一株镰孢霉发酵产物不同极性提取物对兔膝软骨细胞的影响
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摘要
通过研究一株内生梨孢镰孢霉(Fusarium poae)发酵产物中不同极性提取物对兔膝软骨细胞的影响,初步了解其中毒性物质的基本性质,为进一步探讨真菌毒素在大骨节病发病中的可能作用以及分离新的潜在致病物质打下良好基础。本研究以预实验中筛选出的对软骨细胞有较强毒损作用的F.poae菌株进行摇瓶培养;发酵产物先用75%乙醇进行提取,再将乙醇总提取物用不同极性的溶剂进行萃取;将所得各种次级提取物分别作用于体外培养的兔膝软骨细胞,检测与软骨细胞损伤相关的指标,其中细胞存活率采用噻唑蓝(MTT)显色法,Ⅱ型胶原蛋白及蛋白聚糖的含量分别采用免疫组化法和甲苯胺蓝染色法,二者相应的mRNA表达量用半定量RT-PCR进行检测。结果发现,不同极性溶剂提取所得的石油醚、乙醚、乙酸乙酯和水提取物,都能在不同程度上造成培养兔软骨细胞的存活率、Ⅱ型胶原蛋白和蛋白聚糖的含量以及相应mRNA的表达量降低;毒性强弱有随溶剂极性增强而减弱的趋势,以石油醚分部的综合毒损作用最强,其后依次为乙醚、乙酸乙酯和水提取物。镰孢霉发酵产物的各种不同极性提取物中,都存在对软骨细胞相关指标造成程度不等毒损作用的物质。这些结果支持我们关于大骨节病的发病可能和多种真菌毒素的综合作用相关的观点。
In this study, the effects of four solvent extracts of a Fusarium strain on rabbit knee chondrocytes were analyzed to determine the basic properties of the toxic substances in these extracts and to provide clear evidence for further evaluating the possible effects of mycotoxins on the onset of Kashin-Beck disease and isolating new potential pathogens. A strain of Fusarium poae which showed strong toxic effects on cartilage cells in preliminary experiments was cultured and its fermentation products were extracted with 75% ethanol. The total extract was extracted with different polar solvents. All secondary extracts were then applied to cultured rabbit chondrocytes in vitro to determine the indices closely related to chondrocyte damage. Cell viability was determined by thiazole blue staining(MTT assay), and the contents of type Ⅱ collagen and proteoglycan were determined by immunohistochemical method and toluidine blue staining. The m RNA expression of both of them was detected by semi-quantitative reverse transcriptase-polymerase chain reaction(RT-PCR). The results showed that the petroleum ether, ethyl ether, ethyl acetate and water extracts that were extracted from different polar solvents more or less reduced the survival rate of rabbit chondrocytes, the synthesis of type Ⅱ collagen and proteoglycan and the expression of their corresponding m RNA. Toxicity was weakened by increased solvent polarity, and the toxicity of the petroleum ether extract was strongest, followed by the ethyl ether, ethyl acetate and water extracts. Clearly, the various solvent extracts of the fermentation products of this Fusarium strain showed varying degrees of toxicity on related indices of cartilage cells. These results showed that the onset of Kashin-Beck disease might be related to the combined action of multiple mycotoxins.
In this study, the effects of four solvent extracts of a Fusarium strain on rabbit knee chondrocytes were analyzed to determine the basic properties of the toxic substances in these extracts and to provide clear evidence for further evaluating the possible effects of mycotoxins on the onset of Kashin-Beck disease and isolating new potential pathogens. A strain of Fusarium poae which showed strong toxic effects on cartilage cells in preliminary experiments was cultured and its fermentation products were extracted with 75% ethanol. The total extract was extracted with different polar solvents. All secondary extracts were then applied to cultured rabbit chondrocytes in vitro to determine the indices closely related to chondrocyte damage. Cell viability was determined by thiazole blue staining(MTT assay), and the contents of type Ⅱ collagen and proteoglycan were determined by immunohistochemical method and toluidine blue staining. The m RNA expression of both of them was detected by semi-quantitative reverse transcriptase-polymerase chain reaction(RT-PCR). The results showed that the petroleum ether, ethyl ether, ethyl acetate and water extracts that were extracted from different polar solvents more or less reduced the survival rate of rabbit chondrocytes, the synthesis of type Ⅱ collagen and proteoglycan and the expression of their corresponding m RNA. Toxicity was weakened by increased solvent polarity, and the toxicity of the petroleum ether extract was strongest, followed by the ethyl ether, ethyl acetate and water extracts. Clearly, the various solvent extracts of the fermentation products of this Fusarium strain showed varying degrees of toxicity on related indices of cartilage cells. These results showed that the onset of Kashin-Beck disease might be related to the combined action of multiple mycotoxins.
