本文關鍵詞：血清因子白蛋白和補體在中樞神經系統(tǒng)損傷后炎癥反應中的作用研究　出處：《第三軍醫(yī)大學》2009年博士論文　論文類型：學位論文

  更多相關文章： 血腦屏障 血清白蛋白 炎癥 中樞神經系統(tǒng) 小膠質細胞 白細胞介素1-β 腫瘤壞死因子-α 補體 C3 脊髓損傷 星形膠質細胞 膠質瘢痕 膠質纖維酸性蛋白

【摘要】： 目的:在包含創(chuàng)傷在內的中樞神經系統(tǒng)(Central nervous system, CNS)病變中,血腦屏障(Blood-brain barrier, BBB)的開放會引起多種血液成分進入CNS,進而可能在CNS引起相應的效應。而炎癥反應是中樞系統(tǒng)微環(huán)境發(fā)生改變后的重要病理改變。研究發(fā)現(xiàn)BBB受損區(qū)域多伴有持續(xù)活化的炎癥反應存在。而持續(xù)過度的炎癥反應在許多中樞病變中都造成了損害性的后果。因此,探究BBB損傷性CNS病變中炎癥反應持續(xù)激活的機制,設法對其進行調控,有可能達到降低損害,改善愈后的目的。 血清白蛋白是血清中含量最豐富的蛋白質,約占血清蛋白質總量的50%。但在CNS卻近乎缺失。BBB損傷后大量的血清白蛋白進入CNS已被證實,然而其在中樞神經系統(tǒng)引起的效應卻鮮有報道。因此,本實驗將首次研究血清白蛋白作用于中樞神經系統(tǒng)主要的炎癥效應細胞——小膠質細胞后對其活化增殖和分泌促炎癥細胞因子的影響,明確其對中樞炎癥反應的激活作用。 補體系統(tǒng)是存在于人和脊椎動物血清與組織液中一組具有酶活性的蛋白,廣泛參與機體的防御反應和免疫調節(jié),也可介導免疫病理如損傷性炎癥反應,是體內具有重要生物學意義的效應系統(tǒng)和效應放大系統(tǒng)。正常CNS中補體表達水平極低。研究發(fā)現(xiàn)血清中的補體成分在CNS損傷后穿過受損的BBB會介導和加重CNS的炎癥反應;而炎癥反應又是造成CNS損傷后繼發(fā)性損傷和膠質瘢痕形成的重要機制。鑒于C3是補體活化途徑中的中心因子,我們擬利用C3基因敲除小鼠的脊髓損傷模型,觀察抑制補體活化能否減輕SCI小鼠脊髓的繼發(fā)性損傷,促進功能恢復,并初步探討其機制。 方法: 第一部分:小膠質細胞是存在于CNS的天然免疫細胞,當CNS微環(huán)境發(fā)生變化時,小膠質細胞可迅速活化增殖;而促炎癥細胞因子IL-1β和TNF-α是介導CNS炎癥反應的重要炎癥介質。因此,我們以0.5 mg/ml的血清白蛋白刺激N9小膠質細胞株,設PBS對照組,通過RT-PCR和Real-time RT-PCR方法檢測干預后3、6、9、12h小膠質細胞IL-1β和TNF-αmRNA表達情況;分別以0、0.1、0.5、1.0、2.0 mg/ml血清白蛋白和LPS刺激N9小膠質細胞,于24h收集各組細胞上清進行ELISA檢測,測定IL-1β和TNF-α細胞因子的濃度,了解其對小膠質細胞分泌IL-1β和TNF-α作用的量效關系;以0.5 mg/ml的血清白蛋白稀釋液刺激N9小膠質細胞株,設PBS對照組,分別于3、6、12、24h收集血清白蛋白刺激組和PBS對照組細胞上清進行ELISA檢測,測定IL-1β和TNF-α細胞因子的濃度,了解其作用的時間特點;將0.5μl 100 mg/ml血清白蛋白注入C57BL/6J小鼠運動皮層,采用免疫組化法在體觀察白蛋白作用后小膠質細胞的活化程度。 第二部分:為準確判斷干預手段對脊髓損傷后功能結局的影響,我們需要穩(wěn)定、重復性好、易操作的動物模型作為研究平臺。由于目前的基因修飾動物以小鼠為主,因此我們以C57BL/6J小鼠為實驗動物,建立脊髓夾傷模型,并對其特點進行評價,為下一步的研究奠定基礎。我們采用BMS后肢運動評分觀察夾傷致0.1、0.25、0.5 mm的不同分級損傷程度對小鼠后肢功能損傷及恢復的影響;并以HE染色觀察不同損傷程度致脊髓損傷后8w時脊髓的病理表現(xiàn);以HE染色觀察SCI后小鼠脊髓3d、1w、2w、4w時脊髓的病理改變特點;以GFAP免疫組織化學染色觀察SCI后3d、1w、2w、4w、8w時小鼠脊髓星形膠質細胞反應特點;以BDA順行示蹤觀察小鼠SCI后8w神經纖維再生特點及其與膠質瘢痕的關系;以流式細胞計數(shù)法檢測SCI后3d、1w、2w、8w的小膠質細胞/巨噬細胞的活化特點。 