三株链格孢霉发酵产物与硒对兔膝软骨细胞的影响

发布时间：2018-06-25 22:23

本文选题：大骨节病 + 内生真菌　；参考：《西北大学》2015年硕士论文

【摘要】：大骨节病是一种慢性地方性疾病,严重影响病区群众的身心健康,自发现大骨节病至今,其致病因素仍然不清楚。本试验结合低硒学说和真菌毒素中毒假说,研究硒-真菌-软骨细胞之间的相互关系,以期为寻找大骨节病的致病因素提供一些新的线索。本实验选择从青海大骨节病区分离鉴定出的小麦优势真菌内生链格孢霉属菌株,测定硒对它们菌丝生长的影响,并研究不同浓度硒条件下的真菌发酵产物对动物软骨细胞的影响,以及观察硒对链格孢霉发酵产物所致软骨细胞损伤的修复作用。采用合成培养基对链格孢霉菌株进行培养,测定不同硒浓度条件下真菌的生长曲线。菌株培养结束后用75%7醇提取各菌株的发酵产物,采用MTT法、RT-PCR、甲苯胺蓝和免疫组化染色法检测它们对软骨细胞活力、蛋白聚糖和II型胶原的合成及其mRNA的表达水平的影响。对经常规培养的真菌发酵产物作用的软骨细胞进行补硒,测定细胞各个指标的变化。结果显示：不同的硒浓度对不同菌株作用效果不同。对于8-5-B,加硒能够延缓稳定期的到来；硒浓度小于0.2mg/L时,硒对菌株的生物量没有显著的影响。对于13-8-A,高浓度的硒反而加速稳定期的到来。对于conf-1,当硒浓度不大于1.0mg/L时,都表现出有促进菌丝生长的作用。三株菌株的发酵产物都能明显降低软骨细胞的活力,真菌培养无硒和加0.5mg/L硒的发酵产物作用于软骨细胞的活力明显高于其它组,这两组的蛋白聚糖和II型胶原mRNA的表达都高于其它组。此实验结果提示：硒能影响链格孢霉的生长,不同浓度的硒可能影响链格孢霉的发酵产物,在实验所设浓度范围内,低浓度的硒可能促进菌株产毒,高浓度的硒能抑制部分菌株产毒。对链格孢霉发酵产物作用的软骨细胞进行补硒,结果发现一定浓度的硒能减轻真菌发酵产物的损伤作用,补硒能拮抗真菌发酵产物对细胞的蛋白聚糖和II型胶原的mRNA和蛋白表达的抑制作用。因此,环境低水平硒可能通过真菌间接作用于人,加重其对人软骨细胞的损伤作用,增加大骨节病发生的可能性。
[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.
【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R684.1

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