adadoda内生曲霉l -天冬酰胺酶对A549细胞株的细胞毒活性研究

D. Prabavathy
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引用次数: 5

摘要

l -天冬酰胺酶是一类具有广谱抗肿瘤活性的酰胺酶。它催化l -天冬酰胺水解成氨和l -天冬氨酸。目的:分离产l -天冬酰胺酶内生真菌,并对其A549细胞株进行细胞毒性研究。方法:筛选以天冬酰胺为源、酚红为指示剂的改性Czapek-Dox培养基。鉴定阳性区,继代培养获得纯培养物。结果:进行了天冬酰胺酶的定量测定,产量为1.8916 (μmol/ml)。酶的分子量为66 kDa。采用MTT法和吖啶橙/溴化乙啶双染色法观察该酶对A549细胞的诱导死亡类型。处理后的凋亡细胞比例显著增加(P < 0.001),分别为62%和84%。用流式细胞术分析不同浓度的部分纯化样品(80 μg/ml和160 μg/ml)对细胞周期阻滞期的影响。较低浓度(80 μg/ml)在S期停止,细胞累积19%。在较高浓度(160 μg/ml)下,S期细胞数量增加30%,其他细胞周期均有变化。结论:分离得到的天冬酰胺酶对A549细胞株具有明显的细胞毒活性。进一步的动物模型研究、毒性试验和药理学研究将有助于证实这种酶作为抗癌药物的使用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Cytotoxic activity of L-asparaginase isolated from endophytic aspergillus nomius of Justicia adhatoda on A549 cell lines
Introduction: L-Asparaginase belonging to a group of amidase possesses a broad spectrum of antitumor activity. It catalyzes the hydrolysis of L-asparagine to ammonia and L-aspartic acid. Objective: In the present study, L-asparaginase producing endophytic fungi were isolated from Justicia adhatoda and cytotoxicity studied on A549 cell line. Methodology: The screening modified Czapek-Dox media contained asparagine as the source and phenol red as the indicator. The positive zones were identified and sub cultured to obtain pure culture. Results: A quantitative assay of asparaginase was carried out and the level of production was found to be 1.8916 (μmol/ml). The molecular weight of the enzyme was 66 kDa. MTT assay and dual staining with acridine orange/ethidium bromide were done to assess the type of cell death induced by the enzyme in A549 cells. The percentage of apoptotic cells after treatment showed a drastic increase in apoptotic cells (P < 0.001) to 62% and 84%, respectively. Flow cytometry analysis was performed to evaluate the cell cycle arrest phase induced by different concentration of partially purified sample (80 μg/ml and 160 μg/ml). Lower concentration (80 μg/ml) showed arrest at S phase with 19% cells accumulated. At higher concentration (160 μg/ml), it showed an increased cell population at S phase with 30% with alterations in the other phase of cell cycle. Conclusion: The above observations show that the isolated asparaginase exhibited a marked cytotoxic activity against A549 cell lines. Further animal model studies, toxicity assays and pharmacological studies of this asparaginase from the endophyte would help to authenticate the use of this enzyme as an anticancer drug.
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