实验性大鼠急性脊髓损伤NF-κB表达及八肽胆囊收缩素抗炎作用分子机制研究
摘要
急性脊髓损伤(Acute spinal cord injury，ASCI)后炎症反应是脊髓继发性损伤的主要因素并在急性和慢性损伤过程中发挥重要作用。核因子-кB(nuclear factor kappa B,NF-кB)是众多细胞因子和炎症介质表达的主要转录因子，其活化可使肿瘤坏死因子(Tumor necrosis factor-alpha，TNF-a)，白介素-1(Interleukin-1beta，IL-1)等促炎因子基因过度表达。但是在脊髓继发性损伤中NF-кB及其NF-кB的抑制蛋白(Inhibitory subunit of NF-кB，IкB)表达变化规律、特别是IкB在脊髓细胞中的表达是否可作为NF-кB变化的观察指标尚不清楚。此外，胶质原纤维酸性蛋白(Glial fibrillary acidicprotein，GFAP)在ASCI时的变化规律与NF-кB的相互关系也有待于阐明。为此，我们采用了大鼠脊髓挫伤模型，研究ASCI后与急性炎症反应有关的病理改变，从整体水平、器官组织水平、蛋白质水平等不同层面观察NF-кB、IкB、GFAP等细胞因子表达的变化，同时对以硫酸酯化八肽活性片段存在的外源性胆囊收缩素(cholecystokinin-8，CCK-8)在急性创伤性脊髓损伤中对NF-кB、IкB、GFAP表达作用加以评价，为寻求减轻继发性脊髓损伤的新抗炎药物提供理论依据。
1、急性脊髓损伤时NF-кB在继发性损伤中变化规律的研究
本研究目的在于研究NF-кB在大鼠ASCI时细胞胞浆内及胞核内的表达变化规律。SD大鼠90只，雌雄不限，体重200～250g。在1％戊巴比妥钠腹腔麻醉下(40mg／kg)，腹卧于动物立体定向架上。所有动物先行后正中入路T_(8-9)椎板切除，72只动物使用OHBA SIKI打击器(Tokyo，Japan)按Gruner改良法作成T_9脊髓背侧损伤模型，打击强度为25势能克厘米(Vgram-cm force，gcf)为损伤组，其余18只动物为对照组。所有动物分别在伤后1h、6h、12h、24h、72h、5d不同时间点处死。除对组织标本进行病理观察外，应用免疫组化、电泳迁移率变
中 文摘 要
动分析法mlectrophoretic Mobility shift Assay EMsA）及免疫印记法
（Western blot analysis）观察脊髓胞核与胞浆内 w4 B的在各个时相
点的表达。
损伤后lh镜下可见脊髓挫伤处灰质内血管破裂出血，红细胞及
血浆积聚在中央灰质、后角的血管周围，神经细胞肿胀；损伤后6h
出血波及整个灰质，神经细胞、星形细胞明显肿胀，神经细胞核周围
尼氏小体变浅；损伤后 12h，在脊髓灰质中央，依然可见片状出血，
在挫伤灶的边缘可以发现神经元细胞核固缩呈坏死样改变，并有水肿
的小胶质细胞及多形核细胞和巨噬细胞的组织浸润，组织间隙明显增
宽。损伤后24h到72h，在挫伤灶中心，脊髓结构紊乱，有大片灰质
坏死区及大量的巨噬细胞和多形核细胞浸润，在挫伤灶的灰白质交接
处有小出血点，白质水肿加重，白质内有空泡形成，以损伤后72h为
重；损伤后sd，在灰、白质坏死区可观察到泡沫样巨噬细胞、多形核
细胞；在正常组织与挫伤灶交界处，可以观察到血管内血细胞淤积，
血管壁出现白细胞浸润，白质内有空腔形成。
免疫组化结果以NFJB胞浆及细胞核表达的吸光度及阳性面积
作为相对表达量，用均数士标准差（王士S）表示、在检验进行统计学
处理。对照组仅有较弱的阳性反应①0．l士 3．l，368．30士门 ．65人 损
伤后 lh表达开始增强（112．2士4．2，387．41士13 ＊5），P＜0＊1，NF－K B＊
性细胞在病灶内和边缘区可以见到；损伤后 6h增强明显门 25＊士 2．5，
402＊2士14．57），p＜0＊of；损伤后12h 达高峰（175．3士2＊，478．23士
14．45），p＜0刀of，在神经细胞、小胶质细胞、巨噬细胞、多形核细胞、
星形细胞胞浆内均有 NF4 B表达；损伤后 24h表达开始下降门 石士
2．3，440．32士14．84），p＜0．001；sd逐步恢复正常p．5士2．8，379．65士
13＊5），p＞0＊5。细胞核内的 N’F－K B在损伤后 lh（8 ＊上2．l，210．00士
14．40）轻度增强 p＞0＊5；6h后逐步增强（94＊士2．3，233．46士门．45），
p＜0＊1；12h达到高峰（146．0土3．I，298＊4士15．46），p＜0＊of；24h后逐
渐下降（90．0士 2．4，265．13士 16．48），p＜0．00；sd基本恢复正常（61．0士
2．8，205＊3土 15．46），p＞0＊5。
应用 Western blot analysis从蛋白水平测定挫伤脊髓e4 B的胞
浆内表达。抗P65抗血清显示65KDA的明显分子条带吸光度作为相
2
中 文摘 要
对表达量，用均数土标准差G士S）表示、t检验进行统计学处理。对
照组显示一个较弱的表达条带O 刀士2．2），而损伤后 lh NF4B表达
开始增高（09．0士 2．＞ p＜0．of；损伤后 6h增高己很明显OIZ．0士 l．7），
p＜0＊of；损伤后 12h 达高峰门＊ ＊士 1．7），p＜0＊01；持续到损伤后 24h
才开始下降（124＊士2二），p＜0＊of； sd后逐步恢复正常（103＊土1．9），
p＞0刀5。
应用 EMSA检测 N’’F＊ B在细胞核内的表达情况?
