携带usp基因的尿道致病性大肠杆菌强毒株诱导HeLa细胞快速早期凋亡的研究

目的 了解致病基因在尿液分离大肠杆菌中的分布情况,分析由携带usp致病基因的尿道致病性大肠杆菌介导的HeLa细胞快速凋亡.方法 采用PCR检测6种尿道致病性大肠杆菌相关致病基因在28株尿液分离的大肠杆菌的分布;通过黏附试验和锥虫蓝染色对尿液分离的大肠杆菌进行初步致病表型筛选;采用Annexin V/PI法进一步检测大肠杆菌引起HeLa细胞早期凋亡的能力;电子显微镜观察细胞凋亡特征.结果 在28株尿液分离的大肠杆菌中,检测到usp基因阳性6株.通过致病表型筛选试验筛选到2株强致病性大肠杆菌(6N和27N),其中6N菌株携带usp基因,而27N除了没有检测到usp基因,其他致病基因与6N相同.它们可以在4 h破坏大量HeLa细胞,使HeLa死亡;流式细胞仪检测凋亡结果 显示,6N菌株在1.5 h可以诱导20.75%的HeLa细胞发生早期凋亡,而27N只有1.55%的HeLa细胞发生早期凋亡.电镜结果 从形态学证实6N菌株可以快速引起HeLa细胞发生早期凋亡.结论 携带usp基因的尿道致病性大肠杆菌可以诱导HeLa发生快速早期凋亡.

Uropathogenic Escherichia coli with usp gene induces the rapid early stage apoptosis of HeLa cells

Objective To understand the distribution of pathogenic genes in uropathogenic Esche-richia coli (UPEC) from urinary specimen and to analyze the pathogenicity of UPEC and their mechanism of apoptosis to HeLa cells. Methods We have analyzed 6 pathogenic genes among the 28 strains of the clini-cally isolated E. coli from urinary tract infected patients. The 6 pathogenic genes were surveyed by using the PCR amplification of the target genes. The adhesion experiments and tryphan-blue staining was used to screen the phenotype of the pathogenic strains, while Annexin V/PI method was applied to study the strains to cause apoptosis of the HeLa cells, which was further confirmed with electronic microscopy. Results We have detected 6 strains that carried the usp gene. Phenotype screening identified two high virulent isolates (strain 6N and 27N) from the 6 strains. Strain 27N and 6N contained very similar virulence gene profile ex-cept that strain 27N did not contain usp gene. Both strains can destroy HeLa cell within 3 hours causing cell death. Results of apoptosis detected by flow cytometry revealed that strain 6N induced 20.75% of HeLa cells to an early stage apoptosis within 1.5 hours. On the other hand, strain 27N induced only 1.55% of HeLa cells to apeptosis. Conclusion High virulent UPEC strain carrying usp gene can induce HeLa cell rapid early apoptosis.