【摘要】： 哺乳動物的頸動脈體(carotid body, CB)是一個位于頸動脈血管分叉處的血運極其豐富的器官。已經知道,慢性缺氧能引起頸動脈體功能活動增強,而最近的研究表明,頸動脈體支持細胞具有干細胞潛能,缺氧能引起支持細胞表達nestin,而且表達量與缺氧時間呈正相關,但多數(shù)臨床疾病都是慢性間歇性缺氧,在此狀態(tài)下nestin和神經再生標記物GAP-43有何變化還未見研究。高壓氧作為現(xiàn)代醫(yī)療手段中的新成員,對一氧化碳中毒、缺血再灌注等癥狀具有較好的臨床效果,高壓氧時頸動脈體的反應如何尚不清楚。PKC作為細胞信號轉導的中心環(huán)節(jié),在神經傳導、遞質釋放、突觸可塑性、長時程增強效應以及腫瘤發(fā)生、細胞凋亡等方面起重要作用。ERK是細胞因子、生長因子介導細胞增殖效應中最重要的途徑。PKC和ERK信號通路是否在頸動脈體化學感受功能或神經再生過程中發(fā)揮作用還有待研究。 白細胞介素-1β(IL-1β)是調節(jié)機體免疫、炎癥、內分泌和代謝活動急性反應的一種重要的促炎性細胞因子,頸動脈體主細胞本身也表達IL-1β,同時還有其I型受體(IL-1RI)的高表達,提示頸動脈體具有感受促炎性細胞因子刺激的物質基礎。促炎性細胞因子可以促進慢性缺氧時頸動脈體中兒茶酚胺類物質的合成,從而調節(jié)頸動脈體的慢性缺氧感受功能。既然缺氧能引起支持細胞分化成具有感受功能的主細胞,那么促炎性細胞因子是否也有類似的作用?另外,頸動脈體感受促炎性細胞因子調節(jié)的細胞內信號途徑也未見報道,對此問題進行研究具有重要理論意義和應用價值。 在本研究中,綜合應用免疫組織化學技術、Westren blot技術在組織切片以及蛋白水平,分別研究了慢性低壓缺氧、高壓氧以及外源性IL-1β刺激對大鼠頸動脈體主細胞TH表達的影響。還初步觀察了CB在上述刺激條件下細胞內信號分子pPKC-γ、pERK1/2水平以及神經干細胞和神經再生的標記物Nestin和GAP-43的變化。 結果簡要歸納如下: (1)正常條件下,TH、pPKC-γ、pERK1/2、GAP-43、Nestin在大鼠頸動脈體均有表達。TH、pPKC-γ主要在頸動脈體球細胞表達,pERK1/2主要在頸動脈體支持細胞表達,GAP-43在球細胞和支持細胞均有表達。 (2)免疫組織化學結果發(fā)現(xiàn),慢性缺氧和IL-1β刺激都可以引起頸動脈體TH、GAP-43、Nestin表達升高;高壓氧刺激抑制TH、GAP-43的表達,高氣壓刺激無顯著差異;慢性缺氧和IL-1β刺激可以引起pERK1/2表達升高,高壓氧和高氣壓刺激也能引起pERK1/2表達升高;慢性缺氧和IL-1β刺激可以引起pPKC-γ表達升高,高壓氧刺激則抑制其表達,而高氣壓刺激則無明顯作用。 (3)Western blot進一步證實了上述結果。 主要結論:(1)CB可以感受免疫因子的刺激。(2)缺氧和IL-1β刺激能引起CB生理活性增強,而高壓氧則抑制CB生理活性,而且這一效應主要是由于高氧引起的。(3)PKC和ERK信號通路在頸動脈體感受作用中發(fā)揮作用。(4)頸動脈體存在分化再生潛能,缺氧和IL-1β刺激能引起CB增殖分化。
[Abstract]:The carotid body (CB) of mammals is an organ with abundant blood supply located at the bifurcation of carotid arteries. Chronic hypoxia has been known to enhance the function of the carotid body. Recent studies have shown that CSCs have stem cell potential, hypoxia can induce nestin expression in CSCs, and hypoxia can induce nestin expression in CSCs. The expression of nestin and GAP-43 is positively correlated with hypoxia time, but most clinical diseases are chronic intermittent hypoxia. There is no study on the changes of nestin and GAP-43 under this condition. Hyperbaric oxygen, as a new member of modern medical methods, has a good clinical effect on carbon monoxide poisoning, ischemia-reperfusion and other symptoms. As the central link of cell signal transduction, PKC plays an important role in nerve conduction, transmitter release, synaptic plasticity, long-term potentiation, tumorigenesis and apoptosis. ERK is the most important pathway of cytokines and growth factors mediating cell proliferation. Whether the signal pathway plays a role in carotid chemosensory function or nerve regeneration is still to be studied.
Interleukin-1 beta (IL-1 beta) is an important pro-inflammatory cytokine that regulates the acute response of immune, inflammatory, endocrine and metabolic activities. The main cell of carotid body itself also expresses IL-1 beta, and has high expression of type I receptor (IL-1RI), suggesting that the carotid body has a material basis for sensing pro-inflammatory cytokine stimulation. Inflammatory cytokines can promote the synthesis of catecholamines in the carotid body during chronic hypoxia, thus regulating the chronic hypoxic sensory function of the carotid body. The intracellular signaling pathways regulated by inflammatory cytokines have not been reported, so it is of great theoretical significance and application value to study this issue.
In this study, the effects of chronic hypobaric hypoxia, hyperbaric oxygen and exogenous IL-1beta stimulation on TH expression in rat carotid somatic host cells were studied by using immunohistochemical technique and Westren blot technique in histological sections and protein levels. The level of K1/2 and the changes of Nestin and GAP-43 in neural stem cells and nerve regeneration markers.
The results are summarized as follows:
(1) Under normal conditions, TH, pPKC-gamma, pERK1/2, GAP-43 and Nestin were expressed in rat carotid body. TH, pPKC-gamma were mainly expressed in carotid body globular cells, pERK1/2 was mainly expressed in carotid body Sertoli cells, and GAP-43 was expressed in globular cells and Sertoli cells.
(2) Immunohistochemical results showed that both chronic hypoxia and IL-1beta stimulation could induce the expression of TH, GAP-43 and Nestin in carotid artery; hyperbaric oxygen stimulation inhibited the expression of TH, GAP-43, while hyperbaric stimulation had no significant difference; chronic hypoxia and IL-1beta stimulation could induce the expression of pERK1/2, hyperbaric oxygen and hyperbaric stimulation could also induce the expression of pERK1/2. Chronic hypoxia and IL-1 beta stimulation could increase the expression of pPKC-gamma, hyperbaric oxygen stimulation could inhibit the expression of pPKC-gamma, but hyperbaric stimulation had no obvious effect.
(3) Western blot further confirmed the above results.
Main conclusions: (1) CB can sense the stimulation of immune factors. (2) Hypoxia and IL-1 beta stimulation can enhance the physiological activity of CB, while hyperbaric oxygen can inhibit the physiological activity of CB, and this effect is mainly due to hyperoxia. (3) PKC and ERK signaling pathways play a role in carotid somatosensory function. (4) There is differentiation and regeneration potential in carotid bodies. Hypoxia and IL-1 beta stimulation can induce proliferation and differentiation of CB.
【學位授予單位】：西北農林科技大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R363
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本文編號：2211678