第三部分:采用上述建立的小鼠脊髓夾傷模型,以C3-/-小鼠及其野生對照C3+/+小鼠為實驗動物,通過BMS評分評估后肢運動功能、HE染色觀察脊髓病理改變、流式細胞術檢測小膠質細胞/巨噬細胞活化、免疫組化和Western blot技術檢測星形膠質細胞GFAP表達,觀察對比C3基因敲除對小鼠SCI后功能恢復的影響并初步探討其機制。 結果: 第一部分: 1.與PBS對照組細胞相比,0.5 mg/ml的血清白蛋白刺激的小膠質細胞3、6、9、12h的IL-1β和TNF-αmRNA表達均顯著增加; 2.0.1、0.5、1.0、2.0 mg/ml白蛋白刺激24h后小膠質細胞培養(yǎng)上清中IL-1β、TNF-α的濃度隨血清白蛋白濃度的增加而增加,2.0 mg/ml刺激組IL-1β、TNF-α濃度最高;各白蛋白刺激組IL-1β濃度和TNF-α濃度均顯著高于0 mg/ml組;LPS刺激組亦顯著高于未刺激組。 3.0.5 mg/ml的血清白蛋白作用后,小膠質細胞分泌IL-1β隨時間增加而增多,且于6h后各時間點均顯著高于對照組;小膠質細胞分泌TNF-α隨時間增加而增多,且于3h開始及其后時間點均顯著高于對照組。 4.小鼠皮層注射血清白蛋白引起小膠質細胞活化的范圍和數(shù)量明顯強于PBS對照組。 第二部分: 1.成功建立了穩(wěn)定的C57BL/6J小鼠脊髓夾傷動物模型,BMS評分檢測顯示SCI后各組小鼠后肢運動功能有不同程度損傷,之后持續(xù)8w的行為學觀察中雖都有恢復,但不同致傷程度組小鼠恢復情況有顯著性差異。夾傷越輕(至0.5mm),恢復越好;夾傷越重(至0.1mm),恢復越差。 2.不同致傷程度小鼠8w時的脊髓病理改變與行為學結果相符。夾至0.1mm組小鼠的脊髓損傷范圍最廣,可見夾傷處大量炎細胞浸潤形成的瘢痕及微小的空洞形成;隨著夾傷程度的減弱,瘢痕的范圍逐漸減小,殘留的正常組織逐漸增多。 3.損傷后4w的連續(xù)病理觀察發(fā)現(xiàn):傷后3d,脊髓以變性、壞死為主要表現(xiàn);1w時已有大量炎細胞浸潤,并且出現(xiàn)繼發(fā)性損傷區(qū)域,可見損傷兩側水腫形成較多空泡;2w時仍有大量炎細胞浸潤,繼發(fā)損傷和水腫區(qū)域較1w時縮小;4w時僅殘留炎細胞浸潤,繼發(fā)損傷表現(xiàn)明顯減弱,組織進入修復期。 4.損傷后8w的星形膠質細胞連續(xù)觀察發(fā)現(xiàn):傷后3d,星形膠質細胞尚未有明顯的活化表現(xiàn);1w時星形膠質細胞肥大,突起增粗增長,呈明顯的活化狀態(tài),范圍延伸至損傷區(qū)的頭尾兩側;2w時星形膠質細胞的數(shù)量較1w時有所增多,但是細胞及突起的形態(tài)已不如7d時肥大明顯;4w和8w時星形膠質細胞的活化程度和范圍明顯減弱,但在損傷中間處瘢痕組織的周邊仍包繞有較多形態(tài)肥大,突起增多的反應型星形膠質細胞形成膠質瘢痕,而稍遠處的星形膠質細胞形態(tài)已基本接近正常。 5.BDA順行示蹤發(fā)現(xiàn)SCI后10w+2d,夾傷至0.25 mm組小鼠脊髓中,大部分BDA標記的皮質脊髓束到達夾傷處的頭側端,但是無法繼續(xù)下行,在夾傷處頭側端形成多個圓形的終球,僅有少數(shù)BDA著色的神經纖維在原皮質脊髓束位置的背側方通過夾傷處,到達夾傷處尾側端。經GFAP復染后,可見BDA終球形成位置恰為星形膠質細胞肥大增生形成膠質瘢痕處。 6.流式細胞術檢測CD68陽性細胞提示損傷處小膠質/巨噬細胞于傷后3d即明顯活化增殖且3d時活化狀態(tài)已很強,其數(shù)量在7d達到高峰后,一直持續(xù)到傷后8w均存在活化狀態(tài)的小膠質/巨噬細胞。 第三部分: 1.BMS評分顯示C3-/-小鼠夾傷后的后肢功能恢復明顯好于C3+/+組小鼠。 2.HE染色提示夾傷后4w,C3-/-小鼠脊髓損傷處的炎癥細胞浸潤形成瘢痕的范圍明顯少于C3+/+組小鼠,而殘留的正常組織卻要多于C3+/+小鼠。 