【摘要】：大骨節(jié)病(Kaschin-Beck Disease, KBD)是一種地方性骨關(guān)節(jié)病,對(duì)人體的危害極大,至今,KBD發(fā)病原因與發(fā)病機(jī)制仍困擾著科學(xué)家。本實(shí)驗(yàn)從感染青海大骨節(jié)病病區(qū)糧食作物內(nèi)生真菌的代謝產(chǎn)物出發(fā),探討其中究竟有哪種或哪些物質(zhì)能夠?qū)浌羌?xì)胞造成損傷,從而為大骨節(jié)病病因的尋找提供一定的實(shí)驗(yàn)參考。本實(shí)驗(yàn)選取分離自青海大骨節(jié)病區(qū)小麥內(nèi)生鐮孢霉5-5-1菌株代謝產(chǎn)物中對(duì)軟骨細(xì)胞損傷嚴(yán)重的石油醚分部進(jìn)行實(shí)驗(yàn)。通過(guò)硅膠柱層析、薄層層析(TLC)、高效液相色譜(HPLC)、氣相-質(zhì)譜聯(lián)用(GC/MS)等方法,對(duì)石油醚分部的代謝產(chǎn)物進(jìn)行逐級(jí)分離。主要通過(guò)MTT檢測(cè)軟骨細(xì)胞存活率,免疫組化檢測(cè)Ⅱ型膠原蛋白的合成量,甲苯胺藍(lán)染色(toluidine blue, TB)檢測(cè)蛋白聚糖的合成量,半定量RT-PCR實(shí)驗(yàn)檢測(cè)Ⅱ型膠原蛋白與蛋白聚糖mRNA的表達(dá)量來(lái)逐級(jí)篩選能造成兔軟骨細(xì)胞損傷的目標(biāo)物質(zhì)。實(shí)驗(yàn)結(jié)果顯示,從石油醚分部中經(jīng)柱層析粗分離得到的A、B、C、D、E、F、G、H這8組樣品中,E、F、G樣品均能夠抑制軟骨細(xì)胞的存活率、蛋白聚糖和Ⅱ型膠原蛋白的合成及其mRNA的表達(dá),且均呈一定的濃度與時(shí)間效應(yīng)。鑒于相同濃度下F樣品對(duì)兔軟骨細(xì)胞各項(xiàng)指標(biāo)的影響最大,因此對(duì)F樣品再進(jìn)行HPLC分離。根據(jù)HPLC圖譜結(jié)果,將其分為F1、F2、F3三部分。將它們分別作用于兔軟骨細(xì)胞,結(jié)果顯示F1、F2樣品能對(duì)軟骨細(xì)胞的存活率、蛋白聚糖和Ⅱ型膠原蛋白的合成及其mRNA的表達(dá)均產(chǎn)生抑制作用。而F3樣品對(duì)兔軟骨細(xì)胞的各項(xiàng)指標(biāo)均無(wú)影響。對(duì)F1樣品進(jìn)行GC/MS分析,顯示F1中可能存在鐮孢霉毒素蛇形菌素(4,15-diacetoxyscirpenol, DAS),經(jīng)HPLC分析與DAS標(biāo)準(zhǔn)品進(jìn)行對(duì)比驗(yàn)證,確認(rèn)DAS的存在,且不同濃度的DAS對(duì)軟骨細(xì)胞的存活率、蛋白聚糖和Ⅱ型膠原蛋白的合成及其mRNA的表達(dá)均產(chǎn)生抑制作用,呈一定的濃度與時(shí)間效應(yīng),這是關(guān)于該毒素對(duì)軟骨細(xì)胞有毒損作用的首次報(bào)道。同樣對(duì)F2樣品進(jìn)行HPLC與GC/MS分析,發(fā)現(xiàn)其成分較復(fù)雜,有待進(jìn)一步分離純化鑒定。以上實(shí)驗(yàn)結(jié)果表明,鐮孢霉5-5-1菌株代謝產(chǎn)物石油醚提取物分級(jí)分離產(chǎn)物中含有DAS及其他能夠損傷軟骨細(xì)胞的物質(zhì),并證實(shí)了KBD病區(qū)植物內(nèi)生真菌代謝產(chǎn)物中含有多種損傷軟骨細(xì)胞的物質(zhì),這些研究結(jié)果將為大骨節(jié)病病因的研究提供新的參考。
[Abstract]:Kaschin-Beck disease (Kaschin-Beck Disease, KBD) is a kind of endemic osteoarthropathy, which is harmful to human body. Based on the metabolites of endophytic fungi of grain crops in the area infected with Kashin-Beck disease in Qinghai Province, this study was conducted to explore which of these substances could damage chondrocytes. So as to provide a certain experimental reference for the search for the cause of Kaschin-Beck disease. In this experiment, petroleum ether was isolated from the metabolites of endophytic Fusarium falcifera 5-5-1 isolated from Kashin-Beck disease area of Qinghai province. The metabolites of petroleum ether were separated step by silica gel column chromatography, thin layer chromatography (TLC), (TLC), high performance liquid chromatography (HPLC),) and GC-MS (GC/MS). The survival rate of chondrocytes was determined by MTT, the synthesis of collagen 鈪，

本文編號(hào)：2209329