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題 名 | The Development of a Bioassay for Tumor Necrosis Factor α-derived from Porcine Alveolar Macrophages Using MTT Colorimetric Measurement in a Cell Line, PK-15=以豬腎細胞株和MTT呈色反應所建立的生物檢測法進行源自豬肺泡巨噬細胞腫瘤壞死因子α的測定 |
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作 者 | 龐飛; 趙志勳; 邱明堂; 蕭世烜; 鄭謙仁; | 書刊名 | 中華民國獸醫學會雜誌 |
卷 期 | 23:4 1997.08[民86.08] |
頁 次 | 頁320-331 |
分類號 | 437.24 |
關鍵詞 | 腫瘤壞死因子α; 肺泡巨噬細胞; PK-15豬腎細胞株; 豬; Tumor necrosis factor-α; TNF-α; Alveolar macrophage; PK-15 cell line; MTT; Porcine; |
語 文 | 英文(English) |
中文摘要 | 本研究的目的是建立一個高敏感度的豬隻肺泡巨噬細胞腫瘤壞死因子α的生物 測定法。我們採用豬腎細胞株PK-l5作為腫瘤壞死因子之標的細胞,並以MTT呈色反應作為 偵測系統。首先,以96孔微量培養盤進行使用MTT呈色法檢測PK-15豬腎細胞株存活率條件 的評估,其中包含PK-l5豬腎細胞的濃度、MTT的作用時間及溶劑的選擇。結果顯示2.5╳ 10□細胞/孔的PK-15豬腎細胞與0.1毫克/毫升的MTT共同培養4小時,並以異丙醇為溶劑在 波長570微米下有最佳的吸光值。在同一測試條件下,PK-l5豬腎細胞無論是對基因工程產 製的人腫瘤壞死因子α或是源自於豬肺泡巨噬細胞經由脂多糖(LPS)刺激所產生的腫瘤壞 死因子α均有極佳的感受性。基因工程產製的人腫瘤壞死因子α在0.02微克/孔濃度下, 經18小時作用後,對PK-15豬腎細胞具有87%的細胞毒性,如於每孔中同時加入1毫升的兔 子抗基因工程產製的人腫瘤壞死因子α多價抗體(每10毫升約可中和1000單位的人腫瘤壞 死因子α活性),可中和該細胞毒性的82%。將經由1毫克/孔脂多糖刺激12小時後的豬肺泡 巨噬細胞上清液,以5倍連續稀釋方式加入PK-15豬腎細胞培養盤中作用18小時後,對PK- 15豬腎細胞具有83%的細胞毒性,如於每孔中同時加入1毫升的兔子抗基因工程產製的人腫 瘤壞死因子α多價抗體,可中和該細胞毒性的40%。 |
英文摘要 | The objective of the study was to establish a sensitive bioassay for detecting the tumor necrosis factor-a (TNF-α) derived from porcine alveolar macrophages (AM). In this bioassay, we used PK-15 cell line as the target cell and MTT (3-(4,5- dimethyl thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) calorimetric assay as the detective system. We first evaluated the condition of utilizing MTT to measure the viability of PK-15 cell line. Three parameters, including cell concentration of PK-15 cell line, incubation period of MTT, and solvent for solubilizing formazan crystals, were tested in the microplate system. It was shown that PK-15 cell line at a concentration of 2.5 X 10□ cells/well incubated with 0.1 mg/well MTT for 4 h followed by the addition of isopropanol Yielded a high absorbance reading at 570 nm. Under the condition described, a recombinant human tumor necrosis factor-a (rhTNF-α) and lipopolvsaccharide (LPS)-stimulated porcine AM conditioned media were added. It was found that PK-15 cell line was very sensitive to both rhTNF-α and porcine TNF-Α. Following an 18 h incubation, the rhTNF-α at a concentration of 0.02 ng/well caused an 86% c-,- totoxicity. A rabbit antirhTNF-α polyclonal antibody (I μL/well, 10 μL of the antibody may, neutralize the activity of 1000 international units (IU) of rhTNF-α) nearly abolished the rhTNF-α-induced cvtotoxicity. Similarly, a 5-fold diluted porcine AM conditioned medium derived from the stimulation of 1 X 10□ AM/mL with 1 μg/mL of LPS for 12 hours caused an 83% cytotoxicity. The same amount of the rabbit anti-rhTNF-α polvclonal antibody (1 μL/well) reduced the porcine TNF-α-induced cytotoxicity by 40%. |
本系統中英文摘要資訊取自各篇刊載內容。