| KBD (Kashin-Beck disease, KBD) is widely distributed in our country, seriously harm, but its etiology remians unclear up to now. At present, some studies suggest that there is a certain relationship between the occurrence of Alternaria and toxins with KBD.This experiment has isolated a dominant Entophytic Alternaria in wheat from Kaschin-beck disease district of Qinghai province in order to explore its metabolite's effect on rabbit cartilage cells, looking for unknown toxins and its can damage chondrocytes.To explore the pathogeny of kashin-beckfactors provide new inspiration.This experiment selects a strain that endogenous Alternaria isolated from the wheat in endemic Qinghai Kaschin-beck ward, The strain was identified by morphological observation and ITS sequences analysis.We taken strain cultured with liquid medium of wheat, using solvent extraction, silica gel column chromatography, thin layer chromatography, HPLC and other separation and extraction means to test the material obtained by separation on cells.The survival rate of the cells was detected by methylthiazolyldiphenyl-tetrazoliumbromide (MTT), while RT-PCR measured the mRNA expressions of aggrecan and collagen ?, the expressions of aggrecan and collagen ? were examined by toluidine blue (TB) and immunological histological chemistry (IHC) as well. By observing the effects of different grading extract on the rabbit knee cartiage cells, eventually to find significant material damaged cells.The experimental strain (No.13-8-A) was went into as Alternaria alternate, its metabolites in coarse petroleum ether extract isolated ingredients through silica gel column chromatography does not substantiallly inhibit the growh of cartilage cells, for aggrecan and collagen ? expression and its mRNA expression level of inhibition weaker. But under the same treatment concentration, the strain of ether extract rough separation of components of cartilage damage is far stronger than the petroleum ether.HPLC results showed that Alternaria alternate can produce Alternaria mycotoxin -alternariol methyl ether(AME), AME can slightly inhibit the growth of cartiage cells, the toxin damage mainly cartilage cells is mainly characterized by inhibiting the expression of aggrecan and its mRNA expression. Another peak in 11min HPLC separation of collecting material can abviously reduce the cartiage cell survival rate,the expression of aggrecan and collagen II and its mRNA expression.besides AME, its metabolitea have a substance that can inhibit the growth of cartiage cell, given the substance of damaging cartilag cells of poison effect was stronger than the AME known, which is worth futher study. Therefore, Alternaria alternate 13-8-A may contain a large number of products that can damage cartiage cells. |
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