3.流式細胞檢測CD68陽性細胞提示傷后3d、7d,C3-/-小鼠損傷處的小膠質/巨噬細胞活化程度明顯弱于C3+/+組小鼠。 4.免疫組化和Western blot檢測均提示C3-/-小鼠傷后4w GFAP的表達明顯弱于C3+/+組小鼠。 結論: 1.本研究證實血清白蛋白能刺激小膠質細胞的活化增殖,并能促進其分泌IL-1β、TNF-α促炎癥細胞因子,提示血清白蛋白在CNS炎癥反應中可能起重要的活化作用。 2.本研究建立了穩(wěn)定、易操作、重復性好的小鼠SCI模型,既適用于SCI急性期小膠質細胞活化炎癥反應的研究,也可用于SCI慢性期星形膠質細胞形成膠質瘢痕及神經再生的研究。 3.補體系統(tǒng)可能通過加重炎癥反應,并促進星形膠質細胞瘢痕的形成,對小鼠脊髓損傷的修復產生不利影響。 綜上所述,本文通過對血清白蛋白和補體的研究,進一步豐富了血清因子對中樞神經系統(tǒng)損傷后炎癥反應作用的認識,并為通過干預炎癥反應治療中樞神經系統(tǒng)病變提供了理論依據(jù)。
[Abstract]:Objective: the central nervous system in the wound, (Central nervous system, CNS) lesions, blood brain barrier (Blood-brain barrier BBB) open will cause various blood components into the CNS, which may cause the corresponding effect in CNS. Inflammation is an important pathological change of central nervous system micro environment changes. The study found that the presence of inflammation associated with impaired BBB regional persistent activation of inflammation. Excessive and persistent in many central lesions have caused damage of consequences. Therefore, the research of mechanism of BBB damage of CNS lesions in the inflammatory response to sustained activation, trying to regulate its, is likely to reduce the damage, to improve after healing.
Serum albumin is the most abundant protein in serum, total serum protein 50%. in CNS is a near absence of.BBB damage of serum albumin into a CNS has been confirmed, but the effect caused in the central nervous system has been rarely reported. Therefore, this experiment will be the first study of serum albumin in inflammatory effector cells the main central nervous system: after the activation of microglia proliferation and secretion of proinflammatory cytokines and the inflammatory reaction of central nervous system activation.
