四株虫生真菌的鉴定及其醇提取物对Hela细胞的抑制作用

Identification of four entomogenous fungi and the inhibitory effect of their ethanol extracts on Hela cells

摘要:

【背景】虫生真菌是非常重要的自然资源，但被发现和利用的种类相对较少。【目的】鉴定从野外采集的4株虫生真菌，并探讨4株菌的醇提物对宫颈癌Hela细胞的抑制活性。【方法】结合形态学特征与rDNA ITS和β-Tubulin序列分析对4株虫生真菌进行种类鉴定，采用四甲基偶氮唑盐[3-(4,5-Dimethyl-2-Thiazolyl)-2,5-Diphenyl-2-H-Tetrazolium Bromide，MTT]比色法分析它们的菌丝醇提物(M-1、M-2、M-3、M-4)和发酵液醇提物(F-1、F-2、F-3、F-4)对Hela细胞增殖的抑制活性，并进一步利用细胞形态学观察和流式细胞术检测所筛选抑制作用最强的醇提取物的抑制活性。【结果】经鉴定，菌株IS和LS均为环链棒束孢(Isaria cateniannulata)，菌株HLS和HLS2均为粉棒束孢(Isaria farinosa)；MTT实验结果显示8种醇提物对Hela细胞增殖均有一定的抑制作用，而且菌丝醇提物和发酵液醇提物的抑制作用大小排序均为：粉棒束孢HLS>环链棒束孢LS>粉棒束孢HLS2>环链棒束孢IS，其中，粉棒束孢HLS的发酵液醇提物F-3的抑制活性最强(P<0.001)，处理24 h后其IC50值为0.667 8 mg/mL，而其他7种醇提物的IC50值均大于1 mg/mL；细胞形态学观察显示F-3在处理时间24 h时，对Hela细胞生长的抑制作用随着浓度的增加而加大，而对正常细胞BHK-21仅有轻微的毒性作用；流式细胞仪检测发现F-3可诱导Hela细胞凋亡，当处理浓度为1.0 mg/mL时，细胞总凋亡率可达到32.45%。【结论】4株菌对Hela细胞增殖的抑制作用存在着显著差异，这可能与4株菌的地理来源、寄主等因素有关；来源于粉棒束孢HLS株系的发酵液醇提物F-3对正常细胞BHK-21的增殖和生长无显著抑制作用，但对Hela癌细胞的增殖和生长抑制活性最强，而且具有诱导肿瘤细胞凋亡的作用，暗示其具有研究利用价值。

Abstract:

[Background] The entomogenous fungi were important resources from the nature, but few of them were found and utilized comparatively. [Objective] To identify four entomogenous fungi collected from the field, and to explore the inhibitory effect of their ethanol extracts on Hela cells. [Methods] Four entomogenous fungi were identified by morphological characteristics, rDNA ITS and β-tubulin sequence analysis. The inhibitory effect of ethanol extracts from mycelium (M-1, M-2, M-3, M-4) and ferment liquid (F-1, F-2, F-3, F-4) on Hela cell proliferation were analyzed by MTT assay. The inhibitory activity of the ethanol extract with the strongest inhibitory effect was detected by cell morphological observation and flow cytometry. [Results] After identification, strains IS and LS are Isaria catenianulata, strains HLS and HLS2 are Isaria farinose. The results of MTT showed that eight kinds of ethanol extracts inhibited the proliferation of Hela cells to some extent, and the order of inhibitory effect of ethanol extracts from mycelium and ferment liquid was Isaria farinosa HLS>Isaria catenianulata LS>Isaria farinosa HLS2>Isaria catenianulata IS, of which F-3 had the strongest inhibitory activity (P<0.001), and its IC50 value was 0.667 8 mg/mL after 24 hours of treatment, while the IC50 value of the other seven ethanol extracts were higher than 1 mg/mL. The cell morphological observation showed that the inhibitory effect of F-3 on Hela cell growth increased with the increase of the concentration when Hela cells were treated with F-3 for 24 h, however. F-3 has only slight toxic effect on BHK-21 cells; flow cytometry showed that F-3 could induce apoptosis of Hela cells, and the total apoptosis rate could reach 32.45% when the treatment concentration was 1.0 mg/mL. [Conclusion] The inhibitory effect of four entomogenous fungi on Hela cell proliferation is significantly different, which may be related to the geographical origin and host of four entomogenous fungi. F-3 ethanol extract from Isaria farinosa has no significant inhibitory effect on the proliferation and growth of BHK-21 cells, but has the strongest inhibitory effect on Hela cells proliferation and growth and has the effect of inducing tumor cells apoptosis, suggesting that it has the further research value.