引文
Cao J.L.,Xiong Y.M.,Li S.C.,Zhang S.Y.,Bi H.Y.,and Mo D.X.,1998,The experimental study on the effects of six mycotoxins on the cultural chondrocytes,J.Xi'an Med.Univ.,10(1):1-8
Cao Y.,2013,The effect of dominant fungi isolated of Triticum aestivum from a KBD area in Shaanxi province on chondrocyte from rabbit'knees,Thesis for M.S.,Northwest University,Supervisor:Yin H.,pp.12-16(曹阳,2013,陕西省大骨节病区小麦优势真菌对兔膝软骨细胞的影响,硕士学位论文,西北大学,导师:殷红,pp.12-16)
Ding R.,and Guo W.S.,2010,Investigative advancement of pathogenesis of kashin-beck disease,Yixue Zongshu(Medical Recapitulate),16(6):901-903(丁冉,郭万首,2010,大骨节病发病机制的研究进展,医学综述,16(6):901-903)
Griffin D.H.,ed.,1994,Fungal physiology(2nd ed.),Wiley-Liss,Inc.,New York,Chichester,Brisbane,Toronto and Singapore,pp.247-271
Guo X.,2010,New progression and prospect on pathogenesis and control of Kashin-Beck disease,Guowai Yixue(Yixue Dili Fence)(Foreign Medical Science(Section of Medgeography)),31(1):1-4(郭雄,2010,大骨节病发病机制与防治研究的新进展及其展望,国外医学(医学地理分册),31(1):1-4)
Guo X.,2008,Progression and prospect of etiology and pathogenesis of Kashin-Beck disease,Xi'an Jiaotong Daxue Xuebao(Yixueban)(Journal of Xi'an Jiaotong University(Medical Sciences)),29(5):481-488(郭雄,2008,大骨节病病因与发病机制的研究进展及其展望,西安交通大学学报(医学版),29(5):481-488)
Guo X.,Ma W.J.,Zhang F.,Ren F.L.,Qu C.J.,and Lammi M.J.,2014,Recent advances in the research of an endemic osteochondropathy in China:Kashin-Beck disease,Osteoarthritis and Cartilage,22(11):1774-1783
Hu J.T.,He B.,Zhao Y.M.,Cheng B.L.,Zhang F.,Zhang Y.,and Yin H.,2015,Identification of an endophytic fungus in wheat and effect of its fermentation product on rabbit cartilage cells,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(11):2421-2427(胡锦涛,贺兵,赵玉濛,成博伦,张枫,张燕,殷红,2015,一株小麦内生真菌的鉴定及其发酵产物对兔软骨细胞的影响,基因组学与应用生物学,34(11):2421-2427)
Ren J.,2012,Isolation and identification of the fungi isolated from Triticum aestivum from a KBD area in Qinghai,Thesis for M.S.,Northwest University,Supervisor:Yin H.,pp.49(任杰,2012,青海大骨节病区小麦籽粒内生真菌分类鉴定,硕士学位论文,西北大学,导师:殷红,pp.49)
Wang W.W.,ed.,2008,Microbial physiology,Science Press,Beijing,China,pp.193-194(王卫卫,主编,2008,微生物生理学,科学出版社,中国,北京,pp.193-194)
Xie X.Y.,2014,Effects of Alternaria isolated of from wheat in a KBD area on condrocyte from rabbit's knees,Thesis for M.S.,Northwest University,Supervisor:Yin H.,pp.16-42(谢晓燕,2014,青海大骨节病区小麦内生链格孢霉对兔膝软骨细胞的影响,硕士学位论文,西北大学,导师:殷红,pp.16-42)
Zhang F.,Guo X.,Wang S.,Wu S.X.,and Wang L.X.,2012,Gene expression profile of mycotoxin-related environmental response genes in the peripheral blood in Kashin-Beck disease,Xi'an Jiaotong Daxue Xuebao(Yixueban)(Journal of Xi'an Jiaotong University(Medical Sciences)),33(4):400-406(张峰,郭雄,王爽,武世勋,王立新,2012,大骨节病患者外周血真菌毒素环境反应基因表达谱的研究,西安交通大学学报(医学版),33(4):400-406)
