TLR4信号负调控CD36表达影响脑出血血肿吸收及其机制

发布时间：2018-04-25 16:13

本文选题：脑出血 + 血肿吸收　；参考：《第三军医大学》2014年博士论文

【摘要】：脑出血(intracerebralhemorrhage,ICH)是一种常见的中风类型，虽经多年研究，但至今治疗手段有限。在动物实验及临床实践中发现，ICH后脑组织自身具有血肿吸收清除的能力，而且患者临床结局的好坏与血肿吸收速度的快慢呈明显正相关。因此促进内源性血肿吸收成为治疗ICH的新途径。 ICH后血肿成分可导致小胶质细胞激活。在ICH早期，小胶质细胞可以通过吞噬红细胞及红细胞溶解成份来清除血肿。小胶质细胞发挥吞噬功能主要由清道夫受体CD36介导。CD36参与了小胶质细胞吞噬β-淀粉样蛋白、氧化低密度脂蛋白过程，并引起慢性无菌炎症，在阿尔茨海默氏病、动脉粥样硬化的发病中起作用。但CD36在ICH后的表达及其作用尚未阐明。越来越多的证据表明，炎症是ICH引起继发性脑损伤的关键因素。研究显示TLR4参与了无菌性炎症的发生发展，在中枢神经系统炎症反应中具有重要地位。已有研究显示TLR4信号激活产生的TNF-α等炎症因子可以下调巨噬细胞上的CD36的表达，进而影响巨噬细胞吞噬功能。但在ICH过程中，TLR4信号能否调控小胶质细胞的CD36表达，并进而影响吞噬功能尚不清楚。在本研究中，我们首先利用临床ICH病例，观察了CD36缺陷患者血肿吸收及神经功能恢复情况。在此基础上，利用在体和离体ICH模型，观察了CD36在ICH后表达变化规律及其在血肿吸收中的作用；然后我们观察了TLR4信号通路对CD36表达的调控及其对小胶质细胞吞噬功能的影响，探讨了ICH后CD36调控血肿吸收的相关机制。最后观察了TLR4抑制剂TAK242对CD36表达的调控及其对小胶质细胞吞噬作用的影响。第一部分CD36缺陷对脑出血患者血肿吸收及神经功能缺损的影响目的：观察CD36缺陷脑出血患者血肿吸收变化和相应神经功能恢复情况，以明确脑出血过程中CD36的功能。方法：收集199例脑出血患者一般临床及头颅CT资料，并对患者进行NIHHS评分和mRS评分。利用PCR-SSP技术和Western blot技术对患者进行CD36缺陷筛查和分型鉴定，筛查出CD36Ⅰ型缺陷患者做为CD36缺陷组。同时挑选出具有和CD36缺陷组相同部位、相同出血量的CD36正常患者作为正常对照组。对比两组患者的脑出血后血肿吸收速度和相应神经功能评分。结果：我们共筛查18例CD36缺陷患者(9.0%)，其中11例为CD36I型缺陷(5.5%)。与对照组相比，CD36缺陷患者入院时临床基本资料、影像学特征、生化指标无显著差异，但经过相同治疗后，CD36缺陷患者血肿吸收速度显著减慢，NIHSS评分及mRS评分显著增高。结论：CD36缺陷患者血肿吸收减慢，神经功能缺损评分加重，提示CD36参与了血肿吸收，并与神经功能的恢复相关。第二部分，CD36在脑出血后脑组织中表达及其在血肿吸收中的作用目的：观察脑出血后脑细胞CD36表达及其变化；了解CD36缺陷后血肿吸收及小胶质细胞吞噬能力的变化，从而明确CD36在血肿吸收过程中的作用；方法：首先采用小鼠在体脑出血模型，利用Real-time RT-PCR、Western Blot观察脑出血后CD36表达的变化；其次利用免疫荧光化学等方法，观察人和小鼠脑出血后CD36主要在哪种脑细胞表达；再次利用离体脑出血模型，通过流式细胞仪、免疫荧光染色，观察CD36-/-小胶质细胞吞噬能力的变化，最后采用在体脑出血模型，观察CD36-/-小鼠血肿吸收和神经功能缺损等变化，以进一步证实CD36在脑出血血肿吸收中的作用。结果：与Sham组相比，脑出血组第1天CD36表达就有显著差异，到第3天达到高峰，到第7天CD36表达仍维持较高水平。CD36可以在神经元细胞、星型胶质细胞、小胶质细胞上表达，但以小胶质细胞表达为主。小胶质细胞可以直接吞噬红细胞，并且CD36-/-小胶质细胞或使用CD36抗体封闭的小胶质细胞吞噬红细胞的能力显著减弱。与野生型小鼠相比，CD36-/-鼠脑出血后血肿吸收显著减慢，神经功能恢复显著减慢，TNF-α、IL-1β表达显著增多。结论：1. CD36在脑出血后表达显著增高，，在第3天达到最高峰，在第7天仍维持较高水平的表达，提示CD36参与了脑出血的病理过程。而且脑出血后，CD36主要在小胶质细胞表达。 2. CD36-/-小胶质细胞对红细胞吞噬能力降低，使用CD36抗体封闭野生型小胶质细胞也得到类似结果，提示CD36在小胶质吞噬RBC过程中起十分重要作用。 3. CD36-/-鼠脑出血后血肿吸收减慢，神经功能缺损加重，脑含水量增多，TNF-α/IL-1β等炎症因子分泌增多，提示CD36参与了血肿吸收，并与神经功能的预后相关。提示CD36可以作为治疗脑出血的一个新靶点。