以頭部外傷病人急性期血液中的黏附因子、去氧核糖核酸和白血球細胞凋亡來評估病人的預後

Serial Serum Adhesion Molecules, Plasma DNA and Serum Leukocyte Apoptosis Levels as Predictors of Outcome in Acute Traumatic Brain Injury

王宏振(Hung-Chen Wang)

指導教授 ： 關皚麗

高雄醫學大學/醫學院/醫學研究所/博士(2014年)

https://doi.org/10.6832/KMU.2014.00106

摘要

[背景] 頭部外傷是一個疾病，它的過程從一開始受傷後所導致的生化和細胞的變化，轉變成接下來的神經受傷及死亡。這種持續性的變化也就是所謂的”第二次傷害”，它包含了許多發炎反應及細胞凋亡的過程。腦部受傷的程度取決於一開始撞擊的嚴重度以及之後因為神經發炎與細胞凋亡所導致的”第二次傷害”的程度。”第二次傷害”會導致腦水腫、顱內壓力上升和遲發性神經細胞破壞。在許多不同的嚴重疾病時，早期血液中會有高濃度的游離DNA，這代表著許多的細胞死亡；而在外傷病人中，這些游離DNA可能來自受傷的組織。頭部外傷是造成年輕人死亡及殘障最常見的原因，儘管在大腦監測及臨床照顧方面都有持續的進步，但這些神經監測的方法卻不足以評估及量化一開始受傷的嚴重度及接下來的”第二次傷害”過程的程度，因此無法為臨床治療上提供足夠的監測，也無法有效評估病人的預後。到目前為止，有關頭部外傷病人的發炎反應及細胞凋亡之連續性變化的研究甚少，因此發炎反應的因子及細胞凋亡的過程對頭部外傷病人的預後的影響也還不清楚。
[假說] 急性頭部外傷病人早期血液中高濃度的黏附因子、白血球細胞凋亡和游離DNA，可能與腦部第二次傷害的致病原因有關，因此可以用來評估頭部外傷病人的嚴重程度，以及預測頭部外傷病人的預後。
[進行之研究與方法] 這個研究是探討急性頭部外傷病人血液中黏附因子、白血球細胞凋亡和游離DNA的變化。總共收集了88位在24小時內因頭部外傷住院的病人，總共收集了229血液樣本，其中71個血液樣本於頭部外傷24小時內完成收集，另外有81個和77個血液樣本分別於頭部外傷後第4天和第7天收集；另外由150位自願者提供150血液樣本做為控制組。血液中可溶性的黏附因子(sICAM-1、 sVCAM-1、sE-selectin、sL-selectin和sP-selectin)使用商業性酵素免疫分析法(ELISA)測量，血液中白血球細胞凋亡是使用流式細胞儀(Flowcytometry)測量，血液中細胞核及粒線體DNA是使用定量即時聚合酶鏈鎖反應(Quantitative Real-Time PCR)來測量。
[成果] 我們的研究發現和控制組比較，急性頭部外傷病人血液中的黏附因子sICAM-1、sVCAM-1，游離性細胞核和粒線體DNA，以及白血球細胞凋亡呈現顯著上升。而且在急性頭部外傷病人血液中的黏附因子sL-selectin，游離性細胞核DNA，以及白血球細胞凋亡和受傷的嚴重度有顯著相關。在預後不佳的族群中，血液中的游離性細胞核DNA和黏附因子sVCAM-1，從受傷後第一天到第七天呈現顯著上升，在白血球細胞凋亡中的單核球和淋巴球第一天的晚期細胞凋亡有顯著上升。另外，受傷後第一天血液中的游離性細胞核DNA和單核球的晚期細胞凋亡是預後的獨立預測因子。
[結論] 我們研究結果的發現將有助於進一步了解急性頭部外傷病人血液中的黏附因子、白血球細胞凋亡和游離性DNA的變化，可能可以提供治療腦部第二次傷害的策略，發展新的藥物以及增加臨床上可監測的生物標誌。

關鍵字

頭部外傷 ； 黏附因子 ； 白血球細胞凋亡 ； 游離脫氧核醣核酸 ； 預後

並列摘要

[Background] Traumatic brain injury (TBI) is a disease process with an initial injury that induces biochemical and cellular changes, which in turn contribute to continuing neuronal damage and death over time. This continuing damage, known as secondary injury, involves the activation of multiple inflammatory and apoptotic pathways. The extent of brain damage is determined by the severity of primary mechanical injury and the intensity of secondary injury cascades causing neuroinflammation and apoptosis. This leads to cerebral edema, increased intracranial pressure, and delayed cellular destruction. Increased circulating cell-free DNA occurs in a variety of critical conditions associated with cell death, and the early and high concentrations of circulating cell-free DNA in traumatic patients suggest that extracellular DNA may originate from damaged tissues. TBI is the most common cause of death and permanent disability in the early decades of life. Despite substantial progress in cerebral neuromonitoring, clinical and neuromonitoring methods are often not sufficient to evaluate and quantify the severity of the initial injury as well as the ongoing secondary destructive processes, and hence they cannot guide efficient therapeutic measures or effectively evaluate the prognosis of the final outcome. To date, little is known about the time course of inflammatory and apoptotic changes in acute TBI. Furthermore, the mechanisms by which inflammatory factors and apoptotic pathways contribute to the outcomes of TBI patients are still unknown.
[Hypothesis] Early and high serum concentrations of adhesion molecules, leukocyte apoptosis and plasma DNA may be associated with the pathogenesis of secondary brain injury after acute TBI, and may therefore be useful in assessing disease severity and predicting clinical outcome.
[Study Designs and Methods] This prospective study on the time course of serum adhesion molecules, serum leukocyte apoptosis and plasma DNA levels in acute TBI patients enrolled 88 adult patients admitted within 24 hours after the onset of acute TBI. Seventy-one blood samples were taken within 24 hours after the onset of TBI, 81 blood samples on Day 4, and 77 blood samples on Day 7 after the onset of acute TBI, and 150 blood samples from 150 age- and sex-matched volunteer subjects were collected. The expressions of serum soluble intercellular cell adhesion-molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble endothelial-selectin (sE-selectin), soluble leukocyte-selectin (sL-selectin) and soluble platelet-selectin (sP-selectin) levels were assessed using commercially available enzyme-linked immunosorbent assay. The expression of leukocyte apoptosis was determined by flow cytometry. The expressions of plasma nuclear and mitochondrial DNA (nDNA and mtDNA) were measured by quantitative real-time polymerase chain reaction assay for the β-globin and ND2 genes.
[Results] Compared to the volunteer subjects, the patients with acute TBI had markedly increased sICAM-1 and sVCAM-1 levels, plasma nDNA and mtDNA levels, and percentage of leukocyte apoptosis. Leukocyte apoptosis, sL-selectin level and plasma nDNA levels in the patients with acute TBI were significantly correlated with the severity of trauma. Plasma nDNA and sVCAM-1 levels increased significantly from Day 1 to Day 7, and late apoptosis was noted in monocytes and lymphocytes on Day 1 in patients with poor outcomes. Furthermore, plasma nDNA and late apoptosis in monocytes on Day 1 were independently associated with outcome in the acute TBI patients.
[Conclusions] The findings in this study provide further understanding of the role of serum adhesion molecules, leukocyte apoptosis and plasma DNA in acute traumatic brain injury, strategies for secondary brain injury, opportunity for drug development, and possible evaluation markers in the panel of traumatic parameters in clinical practice.

並列關鍵字

Traumatic brain injury ； serum adhesion molecules ； serum leukocyte apoptosis ； plasma DNA ； Outcome

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