本文選題：大骨節(jié)病 + 內(nèi)生真菌��； 參考：《西北大學(xué)》2015年碩士論文

【摘要】：大骨節(jié)病是一種慢性地方性疾病,嚴(yán)重影響病區(qū)群眾的身心健康,自發(fā)現(xiàn)大骨節(jié)病至今,其致病因素仍然不清楚。本試驗結(jié)合低硒學(xué)說和真菌毒素中毒假說,研究硒-真菌-軟骨細(xì)胞之間的相互關(guān)系,以期為尋找大骨節(jié)病的致病因素提供一些新的線索。本實驗選擇從青海大骨節(jié)病區(qū)分離鑒定出的小麥優(yōu)勢真菌內(nèi)生鏈格孢霉屬菌株,測定硒對它們菌絲生長的影響,并研究不同濃度硒條件下的真菌發(fā)酵產(chǎn)物對動物軟骨細(xì)胞的影響,以及觀察硒對鏈格孢霉發(fā)酵產(chǎn)物所致軟骨細(xì)胞損傷的修復(fù)作用。采用合成培養(yǎng)基對鏈格孢霉菌株進(jìn)行培養(yǎng),測定不同硒濃度條件下真菌的生長曲線。菌株培養(yǎng)結(jié)束后用75%7醇提取各菌株的發(fā)酵產(chǎn)物,采用MTT法、RT-PCR、甲苯胺藍(lán)和免疫組化染色法檢測它們對軟骨細(xì)胞活力、蛋白聚糖和II型膠原的合成及其mRNA的表達(dá)水平的影響。對經(jīng)常規(guī)培養(yǎng)的真菌發(fā)酵產(chǎn)物作用的軟骨細(xì)胞進(jìn)行補(bǔ)硒,測定細(xì)胞各個指標(biāo)的變化。結(jié)果顯示：不同的硒濃度對不同菌株作用效果不同。對于8-5-B,加硒能夠延緩穩(wěn)定期的到來；硒濃度小于0.2mg/L時,硒對菌株的生物量沒有顯著的影響。對于13-8-A,高濃度的硒反而加速穩(wěn)定期的到來。對于conf-1,當(dāng)硒濃度不大于1.0mg/L時,都表現(xiàn)出有促進(jìn)菌絲生長的作用。三株菌株的發(fā)酵產(chǎn)物都能明顯降低軟骨細(xì)胞的活力,真菌培養(yǎng)無硒和加0.5mg/L硒的發(fā)酵產(chǎn)物作用于軟骨細(xì)胞的活力明顯高于其它組,這兩組的蛋白聚糖和II型膠原mRNA的表達(dá)都高于其它組。此實驗結(jié)果提示：硒能影響鏈格孢霉的生長,不同濃度的硒可能影響鏈格孢霉的發(fā)酵產(chǎn)物,在實驗所設(shè)濃度范圍內(nèi),低濃度的硒可能促進(jìn)菌株產(chǎn)毒,高濃度的硒能抑制部分菌株產(chǎn)毒。對鏈格孢霉發(fā)酵產(chǎn)物作用的軟骨細(xì)胞進(jìn)行補(bǔ)硒,結(jié)果發(fā)現(xiàn)一定濃度的硒能減輕真菌發(fā)酵產(chǎn)物的損傷作用,補(bǔ)硒能拮抗真菌發(fā)酵產(chǎn)物對細(xì)胞的蛋白聚糖和II型膠原的mRNA和蛋白表達(dá)的抑制作用。因此,環(huán)境低水平硒可能通過真菌間接作用于人,加重其對人軟骨細(xì)胞的損傷作用,增加大骨節(jié)病發(fā)生的可能性。
[Abstract]:Kaschin-Beck disease (KBD) is a chronic endemic disease, which seriously affects the physical and mental health of the people. Based on the theory of selenium deficiency and the hypothesis of mycotoxin poisoning, the relationship between selenium, fungi and chondrocytes was studied in order to provide some new clues for finding the pathogenic factors of Kaschin-Beck disease. In this experiment, endophytic streptomyces of wheat dominant fungi were isolated and identified from Kashin-Beck disease area in Qinghai, and the effect of selenium on the growth of their hyphae was determined. The effects of fungal fermentation products on chondrocytes and the repair of chondrocyte damage induced by streptomyces fermentation were studied. Streptomyces spore strains were cultured in synthetic medium and the growth curves of fungi were measured under different selenium concentrations. The fermentation products of each strain were extracted with 757-alcohol after culture. MTT assay, toluidine blue and immunohistochemical staining were used to detect their effects on chondrocyte viability, synthesis of proteoglycan and type II collagen and their mRNA expression levels. The chondrocytes treated by fungal fermentation products were supplemented with selenium and the changes of cell indexes were measured. The results showed that different selenium concentrations had different effects on different strains. For 8-5-B, adding selenium could delay the arrival of stable period, but when selenium concentration was less than 0.2 mg / L, selenium had no significant effect on the biomass of the strain. For 13-8 A, high concentration of selenium accelerates the arrival of stable phase. For conf-1, when the concentration of selenium is less than 1.0 mg / L, it can promote the growth of hyphae. The fermentation products of the three strains could significantly reduce the viability of chondrocytes. The effects of selenium free culture and 0.5 mg / L selenium on chondrocyte viability were significantly higher than those in other groups. The expression of proteoglycan and type II collagen mRNA in both groups was higher than that in other groups. The results suggest that selenium can affect the growth of Streptomyces and that different concentrations of selenium may affect the fermentation products of Streptomyces. High concentration of selenium can inhibit the production of some strains. The effect of selenium supplementation on chondrocytes of Streptomyces spp. Fermentation products was studied. It was found that certain concentration of selenium could attenuate the damage of fungal fermentation products. Selenium supplementation could antagonize the inhibitory effect of fungal fermentation products on the mRNA and protein expression of proteoglycan and type II collagen. Therefore, environmental low level selenium may indirectly act on human through fungi, increase its damage to human chondrocytes and increase the possibility of Kaschin-Beck disease.
【學(xué)位授予單位】：西北大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R684.1

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