一株内生链格孢霉代谢产物的分离及其对兔软骨细胞的影响

发布时间：2018-09-14 14:02

【摘要】：大骨节病(Kashin-Beck disease,KBD)在我国分布广、危害大,但至今其病因尚未明了。目前,已有一些研究表明,链格孢霉及其毒素与大骨节病的发生存在一定的关系,本实验通过观察分离自青海大骨节病病区小麦内生链格孢霉代谢产物对兔软骨细胞的影响,寻找除已知毒素外对软骨细胞有毒损作用的物质,为探究大骨节病的病因研究提供新的启示。本实验选择一株分离自青海大骨节病区小麦的内生链格孢霉属真菌(编号13-8-A),通过显微形态学观察和ITS序列分析鉴定本实验菌种,将菌株进行麦仁液体发酵,采用溶剂萃取法,硅胶柱色谱层析,薄层色谱层析,高效液相色谱法等分离提取手段,将逐级分离得到的物质进行细胞检测,通过MTT法检测软骨细胞存活率、RT-PCR检测蛋白聚糖和Ⅱ型胶原蛋白mRNA的表达量、甲苯胺蓝染色法和免疫组化染色法检测这两种蛋白的表达。通过观察不同分级提取物对兔膝软骨细胞上述与大骨节病有关指标的影响,以寻找对软骨细胞有明显损伤的物质。经鉴定13-8-A菌株为互隔链格孢霉(Alternaria alternate)。该菌株代谢产物的石油醚提取物通过硅胶柱层析分离的成分基本不抑制软骨细胞的生长,对蛋白聚糖和Ⅱ型胶原蛋白的表达及其mRNA表达量的抑制作用较弱。但在同样处理浓度下,该菌株的乙醚提取物的粗分离成分对软骨细胞的损伤远远强于石油醚。HPLC检测结果显示,该菌株可以产生链格孢霉毒素——链格孢霉单甲基乙醚(AME),标准品AME可以轻微抑制兔软骨细胞的生长,该毒素对软骨细胞的损伤主要表现为抑制蛋白聚糖的表达及其mRNA的表达量；而另外一种HPLC分离保留时间峰值在11min的收集物质可以明显降低软骨细胞存活率、蛋白聚糖和Ⅱ型胶原蛋白的表达及其mRNA表达量。故该菌株代谢产物中还存在除AME外能抑制软骨细胞生长的物质,鉴于该物质对软骨细胞的毒损作用比已知的AME更强,其值得更进一步研究。由此可知,互隔链孢霉13-8-A代谢产物中应该存在多种可以损伤软骨细胞的物质。
[Abstract]:Kaschin-Beck disease (Kashin-Beck disease,KBD) is widely distributed and harmful in China. At present, some studies have shown that streptomyces and its toxins are related to the occurrence of Kaschin-Beck disease (KBD). The effects of endogenous streptomyces metabolites on rabbit chondrocytes were observed in this experiment. In order to provide new inspiration for studying the etiology of Kaschin-Beck disease (Kashin Beck disease), it is necessary to find out the toxic substances that affect chondrocytes in addition to known toxins. A strain of endophytic Streptomyces (No. 13-8-A) isolated from Kashin-Beck disease region in Qinghai was selected in this experiment. The strain was identified by microscopic observation and ITS sequence analysis. Silica gel column chromatography, thin-layer chromatography and high performance liquid chromatography were used to separate and extract the separated substances for cell detection. The expression of proteoglycan and type 鈪，

本文编号：2242916