Inflammatory responses are a major component of secondary injury and play a central role in mediating the pathogenesis of acute and chronic spinal cord injury (SCI). The nuclear factor-kappa B (NF- K B) is a primary transcription factor for the transcription activation of a variety of cytokinin and genes regulating inflammatory. The activation of NF- K B made inflammation genes over expression including tumor necrosis factor-alpha (TNF-a) and Interleukin-lbeta(IL-l). But the expression of NF-KB and inhibitory subunit of NF- K. B(I K B) in SCI is not very clear, exespecially the expression of I K B in spinal cells .Glial fibrillary acidic protein (GFAP) is an important role of SCI .The increase of the number and expression of GFAP-positive cells was correlated to the inflammatory responses and functional recovery of the spinal cord. We used a contusion model in rats to initiate the early biochemical and molecular changes with acute inflammation after SCI and examined the expression of activated NF- K BN I K B
and GFAP with immunohistochemistry western blot analyses and electrophoretic mobility shift assays (EMS A). In order to search the new medicine of relieving inflammatory responses we injected different dose of CCK-8 (Cholecystokinin-octapeptide) to rats before injury and examined the expression of NF- K B^ I K B, GFAP.
1 Expression of activated NF- K B after acute traumatic SCI.
The purpose is observe the expression of NF- K B at cytochylema and nucleolus of SD rat in ASCI. All the animal were divided into control group(l 8) and injury group(72). 1% nembutal was injected in anaesthesia. We applied OHBA SIKI spinal cord beat equipment and finished contusion
model with animal of injury group. Only laminectomy of Tg 9 were finised to control group. All the animal were killed at different time point which included UK 6rK 12rK 24h. 72h and 5d. Immunohistochemistryx western blot analysis^ EMS A experiments were finished . The expression of NF-K B at every time point was recorded .
At Ih after ASCI, There was angiorrhexis at area of contusion of spinal cord. Lots of agminated akaryocyte and plasma could be found at dorsal horn. At 6h after ASCI, nerve cells and astrocyte showed obvious edema. At 12h after SCI, a well defined hemorrhagic zone was observed within the central gray matter of the spinal cord. Within the epicenter of the contusion, gray matter structures appeared necrotic, with polymorphonuclear leukocytes (PMNL) invading the injured parenchyma. Necrotic neurons contained pyknotic nuclei surrounded by an eosinophilic cytoplasm. Severely damaged white matter tracts appeared swollen and edematous. In gray areas bordering the contusion, selective neuronal necrosis was observed within parenchyma containing swollen astrocytic cell bodies. In addition, petechial hemorrhages were observed throughout the gray matter and white matter tracts remote from the contusion. At 24 and after contusion injury, Photomicrographs show necrosis, infiltration of leukocytes, and white matter vacuolization. Severe vacuolization of the white matter was observed after SCI. At 72 hr after SCI, large numbers of macrophages were observed in the white matter. Macrophages and PMNLs were present at the gray/white interface. By 3 d after spinal cord trauma, the lesion remained well defined, and evidence of hemorrhage was observed. These foamy macrophages were seen within necrotic gray and white matter tracts. PMNLs also were dispersed throughout the necrotic areas . By 5 d after spinal cord trauma within areas adjacent to the injury, petechial hemorrhages were detected, and blood vessels demonstrated red blood cell stasis. Blood vessels also contained luminal leukocytes.
