，通过对慢性脊髓损伤大鼠的观察，可见大鼠慢性脊髓损伤后出现慢性脊髓病理改变过程，逐渐出现神经元变性以及脱髓鞘改变。此外，慢性脊髓损伤后在不同时间点， p62在大鼠慢性脊髓损伤后表达持续增加，参与了慢性脊髓损伤后的病理机制过程，提示p62的积聚可能与慢性脊髓损伤的发生发展有关。随着慢性脊髓损伤后p62的表达增加，相应的NFkB、LC3和caspase3的表达也相应增加，且免疫组化结果可见p62与NFkB同时在神经元胞核内表达，进一步提示p62可能参与NFkB介导的脊髓损伤后凋亡反应，在其中发挥一定的作用，但其确切机制仍需要进一步研究。
Object： With the development of technology and the increase of average age of life, theincidence of degenerative spinal disease is becoming higher and higher, especially in chronic spinalcord injury, which effects the life quality in human beings. Chronic mechanical compression of thespinal cord, which is commonly caused by degeneration of the spine, impairs motor and sensoryfunctions insidiously and progressively. Chronic spinal cord injury affects the late life quality andbrings the heavy burdens for families and countries. Yet the exact mechanisms of chronic spinal cordcompression (SCC) remain to be elucidated. Chronci spinal cord injury is caused by multi factors andthe exact mechanism is still unclear. Recently, apoptosis and autophay has been paid more attention to.There are many signaling pathways in the apoptosis process, including the classic Fas mediatedapoptosis. Meanwhile, p62has been demonstrated that it correlates with apoptosis process via NFkBsignaling pathway. To study the role of p62in this condition, we developed a simple animalexperimental model that reproduced the clinical course of mechanical compression of the spinal cord.
Methods： Posterior spinal cord compression device was used to create the rat model throughthe T8lamine.36SD rats were randomly divided into six groups including control group,1-weekgroup,4-week group,8-week group,12-week group and16-week group. A custom-designedcompression device was implanted on the exposed spinal cord of female SD rats between the T-7andT-9vertebrae. A root canal screw was tightened1complete turn (1pitch=0.3mm) every7days for6weeks. After then, the compression device was stable and rats were evaluated for consecuative tenweeks. The placement of the compression device and the degree of compression were validated everyweek using radiography. HE staining was used to assessed the spinal tissue. PCR and Western Blotwere used to qualitilize the expression of p62, NFkB, LC3and caspase3. Immunohistochemistry wasused to indentifiy the localization and expression of p62, NFkB, LC3and caspase3at different timeinterval. After1,4,8,12and16weeks, the compressed T7-9spinal cords were harvested andexamined via TUNEL. Furthermore, a BBB rating scale was also calculated at different time interval.
Results：Lateral projection of the thoracic spine showed a progressively increasing rate of meanspinal cord narrowing in the compression group. No obvious moter and sensory deficit was obvervedin control group and1-week group. BBB scores were observed from week1to week8(18.33±0.82；14.83±0.75;12.67±0.82, P=0.000; p=0.000; p=0.000). Obvious pathological changes wereobserved starting at Week12and16(14.83±0.75；16.0±0.82, p=0.000; p=0.000). Histologically, noobvious changes in both gray and white matters could be seen in control and1-week compressiongroups. At4weeks, the number of neurons in the gray matter of rats with chronic compression of thespinal cord decreased. Demyelination and vacuolus changes were seen in the white matter. At8weeks, in gray matter, the number of neurons in the gray matter progressively decreased. In the white matter,myelin destruction and loss of axons and glia were noted. The pathological examination showedneuronatrophy, increased gap around the neurons, mild demyelinated and vacuolar degeneration inexperimental group at12weeks after surgery, while more remarkably at16weeks after surgery. Thenumber of terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-endlabeling (TUNEL)–positive neurons increased from week1to week16. The PCR analysis showedthat the expression of p62continuously increased from week1to week12, while relatively lower inweek16. In addition, the expression of LC3, NFkB and caspase3increased all the time after surgery.In werstern blot, the expression of p62increased from week1to week12, while a littlbe bit lower inweek16. In addition, the expression of LC3, NFkB and caspase3increased all the time after surgery.