The complement system is present in human serum and tissue fluid in vertebrates and a group of proteins with enzymatic activity, immune defense reaction and widely involved in regulation, but also induce immune pathological injury such as inflammatory reaction, is the effect of system and has important biological significance, the effect should be amplified system. Complement expression in normal CNS level very low. The study found that the complement components in serum through BBB damage after CNS damage will be mediated by inflammation and exacerbation of CNS; inflammation can cause secondary injury and glial scar formation after injury. CNS in C3 is the complement activation factor pathway in our center, quasi knockout the mouse model of spinal cord injury by C3 gene, the secondary injury can reduce the SCI activation inhibition was observed in the mouse spinal cord complement, promote functional recovery, and to explore its mechanism.
Method:
The first part: microglial cells are innate immune cells existed in CNS, when the CNS micro environment changes, microglia can be activated and proliferation; proinflammatory cytokines IL-1 and TNF- alpha beta is an important medium of inflammation mediated inflammatory reaction in CNS. Therefore, we use serum albumin stimulation of 0.5 mg/ml N9 glial cells, the control group with PBS, RT-PCR and Real-time by RT-PCR assay after intervention 3,6,9,12h microglia IL-1 beta and TNF- alpha mRNA expression respectively; the serum albumin and LPS 0,0.1,0.5,1.0,2.0 mg/ml stimulated N9 microglia in 24h culture supernatant was collected for detection of ELISA, IL-1 and TNF- concentration determination of beta alpha cell factor the understanding of the dose-response relationship between the secretion of IL-1 and TNF- alpha beta effects on microglia; the serum albumin of 0.5 mg/ml dilution stimulation of N9 cells, the control group with PBS, respectively, 3,6,12,2 4h serum albumin stimulation group and PBS control group cells were detected by ELISA, IL-1 and TNF- concentration determination of beta alpha cytokines, understand the characteristics of the time effect; 0.5 l 100 mg/ml serum albumin injection in mice C57BL/6J cortex, activation of microglia was detected by immunohistochemical method and albumin in vivo after.
The second part: to accurately determine the effect of interventions on functional outcome after spinal cord injury, we need stability, good reproducibility and easy operation of the animal model as the research platform. Because of a genetically modified animal at present by mice, so we used C57BL/6J mice as experimental animals, a spinal cord injury model, and to evaluate the the characteristics, lay the foundation for the next research. The degree of injury of different grades by BMS hind limb crush induced by 0.1,0.25,0.5 was used to observe the influence of mm and recovery of hindlimb function injury in mice; and HE staining was used to observe the degree of injury caused by the pathological manifestations of spinal cord after spinal cord injury 8W; SCI was detected by HE staining. 3D 1W, 2W mouse spinal cord, 4W, spinal cord pathological features; GFAP immunohistochemical staining after SCI 3D, 1W, 2W, 4W, characteristics of mouse spinal cord astrocytes in response to 8W; to direct BDA The relationship between 8W nerve regeneration and glial scar after SCI in mice was observed by tracer. The activation characteristics of microglia / macrophages in 3D, 1W, 2W and 8W after SCI were detected by flow cytometry.
The third part: using the mouse spinal cord crush model in C3-/- mice and wild-type C3+/+ mice as experimental animal, we used the BMS score to assess hindlimb function, pathological changes of the spinal cord were observed by HE staining, flow cytometry detection of microglia / macrophages activation, expression of astrocyte GFAP immunohistochemistry and Western blot technology, comparative Observation on effects of C3 gene knockout on the functional recovery after SCI mice and explore its mechanism.
Result:
Part one:
1. compared with the PBS control group, the expression of IL-1 beta and TNF- alpha mRNA of 3,6,9,12h in microglia stimulated by 0.5 mg/ml serum albumin increased significantly.
2.0.1,0.5,1.0,2.0 mg/ml albumin after 24h stimulation of microglial cells in the culture supernatant of IL-1 beta, TNF- alpha concentration increased with increasing concentrations of serum albumin, 2 mg/ml stimulation group IL-1 beta, TNF- alpha was the highest; the albumin stimulation group IL-1 beta concentrations and TNF- concentrations were significantly higher than that of 0 mg/ml group; LPS group was significantly higher than that of no stimulation group.