In order to discuss the express and rule of NF- K B in the spinal cord, we carried out Immunohistochemistry research. In control group(90.1+
3.1), Little immunoreactivity of NF- K B can be found. DAB staining was absent when the primary antibody was omitted or replaced with a control antibody of an identical isotype. At 1 h postinjury, NF- K B immunorea
1、急性脊髓损伤时NF-кB在继发性损伤中变化规律的研究
本研究目的在于研究NF-кB在大鼠ASCI时细胞胞浆内及胞核内的表达变化规律。SD大鼠90只，雌雄不限，体重200～250g。在1％戊巴比妥钠腹腔麻醉下(40mg／kg)，腹卧于动物立体定向架上。所有动物先行后正中入路T_(8-9)椎板切除，72只动物使用OHBA SIKI打击器(Tokyo，Japan)按Gruner改良法作成T_9脊髓背侧损伤模型，打击强度为25势能克厘米(Vgram-cm force，gcf)为损伤组，其余18只动物为对照组。所有动物分别在伤后1h、6h、12h、24h、72h、5d不同时间点处死。除对组织标本进行病理观察外，应用免疫组化、电泳迁移率变
中 文摘 要
动分析法mlectrophoretic Mobility shift Assay EMsA）及免疫印记法
（Western blot analysis）观察脊髓胞核与胞浆内 w4 B的在各个时相
点的表达。
损伤后lh镜下可见脊髓挫伤处灰质内血管破裂出血，红细胞及
血浆积聚在中央灰质、后角的血管周围，神经细胞肿胀；损伤后6h
出血波及整个灰质，神经细胞、星形细胞明显肿胀，神经细胞核周围
尼氏小体变浅；损伤后 12h，在脊髓灰质中央，依然可见片状出血，
在挫伤灶的边缘可以发现神经元细胞核固缩呈坏死样改变，并有水肿
的小胶质细胞及多形核细胞和巨噬细胞的组织浸润，组织间隙明显增
宽。损伤后24h到72h，在挫伤灶中心，脊髓结构紊乱，有大片灰质
坏死区及大量的巨噬细胞和多形核细胞浸润，在挫伤灶的灰白质交接
处有小出血点，白质水肿加重，白质内有空泡形成，以损伤后72h为
重；损伤后sd，在灰、白质坏死区可观察到泡沫样巨噬细胞、多形核
细胞；在正常组织与挫伤灶交界处，可以观察到血管内血细胞淤积，
血管壁出现白细胞浸润，白质内有空腔形成。
免疫组化结果以NFJB胞浆及细胞核表达的吸光度及阳性面积
作为相对表达量，用均数士标准差（王士S）表示、在检验进行统计学
处理。对照组仅有较弱的阳性反应①0．l士 3．l，368．30士门 ．65人 损
伤后 lh表达开始增强（112．2士4．2，387．41士13 ＊5），P＜0＊1，NF－K B＊
性细胞在病灶内和边缘区可以见到；损伤后 6h增强明显门 25＊士 2．5，
402＊2士14．57），p＜0＊of；损伤后12h 达高峰（175．3士2＊，478．23士
14．45），p＜0刀of，在神经细胞、小胶质细胞、巨噬细胞、多形核细胞、
星形细胞胞浆内均有 NF4 B表达；损伤后 24h表达开始下降门 石士
2．3，440．32士14．84），p＜0．001；sd逐步恢复正常p．5士2．8，379．65士
13＊5），p＞0＊5。细胞核内的 N’F－K B在损伤后 lh（8 ＊上2．l，210．00士
14．40）轻度增强 p＞0＊5；6h后逐步增强（94＊士2．3，233．46士门．45），
p＜0＊1；12h达到高峰（146．0土3．I，298＊4士15．46），p＜0＊of；24h后逐
渐下降（90．0士 2．4，265．13士 16．48），p＜0．00；sd基本恢复正常（61．0士
2．8，205＊3土 15．46），p＞0＊5。
应用 Western blot analysis从蛋白水平测定挫伤脊髓e4 B的胞
浆内表达。抗P65抗血清显示65KDA的明显分子条带吸光度作为相
2
中 文摘 要
对表达量，用均数土标准差G士S）表示、t检验进行统计学处理。对
照组显示一个较弱的表达条带O 刀士2．2），而损伤后 lh NF4B表达
开始增高（09．0士 2．＞ p＜0．of；损伤后 6h增高己很明显OIZ．0士 l．7），
p＜0＊of；损伤后 12h 达高峰门＊ ＊士 1．7），p＜0＊01；持续到损伤后 24h
才开始下降（124＊士2二），p＜0＊of； sd后逐步恢复正常（103＊土1．9），
p＞0刀5。
应用 EMSA检测 N’’F＊ B在细胞核内的表达情况?
