| Objective To observe different mRNA level of Candida albican ALS gene family between yeast and hyphal cells,to observe that between plantonic and biofilm cells,so that to provide valuable data for the ALS genes appear important for virulence.Methods Four C.albicans standard strains and eleven clinical strains were induced to form yeast form in the condition cultured in GSB culture medium for 48 hours at 25℃,at planktonic condition.Germ tubes were induced by cultured in RPMI-1640 medium for 3 hours at 37℃,PH value at 7.0 with the shake speed of 150rpm.Biofilm models in vitro were formed on 6-well tieeue culture plate in RPMI-1640 media.after 48 hours' incubation,the biofilm -grown cells were harvest.Hot- phenol method was performed to isolate total RNA from the cells of the four different biological forms separately.The purity of RNA was measured by absorption at 260/280nm with spectrophotometer.A semi-quantitative RT-PCR two-step method was developed to determine ALS2 and ALS3 genes against the housekeeping gene ACT1.The identity of cloned fragments was confirmed with agarose gel electrophoresis.Gene expression levels were assayed by gel documentation system and QUANTITY ONE software.The paired t test was used to analyze the experimental data.Result The RT-PCR result showed that the expression levels of ALS2 mRNA(P=0.005,P＜0.01) and ALS3 mRNA(P=0.0022,P＜0.01) of the standard strains were higher in germ tube compared with yeast form.The expression levels ofALS2 mRNA and ALS3 mRNA of the clinical strains were higher in germ tube compared with yeast form(P=0.000,P＜0.01).The expression of ALS3 mRNA was negative or at low level in yeast form in the fifteen strains.The expression levels of ALS2 mRNA(P=0.0276,P＜0.05) and ALS3 mRNA(P=0.0405,P＜0.05) of the four standard strains in biofilm increased compared with planktonic cells. There were also increases in biofilm cells compared with the planktonic cells in the expression levels of ALS2 mRNA(P=0.0004,P＜0.01) and ALS3 mRNA(P=0.0005,P＜0.01) of the clinical strains.Conclusion The ALS2 mRNA and ALS3mRNA expression may play an important role in Candida albicans transformation from yeast to germ tube.ALS2 mRNA and ALS3 mRNA may have function in Candida albicans biofilm formatiom in vitro. |