Conclusions： This practical model accurately reproduces characteristic features of clinicalchronic SCC, including progressive motor and sensory disturbances after a latency and insidiousneuronal loss. In addition, we found that the expression of p62increased in the chronic compressionprocess and may play an important role in the chronic spinal cord injury. It indicated that theaccumulation of p62may contribute to the development of chronic spinal cord injury. Finally, withthe increased expression of p62, the expression of NFkB, LC3and caspase3increased. In addition,Immunohistochemistry analysis showed that the expression of p62and NFkB could be seen both inthe nucleus. It indicated that p62may take part in the NFkB signaling pathway and play an importantrole. However, futher study should be carried out to clarify the exact mechanism of the chronic spinalcord injury.
研究方法:采用后路慢性压迫装置造成SD大鼠T8的脊髓慢性损伤。将36只SD大鼠分为对照组，1周、4周、8周、12周和16周模型组。首先，通过自制的后路慢性脊髓压迫装置，固定在大鼠的T7至T9水平，通过间断的螺钉拧入，连续6周每周拧入1个螺距0.3mm，造成脊髓慢性压迫，6周后停止拧入，持续观察。对出现术后并发症或神经功能急性损伤大鼠采取废弃处理，及时补充每组的模型动物数量。慢性压迫螺钉每周拧入一个螺距0.3mm，连续拧入6周，后10周采取静态观察，不再拧入。每4周进行X线检测螺钉压迫程度，通过HE染色观察大体形态变化，采用PCR和Western Blot检测p62、NFkB、LC3、caspase3在造模后不同时间点的表达量，通过免疫组化观察上述指标在不同时间点的表达变化情况，通过TUNEL检测术后不同时间点的凋亡进展。活体动物神经功能评分方面，采用BBB评分进行神经功能评价，观察术后不同时间点的大鼠神经功能变化情况。
结果：造模后复查侧位片提示慢性压迫装置在位，造成椎管内压迫。大鼠的运动和感觉功能障碍出现在造模后1周和8周时，神经功能评分降低(18.33±0.82；14.83±0.75；12.67±0.82)，与对照组相比有统计学意义(P=0.000；P=0.000；P=0.000)，提示慢性压迫造成了神经功能障碍。至造模后12周及16周神经功能BBB评分略有升高(14.83±0.75；16.0±0.82，P=0.000；P=0.000)，但与对照组相比，均有统计学意义(P<0.05)。组织学方面，造模后1周时，HE染色切片观察未见明显神经元变性以及白质的髓鞘改变；4周时出现轻度的神经元变形以及白质的脱髓鞘改变，出现少量空泡样变性；造模后8周时出现神经元固缩变性，白质空泡样改变，脱髓鞘改变。造模后12周时，出现较为明显的神经元变性，空泡样变以及空泡样变；造模后16周时，神经元广泛变性，空泡样变加重，脱髓鞘改变更加明显。TUNEL凋亡检测发现从造模后1周开始出现阳性细胞，持续表达至造模后16周，逐渐出现核固缩碎裂等典型的凋亡特征。PCR结果提示，造模后p62的表达持续增加，造模后至12周时，p62的表达持续增加，至造模后16周时，表达相对值较12周略有下降；LC3、NFkB以及caspase3结果均提示造模后1周至16周，上述基因的表达量均持续增加；WB结果提示，p62的表达量至造模后12周时表达量持续增加，12周至16周时增加不明显，LC3、NFkB以及casepase3的表达量在造模后各时间点均显著增加。
结论:大鼠的后路慢性压迫装置能够较稳定的模拟慢性脊髓损伤疾病，可重复性好，通过对慢性脊髓损伤大鼠的观察，可见大鼠慢性脊髓损伤后出现慢性脊髓病理改变过程，逐渐出现神经元变性以及脱髓鞘改变。此外，慢性脊髓损伤后在不同时间点， p62在大鼠慢性脊髓损伤后表达持续增加，参与了慢性脊髓损伤后的病理机制过程，提示p62的积聚可能与慢性脊髓损伤的发生发展有关。随着慢性脊髓损伤后p62的表达增加，相应的NFkB、LC3和caspase3的表达也相应增加，且免疫组化结果可见p62与NFkB同时在神经元胞核内表达，进一步提示p62可能参与NFkB介导的脊髓损伤后凋亡反应，在其中发挥一定的作用，但其确切机制仍需要进一步研究。
Object： With the development of technology and the increase of average age of life, theincidence of degenerative spinal disease is becoming higher and higher, especially in chronic spinalcord injury, which effects the life quality in human beings. Chronic mechanical compression of thespinal cord, which is commonly caused by degeneration of the spine, impairs motor and sensoryfunctions insidiously and progressively. Chronic spinal cord injury affects the late life quality andbrings the heavy burdens for families and countries. Yet the exact mechanisms of chronic spinal cordcompression (SCC) remain to be elucidated. Chronci spinal cord injury is caused by multi factors andthe exact mechanism is still unclear. Recently, apoptosis and autophay has been paid more attention to.There are many signaling pathways in the apoptosis process, including the classic Fas mediatedapoptosis. Meanwhile, p62has been demonstrated that it correlates with apoptosis process via NFkBsignaling pathway. To study the role of p62in this condition, we developed a simple animalexperimental model that reproduced the clinical course of mechanical compression of the spinal cord.