Inflammatory responses are a major component of secondary injury and play a central role in mediating the pathogenesis of acute and chronic spinal cord injury (SCI). The nuclear factor-kappa B (NF- K B) is a primary transcription factor for the transcription activation of a variety of cytokinin and genes regulating inflammatory. The activation of NF- K B made inflammation genes over expression including tumor necrosis factor-alpha (TNF-a) and Interleukin-lbeta(IL-l). But the expression of NF-KB and inhibitory subunit of NF- K. B(I K B) in SCI is not very clear, exespecially the expression of I K B in spinal cells .Glial fibrillary acidic protein (GFAP) is an important role of SCI .The increase of the number and expression of GFAP-positive cells was correlated to the inflammatory responses and functional recovery of the spinal cord. We used a contusion model in rats to initiate the early biochemical and molecular changes with acute inflammation after SCI and examined the expression of activated NF- K BN I K B
and GFAP with immunohistochemistry western blot analyses and electrophoretic mobility shift assays (EMS A). In order to search the new medicine of relieving inflammatory responses we injected different dose of CCK-8 (Cholecystokinin-octapeptide) to rats before injury and examined the expression of NF- K B^ I K B, GFAP.
1 Expression of activated NF- K B after acute traumatic SCI.
The purpose is observe the expression of NF- K B at cytochylema and nucleolus of SD rat in ASCI. All the animal were divided into control group(l 8) and injury group(72). 1% nembutal was injected in anaesthesia. We applied OHBA SIKI spinal cord beat equipment and finished contusion
model with animal of injury group. Only laminectomy of Tg 9 were finised to control group. All the animal were killed at different time point which included UK 6rK 12rK 24h. 72h and 5d. Immunohistochemistryx western blot analysis^ EMS A experiments were finished . The expression of NF-K B at every time point was recorded .
At Ih after ASCI, There was angiorrhexis at area of contusion of spinal cord. Lots of agminated akaryocyte and plasma could be found at dorsal horn. At 6h after ASCI, nerve cells and astrocyte showed obvious edema. At 12h after SCI, a well defined hemorrhagic zone was observed within the central gray matter of the spinal cord. Within the epicenter of the contusion, gray matter structures appeared necrotic, with polymorphonuclear leukocytes (PMNL) invading the injured parenchyma. Necrotic neurons contained pyknotic nuclei surrounded by an eosinophilic cytoplasm. Severely damaged white matter tracts appeared swollen and edematous. In gray areas bordering the contusion, selective neuronal necrosis was observed within parenchyma containing swollen astrocytic cell bodies. In addition, petechial hemorrhages were observed throughout the gray matter and white matter tracts remote from the contusion. At 24 and after contusion injury, Photomicrographs show necrosis, infiltration of leukocytes, and white matter vacuolization. Severe vacuolization of the white matter was observed after SCI. At 72 hr after SCI, large numbers of macrophages were observed in the white matter. Macrophages and PMNLs were present at the gray/white interface. By 3 d after spinal cord trauma, the lesion remained well defined, and evidence of hemorrhage was observed. These foamy macrophages were seen within necrotic gray and white matter tracts. PMNLs also were dispersed throughout the necrotic areas . By 5 d after spinal cord trauma within areas adjacent to the injury, petechial hemorrhages were detected, and blood vessels demonstrated red blood cell stasis. Blood vessels also contained luminal leukocytes.
In order to discuss the express and rule of NF- K B in the spinal cord, we carried out Immunohistochemistry research. In control group(90.1+
3.1), Little immunoreactivity of NF- K B can be found. DAB staining was absent when the primary antibody was omitted or replaced with a control antibody of an identical isotype. At 1 h postinjury, NF- K B immunorea
引文
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2． Bethea JR, Marca Castro,Robert W,et,al. Traumatic spinal cord injury induces nuclear factor-B activation. The J Neuroscience, 1998, 18:3251-3260
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4． Tator CH, Fehlings MG. Review of the secondary injury theory mechanisms. J Neurosurg, 1991, 75:15-26
5． Salzman SK, Acosta R, Beck G, et al. Spinal endothelin content is elevated after moderate local trauma in the rat to Ievels associated with locomotor dysfunction after intrathecal injection.J Neurotrama, 1996, 13:93-101
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2． Bethea JR, Marca Castro,Robert W,et,al. Traumatic spinal cord injury induces nuclear factor-B activation. The J Neuroscience, 1998, 18:3251-3260
3． Dignam JD, Lebovitz RM, Roeder RG .Accurate transcription initiation by RNA polymerase Ⅱ in a soluble extract from isolated mammalian nuclei.Nucleic Acids Res, 1983, 11:1475-1489
4． Tator CH, Fehlings MG. Review of the secondary injury theory mechanisms. J Neurosurg, 1991, 75:15-26
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