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

龐飛(Victor Fei Pang) ； 趙志勳(Jihn-Shiun Chao) ； 邱明堂(Ming-Tang Chiou) ； 蕭世烜(Shih-Suan Hsiao) ； 鄭謙仁(Chian-Ren Jeng)

《中華民國獸醫學會雜誌》 23卷4期 (1997/08) Pp. 320-331

https://doi.org/10.7009/JTCSVS.199708.0320

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-15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-15豬腎細胞的濃度、MTT的作用時間及溶劑的選擇。結果顯示2.5×10^4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-15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，如於每孔中同特加入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%。

關鍵字

腫瘤壞死因子α ； 肺泡巨噬細胞 ； PK-15豬腎細胞株 ； MTT ； 豬

並列摘要

The objective of the study was establish a sensitive bioassay for detecting the tumor necrosis factor-α (TNF-a) 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) colorimetric 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 2.5×10^4 cells/well incubated with 0.1mg/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-α (rhTNF-α) and lipopolysaccharide (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 0.02ng/well caused an 86% cytotoxicity. A rabbit anti-rhTNF-α polyclonal antibody (1μL/well, 10μL of the antibody may neutralize the activity of 1000 international units (IU) of rhTNF-α) nearly abolished the rhTNF-α-induced cytotoxicity. Similarly, a 5-fold diluted porcine AM conditioned medium derived from the stimulation of 1×10^6 AM/mL with 1μg/mL of LPS for 12 hours caused an 83% cytotoxicity. The same amount of the rabbit anti-rhTNF-α polyclonal antibody (1μL/well) reduced the porcine rhTNF-α-induced cytotoxicity by 40%.

並列關鍵字

Tumor necrosis factor-a (TNF-a) ； Alveolar macrophage ； PK-15 cell line ； MTT ； Porcine