寡核苷酸转化变形链球菌方法的筛选

The optimized condition for delivery gtfB antisense oligonucleotide into Streptococcus mutans

目的:筛选一种适合变形链球菌的寡核苷酸转运方法,为利用反义寡核苷酸抑制变形链球菌基因表达打下基础。方法:分别采用阳离子聚合体So-fastTM,及阳离子脂质体为载体,以FITC标记的寡核苷酸转化变形链球菌,流式细胞术检测摄取率,并与自然转化和电穿孔转化法的摄取率进行比较。利用生长曲线和琼脂平皿记数法,观察转化试剂和(或)转化方法对细菌增殖能力的影响。结果:So-fastTM转化组的摄取率最高,近80%,其次为电穿孔组(70.5%)和脂质体转化组(17.3%),自然转化组的摄取率最低,约4%。So-fastTM组和对照组的菌落计数和生长曲线均无明显差别(P>0.05),电穿孔组与未穿孔组的菌落计数差别明显(P<0.01)。结论:So-fastTM有明显的促转化作用,并且不影响受试菌的增殖,可做为寡核苷酸转化变形链球菌的载体。

Objective： To screen out an appropriate delivery system for transformation of Streptococcus mutans with antisense phosphorothioate oligodeoxyribonucleotides （PS-ODN） to regulate gene expressions. Methods： To delivery FITC labeled PS-ODN into S. mutans using polycation So-fast^TM and cationic liposomes lipofectamine2000 as transfer carrier; natural transformation and electroporation was also applied; the uptake rate was recorded by FACS. The effects of various treatments on viability and recovery of S. mutans were determined by growth curve and colony number counting. Results： The largest uptake rate was achieved by the combination of FITC-labeled PS-ODN with So-fast^TM, in which about 80% of bacterial cells had integrated antisense oligonucleotides, followed by electroproation（70.5% ） , encapsulated PS-ODN with lipofectamine2000（ 17.3% ） and free PS-ODN（4% ）. The results of growth curve and cloneforming units （CFU） counting showed that So-fast^TM had no significant effects on proliferation and recovery of S. mutans （ P 〉 0.05 ）. Conclusion ： So-fast^TM can greatly improve the penetration of PS-ODN into S. mutans and can be used as appropriate delivery system of PS-ODN for S. mutans. No matter what approaches were adopted, the uptake rate reached the maximum at 5 μmol/L and 2 h-exposure. The penetration can not be enhanced by increasing the PS-ODN concentration and the transformation time.