https://scholars.lib.ntu.edu.tw/handle/123456789/144415
標題: | 利用cDNA微陣列分析初期敗血症病患週邊血液單核球的基因表現差異:革蘭氏陽性與革蘭氏陰性敗血症之比較 Differentiate Gram Positive from Gram Negative Sepsis:Gene Expression Analysis in Peripheral Blood Mononuclear Cells from Patients with Early Sepsis |
作者: | 蔡幸真 Tsai, Hsing-Chen |
關鍵字: | cDNA微陣列;敗血症;基因表現;sepsis;gene expression;cDNA microarray | 公開日期: | 2004 | 摘要: | 敗血症(sepsis)乃重症病患之主要死因。早期得知致病原並給予正確抗生素治療,對病患預後有決定性之影響。目前無論由初期臨床症狀或等待血液培養結果,欲早期得知致病原皆有其困難性。釵h證據顯示,革蘭氏陰性菌與革蘭氏陽性菌所產生之敗血症,其臨床表現、致病機轉、宿主反應及對於抗細胞激素的療效方面皆有明顯之不同。因此,我們假設敗血症病患,其免疫細胞對於革蘭氏陽性和陰性菌之感染應有不同的基因表現。本研究中,我們嘗試運用基因微陣列技術(cDNA microarray),分析革蘭氏陽性菌及陰性菌敗血症病患之週邊血液單核球的基因表現差異,期能發展敗血症致病原的早期診斷工具,進而探討敗血症複雜之致病機轉。 我們前瞻性地收集三組血液檢體:革蘭氏陽性菌組、革蘭氏陰性菌組及無敗血症對照組。革蘭氏陽性菌組及陰性菌組,乃自符合敗血症定義、且處於感染早期的病人身上抽血,並依據血液培養結果作分類,所有病患必須由血液培養證實為菌血症;對照組則為至台大健康管理中心接受健檢且大於40歲的成人。抽血後立即分離出週邊血液單核球,萃取出核醣核酸並進行增量反應(amplification),接著使用由台大微陣列核心實驗室所製備之人類cDNA微陣列薄膜(9600點)及血管新生基因晶片(AngioChip)作雜交呈色。所得到的結果經高解析度(3000dpi)平台掃描器掃描,以影像處理軟體(GenePix,Axon instruments )量化基因表現強度後,將不同微陣列薄膜的資料以基因表現強度總和(total intensity)作校正,經對數轉換(log transformation)及標準化(standardization)等步驟,再以student t-test進行分析,尋找不同組別間有差異表現的基因群。 結果顯示,在敗血症組(革蘭氏陽性菌組,n=6;革蘭氏陰性菌組,n=6)及對照組(n=10)間有明顯不同的基因表現圖譜,2000多個基因達統計上的顯著差異。革蘭氏陽性菌及陰性菌兩組的基因表現圖譜大致相似,但仍有60個基因在兩組間達到統計顯著差異,其分類包括訊息傳遞分子(如phosphatidylinositol 3-kinase catalytic subunit, NF-kB p65 subunit, zinc finger proteins)、細胞接合因子(Thrombomodulin, Platelet glycoprotein IX, MAdCAM-1)、蛋白分解系統(26S proteasome subunit, ubiquitin specific protease 8及von Hippel-Lindau syndrome, pVHL)、趨化激素(Homo sapiens CC chemokine gene cluster)與轉錄轉譯相關基因等等。其中pVHL在同步定量聚合酶連鎖反應中,革蘭氏陰性菌組之平均表現量為對照組之2.10倍,革蘭氏陽性菌組為1.52倍,趨勢與基因微陣列的結果相仿。 本研究初步顯示,不論是革蘭氏陽性菌或陰性菌感染,敗血症病患之週邊血液單核球的多數基因均有一致的表現方向,但仍有少數基因屬菌原差異之表現,符合先前敗血症雖是病患本身免疫系統針對不同細菌入侵的共同反應,但針對革蘭氏陽性和陰性菌感染,仍有不同基因表現的假設。後續研究菌原差異表現之基因將有助於吾人探討敗血症病因學之複雜機轉,發展敗血症致病原之早期診斷工具,及提供未來敗血症治療之對策。 Sepsis is one of the leading causes of death in critically ill patients. Early identification of pathogens and treatment with appropriate antibiotics are crucial for patients’outcome. However, difficulties remain for clinicians to timely identify the causative pathogens of sepsis, either by presenting symptoms or by laboratory studies. Plenty of evidences disclosed that Gram-positive sepsis differed substantially from Gram-negative sepsis in clinical manifestations, immunopathogenesis, host response, and response to the treatment with anti-inflammatory agents. Hence, we proposed that gene expression profiles involving the response of host immune system were different between Gram-positive and Gram-negative sepsis. We used cDNA microarry technique to analyze the gene expression patterns of peripheral blood mononuclear cells (PBMC) from patients in the early phase of Gram-positive or Gram-negative sepsis. Once identifying pathogen-specific genes, we anticipated to develop a diagnostic tool for early identification of the causative pathogen, and to explore the complex pathogenesis of sepsis. Blood samples were prospectively collected from three groups of subjects: the Gram-positive group, the Gram-negative group, and the non-sepsis control group. Patients who fit the