第三部分TLR4信号激活并负调控CD36表达影响脑出血血肿吸收及机制目的：观察TLR4-/-、MyD88-/-鼠脑出血后CD36表达变化，同时观察TLR4信号通路对血肿吸收和对小胶质细胞吞噬红细胞能力的影响，进而研究TLR4信号通路对CD36表达调控的机制。方法：使用在体脑出血模型，用Western blot检测TLR4-/-、MyD88-/-鼠脑出血后CD36表达，并观察TLR4-/-、MyD88-/-鼠血肿吸收情况；使用离体脑出血模型，用流式细胞仪检测TLR4-/-、MyD88-/-小胶质细胞CD36表达和吞噬能力的变化；利用离体脑出血模型，用Western blot和流式细胞仪观察炎症因子对小胶质细胞CD36表达和吞噬能力的影响。结果：与野生型小鼠相比，TLR4-/-、MyD88-/-鼠脑出血后CD36表达显著增高，血肿吸收显著增快。与野生型小胶质细胞相比，TLR4-/-、MyD88-/-小胶质细胞CD36表达显著增加，吞噬能力增强，而且这种作用可以被CD36抗体阻断。TLR4信号通路下游主要炎症因子TNF-α、IL-1β可以抑制小胶质细胞CD36表达，减弱其吞噬能力，TNF-α作用更加明显。结论：1. TLR4-/-、MyD88-/-鼠脑出血后CD36表达增高，血肿吸收增快，提示TLR4信号途径参与CD36表达的调控。 2.在离体脑出血模型中,TLR4-/-、MyD88-/-小胶质细胞CD36表达增高，吞噬红细胞能力增强。而使用CD36抗体后，可以减弱该作用，进一步证实TLR4信号途径参与CD36表达的调控。 3.在离体脑出血模型中，TLR4信号通路下游炎症因子TNF-α、IL-1β可以抑制小胶质细胞的CD36表达，并且可以削弱小胶质细胞的吞噬能力，其中以TNF-α为主，提示TLR4信号通路通过TNF-α、IL-1β负调控CD36的表达。第四部分TLR4抑制剂促进CD36表达，影响脑出血血肿吸收目的：观察TLR4抑制剂TAK242对小胶质细胞CD36表达和吞噬能力的影响，观察TAK242对神经元的保护作用及可能机制。观察TAK242对小鼠脑出血后血肿吸收的影响。方法：使用离体脑出血模型，用Western blot和流式细胞仪检测TAK242对小胶质细胞CD36表达和吞噬能力的影响；利用神经元、小胶质细胞共培养技术，观察TAK242对神经元的保护作用；在离体脑出血模型中，用Real-time RT-PCR检测TAK242对过氧化氢酶表达的影响，并测定过氧化氢浓度；同时使用在体脑出血模型，并观察TAK242对血肿吸收的影响。结果：在离体脑出血模型中，与对照组相比，TAK242增加了小胶质细胞CD36表达和吞噬能力，而炎症因子TNF-α、IL-1β可以减弱TAK242这一作用；在神经元、小胶质细胞共培养体系中，TAK242显著减少了神经元凋亡/坏死数量，并且增加了小胶质细胞的过氧化氢酶表达，减少过氧化氢分泌；在小鼠脑出血模型中，与对照组相比，TAK242可以促进脑出血血肿的吸收。结论：1.TAK242可以增强小胶质细胞CD36表达和吞噬红细胞能力，而TNF-α和IL-1β可以减弱TAK242作用，证明TAK242是通过抑制TLR4信号通路进而影响CD36表达。 2. TAK242增加小胶质细胞的过氧化氢酶表达，减少过氧化氢分泌，对神经元细胞具有保护作用。 3. TAK242不仅具有神经保护作用，而且促进了血肿的吸收，可能成为治疗脑出血的新药物。本研究在观察CD36在脑出血血肿吸收中作用的基础上，同时探讨了TLR4信号调控CD36表达,进而影响血肿吸收。进一步明确了脑出血血肿吸收的调控机制，也为通过促进血肿吸收治疗ICH这一全新理念提供实验依据，可望为ICH治疗提供新方法。
[Abstract]:Cerebral hemorrhage ( ICH ) is a common type of stroke . Although it has been studied for many years , the treatment has been limited . In animal experiments and clinical practice , it has been found that the brain tissue itself has the ability to absorb and remove hematoma , and the clinical outcome of the patient is positively correlated with the fast and slow speed of hematoma absorption . Therefore , it is a new way to promote the absorption of endogenous hematoma .

