【摘要】：大骨節(jié)病(Kashin-Beck disease,KBD)在我國分布廣、危害大,但至今其病因尚未明了。目前,已有一些研究表明,鏈格孢霉及其毒素與大骨節(jié)病的發(fā)生存在一定的關(guān)系,本實驗通過觀察分離自青海大骨節(jié)病病區(qū)小麥內(nèi)生鏈格孢霉代謝產(chǎn)物對兔軟骨細(xì)胞的影響,尋找除已知毒素外對軟骨細(xì)胞有毒損作用的物質(zhì),為探究大骨節(jié)病的病因研究提供新的啟示。本實驗選擇一株分離自青海大骨節(jié)病區(qū)小麥的內(nèi)生鏈格孢霉屬真菌(編號13-8-A),通過顯微形態(tài)學(xué)觀察和ITS序列分析鑒定本實驗菌種,將菌株進行麥仁液體發(fā)酵,采用溶劑萃取法,硅膠柱色譜層析,薄層色譜層析,高效液相色譜法等分離提取手段,將逐級分離得到的物質(zhì)進行細(xì)胞檢測,通過MTT法檢測軟骨細(xì)胞存活率、RT-PCR檢測蛋白聚糖和Ⅱ型膠原蛋白mRNA的表達(dá)量、甲苯胺藍(lán)染色法和免疫組化染色法檢測這兩種蛋白的表達(dá)。通過觀察不同分級提取物對兔膝軟骨細(xì)胞上述與大骨節(jié)病有關(guān)指標(biāo)的影響,以尋找對軟骨細(xì)胞有明顯損傷的物質(zhì)。經(jīng)鑒定13-8-A菌株為互隔鏈格孢霉(Alternaria alternate)。該菌株代謝產(chǎn)物的石油醚提取物通過硅膠柱層析分離的成分基本不抑制軟骨細(xì)胞的生長,對蛋白聚糖和Ⅱ型膠原蛋白的表達(dá)及其mRNA表達(dá)量的抑制作用較弱。但在同樣處理濃度下,該菌株的乙醚提取物的粗分離成分對軟骨細(xì)胞的損傷遠(yuǎn)遠(yuǎn)強于石油醚。HPLC檢測結(jié)果顯示,該菌株可以產(chǎn)生鏈格孢霉毒素——鏈格孢霉單甲基乙醚(AME),標(biāo)準(zhǔn)品AME可以輕微抑制兔軟骨細(xì)胞的生長,該毒素對軟骨細(xì)胞的損傷主要表現(xiàn)為抑制蛋白聚糖的表達(dá)及其mRNA的表達(dá)量；而另外一種HPLC分離保留時間峰值在11min的收集物質(zhì)可以明顯降低軟骨細(xì)胞存活率、蛋白聚糖和Ⅱ型膠原蛋白的表達(dá)及其mRNA表達(dá)量。故該菌株代謝產(chǎn)物中還存在除AME外能抑制軟骨細(xì)胞生長的物質(zhì),鑒于該物質(zhì)對軟骨細(xì)胞的毒損作用比已知的AME更強,其值得更進一步研究。由此可知,互隔鏈孢霉13-8-A代謝產(chǎn)物中應(yīng)該存在多種可以損傷軟骨細(xì)胞的物質(zhì)。
[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，

本文編號：2242916