definition of sepsis had their blood sampled in the early phase (within 24 hours) of disease, and were later classified into Gram-positive or -negative group based on the result of blood culture. Only those with positive blood culture results were enrolled into the study. Non-sepsis control group included people who were older than 40 years of age and were undertaking health examinations in the Health Management Center of NTUH. The peripheral blood mononuclear cells were immediately isolated after blood sampling, followed by extraction of total RNA and amplification. cDNA microarrays with 9600 non-redundant EST clones and with 346 angiogenesis-related genes were used. The signals developed during the hybridization process were scanned with a high-resolution (3000dPi) platform scanner, and digitalized with image processing software (GenePix, Axon instruments). To standardize and normalize the gene expression on each membrane for comparison, the intensities of genes were rescaled with the sum of total signal intensities on the individual membrane, followed by log transformation and standardization. The difference of gene expression was analyzed with student t test. The results demonstrated a substantial difference of gene expression profile among the Gram-positive (n=6), the Gram-negative (n=6) and the control groups (n=10). More than 2000 genes were differentially expressed between sepsis and non-sepsis patients. Among these, sixty genes were statistically different between the Gram-positive and the Gram-negative sepsis, including those involved in signal transduction pathway (eg. NF-kB p65 subunit, zinc finger proteins and phosphatidylinositol 3-kinase catalytic subunit), cell-cell interaction (eg. Thrombomodulin, Platelet glycoprotein IX, MAdCAM-1), ubiquitin-proteasome proteolysis (eg. 26S proteasome subunit, ubiquitin specific protease 8 and von Hippel-Lindau syndrome), chemokines (Homo sapiens CC chemokine gene cluster), and genes participating in transcription or translation processes. We later quantified the expression of von Hippel-Lindau syndrome gene by RT-QPCR and revealed a coherent trend found by cDNA microarray. The mean expression amount of VHL gene was 2.10 folds of control group in Gram-negative group, and it was 1.52 folds of controls in Gram-positive group. Our preliminary results had shown that most genes were regulated similarly in either Gram-positive or Gram-negative sepsis, which was in agree with the assumption of final common pathway in the pathogenesis of sepsis. However, several genes did have a pathogen-specific expression, which was also in agree with our hypothesis that Gram-positive and Gram-negative sepsis differed in the pathogenesis. Further studies in these pathogen-specific genes may help to elucidate the complex pathogenic mechanism of sepsis, to develop a rapid diagnostic tool for identifying causative pathogens and, perhaps, to assist establishing strategies for treating sepsis. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/55459 | 其他識別: | zh-TW |
顯示於: | 臨床醫學研究所 |
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