After the action of serum albumin of 3.0.5 mg/ml, the secretion of IL-1 beta in microglia increased with time and increased at all time points after 6h. The secretion of TNF- alpha increased by time and increased at the beginning and after time of 3H.
4. the range and number of microglia activation caused by the injection of serum albumin in mice was significantly stronger than that in the PBS control group.
The second part:
1. successfully established stable C57BL/6J mouse spinal cord clamp injury animal model, BMS score showed hindlimb motor function after SCI mice have different degrees of injury, after 8W behavioral observations have been restored, but different injury degree of mice recovery were significantly different. Pinch the lighter (to 0.5mm), the better; crush the heavier (to 0.1mm), the worse the recovery.
The pathological changes of the spinal cord and the behavior of 2. different injury degree of mice 8W learning results. To clip the mice of 0.1mm spinal cord injury range widely, scar visible clip wound infiltration of inflammatory cells and the formation of tiny hole formation; with the weakening of crush degree, scar area gradually reduced, normal tissue residue gradually increased.
Observed continuous pathology in 3. 4W after injury: 3D after injury to spinal cord degeneration, necrosis, as the main performance; 1W has a large number of inflammatory cell infiltration, and a secondary injury area, visible damage on both sides of the edema and vacuolar 2W; there is still a large number of inflammatory cell infiltration, secondary injury and edema region is 1W reduced 4W; only the residual inflammatory cell infiltration, secondary injury decreased tissue into the repair period.
4. 8W after injury of astrocytes in continuous observation: 3D after injury, astrocytes had no obvious change. 1W; astrocyte hypertrophy, thickening of neurite growth were activated obviously, extends to the damage zone on both sides of head and tail; 2W the number of astrocytes was 1W but obviously increased, mast cells and processes the form as 7d; 4W and 8W activation degree and range of astrocytes decreased significantly, but in the middle of the scar tissue around the injury is wrapped with morphological hypertrophy, prominences reactive astrocytes glial scar formation, and a distance astrocyte morphology has been close to normal.
5.BDA anterograde tracing found SCI 10w+2d after crush injury to 0.25 mm mice in the spinal cord, the head side of the corticospinal tract most BDA markers to a wound, but cannot continue downward, forming a plurality of round ball in the final clip wound side head, only a small number of BDA nerve fibers in the dorsal lateral color original corticospinal tract position by clamping the wound, wound to clamp the caudal. After stained by GFAP, visible BDA final ball formation position exactly astrocyte hyperplasia formation of glial scar.
6. flow cytometry detection of CD68 positive cells suggested that the microglia / macrophages were activated and proliferated at 3D after injury, and the activation state was very strong at 3D. The number of microglia / macrophages increased to a peak at 7d and lasted until 8W after injury.
The third part:
The 1.BMS score showed that the recovery of hind limb function in C3-/- mice was better than that in group C3+/+.
2.HE staining showed that the infiltration of inflammatory cells in spinal cord injury site of 4W C3-/- mice after clipping was significantly less than that of C3+/+ group, while residual normal tissue was more than that of C3+/+ mice.
The detection of CD68 positive cells by 3. flow cytometry showed that the activation degree of microglia / macrophage in 3D, 7d and C3-/- mice after injury was significantly weaker than that of C3+/+ mice.
4. immunohistochemistry and Western blot showed that the expression of 4W GFAP in C3-/- mice was significantly weaker than that of C3+/+ mice.
Conclusion:
1., this study confirms that serum albumin can stimulate the activation and proliferation of microglia, and promote its secretion of IL-1 beta, TNF- alpha and proinflammatory cytokines, suggesting that serum albumin may play an important role in CNS inflammatory response.
2., a stable, easy to operate and reproducible mouse SCI model was established in this study. It is not only suitable for the study of microglia activation and inflammatory response in acute phase of SCI, but also for SCI chronic astrocytes forming glial scar and nerve regeneration.
The 3. complement system may increase the inflammatory response and promote the formation of astrocyte scar, which has an adverse effect on the repair of spinal cord injury in mice.
To sum up, through the study of serum albumin and complement, we further enrich the understanding of the role of serum factors in the inflammatory response after central nervous system injury, and provide a theoretical basis for the intervention of inflammatory response in the treatment of central nervous system diseases.

【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R363

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