2 . CD36 - / - microglial cells decreased the phagocytosis of erythrocytes . Similar results were obtained by using CD36 antibody to close wild - type microglial cells , suggesting that CD36 plays an important role in the RBC process .

More and more evidence shows that inflammation is a key factor in ICH - induced secondary brain injury . The study shows that it is involved in the development of aseptic inflammation and plays an important role in the inflammatory response of the central nervous system .

In this study , we first used the clinical ICH cases to observe the absorption of hematoma and the recovery of neurological function in patients with CD36 deficiency . On the basis of this , the change rule of CD36 in ICH and its role in the absorption of hematoma were observed by using in vivo and ex vivo ICH models .
Then we observed the regulation of the expression of CD36 and its influence on the phagocytic function of microglial cells , and discussed the mechanism of CD36 - regulated hematoma absorption after ICH . Finally , the regulation of CD36 expression and its effect on the phagocytosis of CD36 were observed .

The effect of CD36 deficiency on hematoma absorption and neurological deficit in patients with cerebral hemorrhage

Objective : To observe the changes of hematoma absorption and the recovery of corresponding neurological function in patients with cerebral hemorrhage of CD36 , so as to clarify the function of CD36 in the course of cerebral hemorrhage .

Methods : 199 patients with cerebral hemorrhage were collected from clinical and skull CT data and NIHHS and mRS were scored . CD36 patients were identified by PCR - SSP and Western blot .

Results : We screened 18 patients with CD36 deficiency ( 9.0 % ) , 11 of them were CD36 I defect ( 5.5 % ) . Compared with the control group , the clinical basic data , imaging characteristics and biochemical indexes were not significantly different in patients with CD36 deficiency , but after the same treatment , the absorption rate of hematoma in patients with CD36 was significantly decreased , NIHSS score and mRS score were significantly increased .

Conclusion : The absorption of hematoma in patients with CD36 deficiency is slow and the score of neurological deficit is aggravated , suggesting that CD36 is involved in the absorption of hematoma and is related to the recovery of neurological function .

The second part , the expression of CD36 in brain tissue after intracerebral hemorrhage and its role in the absorption of hematoma

Objective : To observe the expression of CD36 and its changes in brain cells after intracerebral hemorrhage .
To understand the changes of the absorption of hematoma and the phagocytic ability of microglial cells after CD36 defects , so as to clarify the role of CD36 in the absorption of hematoma ;

Methods : The expression of CD36 in intracerebral hemorrhage was observed by Real - time RT - PCR and Western Blot .
Secondly , the expression of CD36 was observed in the brain cells of human and mouse after intracerebral hemorrhage by immunofluorescence chemistry .
The changes of the phagocytic ability of CD36 - / - microglial cells were observed by flow cytometry and immunofluorescence staining , and the changes of CD36 - / - mouse hematoma absorption and neurological deficit were observed by flow cytometry and immunofluorescence staining to further confirm the role of CD36 in the absorption of hematoma hematoma .

Results : Compared with the Sham group , the expression of CD36 in the first day of the cerebral hemorrhage group was significantly different , and the expression of CD36 on the 7th day was still higher than that in the wild type mice .

Conclusion : 1 . CD36 expression increased significantly after intracerebral hemorrhage , reached the highest peak on Day 3 , and maintained a high level of expression on Day 7 , suggesting that CD36 was involved in the pathological process of cerebral hemorrhage . After intracerebral hemorrhage , CD36 was mainly expressed in microglial cells .

In the early ICH , microglial cells can remove hematoma by phagocytizing red blood cells and red blood cell lysis components . CD36 plays a role in the phagocytosis of 尾 - amyloid , oxidized low - density lipoprotein , and causes chronic aseptic inflammation , plays a role in the pathogenesis of Alzheimer ' s disease and atherosclerosis . However , the expression of CD36 after ICH and its role have not yet been clarified .