Zhang F.,Zhang L.,Zhang C.Y.,Zhao S.M.,Cai C.,Zhang Y.,and Yin H.,2015,Isolation and identification of a T-2 toxin-producing Fusarium poae strain and studies on its culture conditions,Weishengwuxue Tongbao(Microbiology China),42(2):340-348(张枫,张蕾,张重阳,赵胜明,蔡晨,张燕,殷红,2015,一株产T-2毒素镰孢菌的分离,鉴定及其产毒条件的研究,微生物学通报,42(2):340-348)
Zhang K.,Influences of dominant fungi isolated from maize and their fermentative products on cultured cartilage cells,Thesis for M.S.,Northwest University,Supervisor:Yin H.,pp.9-16(张凯,2013,玉米主要真菌及其发酵产物对培养软骨细胞的影响,硕士学位论文,西北大学,导师:殷红,pp.9-16)
Zhang Y.,Zhang F.,Xie X.Y.,He B.,Zhao Y.M.,Hu J.T.,and Yin H.,2015,Effect of selenium and metabo lites of a Fusarium ssp.isolated from wheat head on chondrocytes,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(10):2067-2074(张燕,张枫,谢晓燕,贺兵,赵玉濛,胡锦涛,殷红,2015,青海大骨节病区小麦内生镰刀菌和硒对兔膝软骨细胞的影响,基因组学与应用生物学,34(10):2067-2074)
Zhao Y.M.,Xie X.Y.,He B.,Hu J.T.,Cheng B.L.,Zhang F.,Zhang Y.,and Yin H.,Effect of metabolite in an entophytic Alternaria alternate of wheat on rabbit cartilage cells,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(4):924-930(赵玉濛,谢晓燕,贺兵,胡锦涛,成博伦,张枫,张燕,殷红,2016,一株小麦内生链格孢霉代谢产物对兔膝软骨细胞的影响,基因组学与应用生物学,35(4):924-930)
Cao Y.,2013,The effect of dominant fungi isolated of Triticum aestivum from a KBD area in Shaanxi province on chondrocyte from rabbit'knees,Thesis for M.S.,Northwest University,Supervisor:Yin H.,pp.12-16(曹阳,2013,陕西省大骨节病区小麦优势真菌对兔膝软骨细胞的影响,硕士学位论文,西北大学,导师:殷红,pp.12-16)
Ding R.,and Guo W.S.,2010,Investigative advancement of pathogenesis of kashin-beck disease,Yixue Zongshu(Medical Recapitulate),16(6):901-903(丁冉,郭万首,2010,大骨节病发病机制的研究进展,医学综述,16(6):901-903)
Griffin D.H.,ed.,1994,Fungal physiology(2nd ed.),Wiley-Liss,Inc.,New York,Chichester,Brisbane,Toronto and Singapore,pp.247-271
Guo X.,2010,New progression and prospect on pathogenesis and control of Kashin-Beck disease,Guowai Yixue(Yixue Dili Fence)(Foreign Medical Science(Section of Medgeography)),31(1):1-4(郭雄,2010,大骨节病发病机制与防治研究的新进展及其展望,国外医学(医学地理分册),31(1):1-4)
Guo X.,2008,Progression and prospect of etiology and pathogenesis of Kashin-Beck disease,Xi'an Jiaotong Daxue Xuebao(Yixueban)(Journal of Xi'an Jiaotong University(Medical Sciences)),29(5):481-488(郭雄,2008,大骨节病病因与发病机制的研究进展及其展望,西安交通大学学报(医学版),29(5):481-488)
Guo X.,Ma W.J.,Zhang F.,Ren F.L.,Qu C.J.,and Lammi M.J.,2014,Recent advances in the research of an endemic osteochondropathy in China:Kashin-Beck disease,Osteoarthritis and Cartilage,22(11):1774-1783
