摘要
目的探讨不同龋敏感儿童口腔变异链球菌HtrA mRNA和蛋白质的表达情况与乳牙龋坏程度和HtrA的关系。方法以高龋、中龋和无龋儿童口腔中分离得到的变异链球菌为实验菌株,复苏,增菌,分离纯化核酸,采用逆转录PCR(RT-PCR)法,提取变异链球菌临床分离株总RNA,凝胶电泳检测RNA完整性,合成cDNA,PCR扩增,将扩增产物凝胶成像系统下观察记录结果,将目的基因HtrA及内参的电泳图像利用凝胶定量分析软件Gel-Pro analyzer 4.0进行灰度扫描,分析计算基因相对表达值;采用蛋白质印迹(Western blot)法,验证变异链球菌临床分离株总蛋白,结果用Bio-Rad凝胶摄像分析系统扫描入计算机中,凝胶定量分析软件Quantity One 4.4.0分析其灰度值,计算蛋白的相对表达水平。结果高龋、中龋及无龋儿童口腔变异链球菌临床分离株HtrA mRNA的表达和HtrA蛋白的表达均存在差异,从高到低为高龋组、中龋组、无龋组,差异有统计学意义(P<0.05)。结论不同龋敏感儿童口腔变异链球菌临床分离菌株HtrA mRNA的表达和HtrA蛋白的表达均存在差异,龋敏感性越高,HtrA mRNA的表达和HtrA蛋白的表达越高。
Objective To evaluate the relationship between mRNA and protein expression of HtrA and Streptococcus mutans isolated from the children with different caries experience and to provide the theoretical and experimental basis on prediction of dental caries in deciduous teeth.Methods The strains of Streptococcus mutans isolated from children with different carious experiences in the preliminary experiments were divided into three groups：high caries-susceptible group,middle caries-susceptible group,caries-free group.All strains were reanimated on the agar plate of MS,and after smear pure culture examination,typical bacteria were incubated in BHI,then purified nucleic acid and extracted all the RNA of streptococcus mutans by reverse transcription PCR and detected it by agarose gel electrophoresis integrality.Synthetic cDNA and take further PCR amplification with cDNA products.Observe records results by Gel imaging system.HtrA of target gene and electrophoresis image were gray scan by Gel quantitative software Gel-Pro analyzer 4.0 was used to analyze relative expression value of gene.After purifying protein,collected total protein of Streptococcus mutans strains by Western Blot method,then tested the concentration of total protein sample.The results of Chemiluminescence imaging were scanned into computer by Bio-Rad analyzing system,calculated the gray value by software Quantity One 4.4.0 which showed the relative expression level of protein.Results There were significant differences in HtrA mRNA and protein expression of different Streptococcus mutans isolated from the children with different caries susceptibility.high caries-susceptible group middle caries-susceptible group caries-free group（P0.05）.Conclusion There were significant differences in HtrA mRNA and protein expression of different Streptococcus mutans isolated from the children with different caries susceptibility.The higher caries susceptibility the group was,the more HtrA mRNA and protein the strain express.
出处
《重庆医学》
CAS
CSCD
北大核心
2013年第24期2834-2836,共3页
Chongqing medicine
基金
四川省卫生厅课题基金资助项目(100247)
关键词
龋齿
乳牙
变异链球菌
HTRA
龋敏感
mRNA
蛋白
dental caries
deciduous teeth
streptococcus mutants
HtrA
caries experience
mRNA
protein