Conclusion CD36 can be used as a new target for the treatment of cerebral hemorrhage .

The signal activation and negative regulation of CD36 expression in the third part affect the absorption and mechanism of hematoma hematoma .

Objective : To observe the changes of CD36 expression after intracerebral hemorrhage in mice after intracerebral hemorrhage , and observe the effect of the signal pathway on the absorption of hematoma and the ability to phagocytize the red blood cell of microglial cells .

Methods : Using the model of intracerebral hemorrhage , Western blot was used to detect the expression of CD36 after intracerebral hemorrhage , and observe the absorption of CD36 - / - , MyD88 - / - mouse hematoma .
Using isolated intracerebral hemorrhage model , the expression of CD36 and the phagocytic ability were detected by flow cytometry .
The effects of inflammatory factors on CD36 expression and phagocytic ability of microglial cells were observed by Western blot and flow cytometry .

Results : Compared with wild type mice , the expression of CD36 was significantly increased after intracerebral hemorrhage , and the absorption of hematoma was increased significantly . Compared with wild type microglial cells , the expression of CD36 was increased and the phagocytic ability was enhanced . The main inflammatory factors TNF - 伪 and IL - 1尾 downstream of the signal pathway could inhibit the expression of CD36 in microglial cells , weaken its phagocytic ability and play a more obvious role in TNF - 伪 .

Conclusion : 1 . After intracerebral hemorrhage , the expression of CD36 increased after intracerebral hemorrhage , and the absorption of hematoma increased rapidly , suggesting that the signal pathway involved in the regulation of CD36 expression .

2 . In the model of ex vivo cerebral hemorrhage , the expression of CD36 , the expression of CD36 and the ability of phagocytosis increased . After using CD36 antibody , the function could be weakened , and the pathway involved in the regulation of CD36 expression was further confirmed .

3 . In the model of isolated intracerebral hemorrhage , the downstream inflammatory cytokines TNF - 伪 and IL - 1尾 can inhibit the expression of CD36 in microglial cells , and the phagocytic ability of microglial cells can be weakened . The expression of TNF - 伪 and IL - 1尾 negatively regulates the expression of CD36 through TNF - 伪 and IL - 1尾 .

Fourthly , the expression of CD36 and the absorption of CD36 in cerebral hemorrhage were affected .

Objective : To observe the effect of TAK242 inhibitor TAK242 on the expression and phagocytic ability of CD36 in mice , and observe the protective effect and possible mechanism of TAK242 on neurons .

Methods : The effect of TAK242 on CD36 expression and phagocytic ability of microglial cells was detected by Western blot and flow cytometry using isolated intracerebral hemorrhage model .
The protective effect of TAK242 on neurons was observed by co - culture with neurons and microglial cells .
In vitro cerebral hemorrhage model , the effect of TAK242 on catalase expression was detected by Real - time RT - PCR , and the concentration of hydrogen peroxide was measured .
The effect of TAK242 on hematoma absorption was observed .

Results : Compared with the control group , TAK242 increased the expression and phagocytic ability of CD36 in microglial cells compared with the control group , while the inflammatory factors TNF - 伪 and IL - 1尾 could attenuate the role of TAK242 .
In the co - culture system of neurons and microglial cells , TAK242 significantly reduces the number of neuron apoptosis / necrosis and increases the expression of catalase in microglial cells and reduces the secretion of hydrogen peroxide ;
Compared with the control group , TAK242 can promote the absorption of hematoma in intracerebral hemorrhage .

Conclusion : 1 . TAK242 can enhance the expression of CD36 in microglial cells and the ability of phagocytizing red blood cells , and TNF - 伪 and IL - 1尾 can attenuate the role of TAK242 , which proves that TAK242 can inhibit the expression of CD36 by inhibiting the signaling pathway .

2 . TAK242 increases the expression of catalase in microglial cells , reduces the secretion of hydrogen peroxide , and has protective effect on neuronal cells .

3 . TAK242 has not only nerve protection , but also promotes the absorption of hematoma , which may become a new drug for the treatment of cerebral hemorrhage .

Based on the observation of the role of CD36 in the absorption of hematoma in intracerebral hemorrhage , the expression of CD36 and the absorption of the hematoma were discussed . The mechanism of the absorption of hematoma was further clarified , and the experimental basis was provided for the new idea of promoting the absorption of hematoma , which could provide a new method for ICH therapy .

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R743.34

【共引文献】