Hu J.T.,He B.,Zhao Y.M.,Cheng B.L.,Zhang F.,Zhang Y.,and Yin H.,2015,Identification of an endophytic fungus in wheat and effect of its fermentation product on rabbit cartilage cells,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(11):2421-2427(胡锦涛,贺兵,赵玉濛,成博伦,张枫,张燕,殷红,2015,一株小麦内生真菌的鉴定及其发酵产物对兔软骨细胞的影响,基因组学与应用生物学,34(11):2421-2427)
Ren J.,2012,Isolation and identification of the fungi isolated from Triticum aestivum from a KBD area in Qinghai,Thesis for M.S.,Northwest University,Supervisor:Yin H.,pp.49(任杰,2012,青海大骨节病区小麦籽粒内生真菌分类鉴定,硕士学位论文,西北大学,导师:殷红,pp.49)
Wang W.W.,ed.,2008,Microbial physiology,Science Press,Beijing,China,pp.193-194(王卫卫,主编,2008,微生物生理学,科学出版社,中国,北京,pp.193-194)
Xie X.Y.,2014,Effects of Alternaria isolated of from wheat in a KBD area on condrocyte from rabbit's knees,Thesis for M.S.,Northwest University,Supervisor:Yin H.,pp.16-42(谢晓燕,2014,青海大骨节病区小麦内生链格孢霉对兔膝软骨细胞的影响,硕士学位论文,西北大学,导师:殷红,pp.16-42)
Zhang F.,Guo X.,Wang S.,Wu S.X.,and Wang L.X.,2012,Gene expression profile of mycotoxin-related environmental response genes in the peripheral blood in Kashin-Beck disease,Xi'an Jiaotong Daxue Xuebao(Yixueban)(Journal of Xi'an Jiaotong University(Medical Sciences)),33(4):400-406(张峰,郭雄,王爽,武世勋,王立新,2012,大骨节病患者外周血真菌毒素环境反应基因表达谱的研究,西安交通大学学报(医学版),33(4):400-406)
Zhang F.,Zhang L.,Zhang C.Y.,Zhao S.M.,Cai C.,Zhang Y.,and Yin H.,2015,Isolation and identification of a T-2 toxin-producing Fusarium poae strain and studies on its culture conditions,Weishengwuxue Tongbao(Microbiology China),42(2):340-348(张枫,张蕾,张重阳,赵胜明,蔡晨,张燕,殷红,2015,一株产T-2毒素镰孢菌的分离,鉴定及其产毒条件的研究,微生物学通报,42(2):340-348)
Zhang K.,Influences of dominant fungi isolated from maize and their fermentative products on cultured cartilage cells,Thesis for M.S.,Northwest University,Supervisor:Yin H.,pp.9-16(张凯,2013,玉米主要真菌及其发酵产物对培养软骨细胞的影响,硕士学位论文,西北大学,导师:殷红,pp.9-16)
Zhang Y.,Zhang F.,Xie X.Y.,He B.,Zhao Y.M.,Hu J.T.,and Yin H.,2015,Effect of selenium and metabo lites of a Fusarium ssp.isolated from wheat head on chondrocytes,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(10):2067-2074(张燕,张枫,谢晓燕,贺兵,赵玉濛,胡锦涛,殷红,2015,青海大骨节病区小麦内生镰刀菌和硒对兔膝软骨细胞的影响,基因组学与应用生物学,34(10):2067-2074)
Zhao Y.M.,Xie X.Y.,He B.,Hu J.T.,Cheng B.L.,Zhang F.,Zhang Y.,and Yin H.,Effect of metabolite in an entophytic Alternaria alternate of wheat on rabbit cartilage cells,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(4):924-930(赵玉濛,谢晓燕,贺兵,胡锦涛,成博伦,张枫,张燕,殷红,2016,一株小麦内生链格孢霉代谢产物对兔膝软骨细胞的影响,基因组学与应用生物学,35(4):924-930)