论文部分内容阅读
目的观察烟曲霉分生孢子静止期及其在膨胀生发过程中表面形态的改变。观察纤连蛋白受体在孢子表面分布状态及其在膨胀生发过程中的变化。方法取培养成熟的烟曲霉,磷酸盐缓冲液冲洗后,通过3.5～4.0μm的钢丝网过滤提取制备纯净分生孢子悬液(108细胞/mL)。加入细胞培养液(RPMI1640+10%FBS),分别在0、2、4、6h取材固定,进行扫描电镜观察。另取定量分生孢子悬液(108细胞/mL)孵育在包被有定量纤连蛋白(50μg/mL)的微量测定细胞培养板中,37℃3h;用磷酸盐缓冲液冲洗3次,再分别加入兔抗人-纤连蛋白抗体和羊抗兔IgG-FITC孵育(37℃1h),胰酶消化后冲洗固定立即送共距焦免疫荧光电镜检查。结果静止期分生孢子表面有特征性的小棘状层形式,即有许多刺猬毛状的突起分布在其表面。孢子膨胀生发过程中,刺猬毛样小棘状层结构逐渐降解脱落,大约4h后完全降解脱落。孢子变成由条索状纤维质构成的光滑细胞壁球体;通过共距焦免疫荧光电镜发现FITC均匀地分布粘附在分生孢子表面刺猬毛样棘状突起上。结论静止期分生孢子的表面可表达出高水平纤连蛋白受体。带有放射荧光标记的纤连蛋白配体-受体均匀地分布在静止期分生孢子最外面小棘状层。孢子膨胀过程中表面小棘状层逐渐脱落。
Objective To observe the quiescent period of Aspergillus fumigatus conidia and its surface morphological changes during swollen hair growth. To observe the distribution of fibronectin receptors on the spore surface and its changes in the expansion of hair growth. Methods After the culture of Aspergillus fumigatus was matured, rinsed with phosphate buffer, the pure conidia suspension (108 cells / mL) was prepared by filtration through 3.5 ~ 4.0μm steel mesh. Cell culture medium (RPMI1640 + 10% FBS) was added and fixed at 0, 2, 4, and 6 h respectively for scanning electron microscopy. Another quantitative conidia suspension (108 cells / mL) was incubated in a microtiter plate coated with quantitative fibronectin (50 μg / mL) and incubated at 37 ° C for 3 h; rinsed three times with phosphate buffered saline Rabbit anti-human fibronectin antibody and goat anti-rabbit IgG-FITC were incubated respectively at 37 ℃ for 1 hour. After digestion with trypsin, the cells were washed and fixed, and then sent to confocal immunofluorescence electron microscopy. Results The quiescent conidiospores were characterized by a small spicular layer, that is, many hedgehog hairlike protrusions distributed on the surface. During the process of spore expansion and germination, the spiny layer structure of hedgehog hair gradually degrade and fall off, and completely degrade after about 4 hours. The spore became a smooth cell wall spheroid composed of cord-like fibers. The FITC was evenly distributed on the surface of sporophyte of hedgehog hair on the surface of conidia by confocal immunofluorescence microscopy. Conclusion The surface of quiescent conidia can express high level of fibronectin receptor. Fibronectin ligands with radio-fluorescence labeling were uniformly distributed on the outermost small spinous layer of quiescent conidia. Small spiny layer on the surface of spores gradually shedding.