Methods： Posterior spinal cord compression device was used to create the rat model throughthe T8lamine.36SD rats were randomly divided into six groups including control group,1-weekgroup,4-week group,8-week group,12-week group and16-week group. A custom-designedcompression device was implanted on the exposed spinal cord of female SD rats between the T-7andT-9vertebrae. A root canal screw was tightened1complete turn (1pitch=0.3mm) every7days for6weeks. After then, the compression device was stable and rats were evaluated for consecuative tenweeks. The placement of the compression device and the degree of compression were validated everyweek using radiography. HE staining was used to assessed the spinal tissue. PCR and Western Blotwere used to qualitilize the expression of p62, NFkB, LC3and caspase3. Immunohistochemistry wasused to indentifiy the localization and expression of p62, NFkB, LC3and caspase3at different timeinterval. After1,4,8,12and16weeks, the compressed T7-9spinal cords were harvested andexamined via TUNEL. Furthermore, a BBB rating scale was also calculated at different time interval.
Results：Lateral projection of the thoracic spine showed a progressively increasing rate of meanspinal cord narrowing in the compression group. No obvious moter and sensory deficit was obvervedin control group and1-week group. BBB scores were observed from week1to week8(18.33±0.82；14.83±0.75;12.67±0.82, P=0.000; p=0.000; p=0.000). Obvious pathological changes wereobserved starting at Week12and16(14.83±0.75；16.0±0.82, p=0.000; p=0.000). Histologically, noobvious changes in both gray and white matters could be seen in control and1-week compressiongroups. At4weeks, the number of neurons in the gray matter of rats with chronic compression of thespinal cord decreased. Demyelination and vacuolus changes were seen in the white matter. At8weeks, in gray matter, the number of neurons in the gray matter progressively decreased. In the white matter,myelin destruction and loss of axons and glia were noted. The pathological examination showedneuronatrophy, increased gap around the neurons, mild demyelinated and vacuolar degeneration inexperimental group at12weeks after surgery, while more remarkably at16weeks after surgery. Thenumber of terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-endlabeling (TUNEL)–positive neurons increased from week1to week16. The PCR analysis showedthat the expression of p62continuously increased from week1to week12, while relatively lower inweek16. In addition, the expression of LC3, NFkB and caspase3increased all the time after surgery.In werstern blot, the expression of p62increased from week1to week12, while a littlbe bit lower inweek16. In addition, the expression of LC3, NFkB and caspase3increased all the time after surgery.
Conclusions： This practical model accurately reproduces characteristic features of clinicalchronic SCC, including progressive motor and sensory disturbances after a latency and insidiousneuronal loss. In addition, we found that the expression of p62increased in the chronic compressionprocess and may play an important role in the chronic spinal cord injury. It indicated that theaccumulation of p62may contribute to the development of chronic spinal cord injury. Finally, withthe increased expression of p62, the expression of NFkB, LC3and caspase3increased. In addition,Immunohistochemistry analysis showed that the expression of p62and NFkB could be seen both inthe nucleus. It indicated that p62may take part in the NFkB signaling pathway and play an importantrole. However, futher study should be carried out to clarify the exact mechanism of the chronic spinalcord injury.
引文
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