相对定量2^(-△△CT)法分析Caspase-3和Caspase-9在氟诱导大鼠肾脏中的表达

Relative quantitative detection of Caspase-3 and Caspase-9 mRNA expression of kidney in rats induced with fluoride

目的检测caspase-3和caspase-9基因在氟诱导大鼠肾脏组织中的表达。方法 48只雄性SD大鼠分为4组(对照组、低氟组、中氟组和高氟组),每组12只,分别加入不同浓度的氟化钠染毒(0、50、100和200 mg/L)。120 d后,处死大鼠,提取各组大鼠肾组织总RNA,实时荧光定量PCR法检测各染氟组caspase-3、caspase-9基因的表达,选择β-actin作为内参基因,使用相对定量2-△△CT法分析。结果对照组和3个染氟组(低、中和高氟组)caspase-3基因表达量分别是1.01±0.01、1.41±0.68、2.31±0.95和2.69±0.49,其中中氟组和高氟组表达显著增高(P<0.05)。caspase-9的基因表达量分别是1.01±0.01、1.30±0.26、2.63±0.56和3.97±0.60,中氟组和高氟组表达显著增高(P<0.01)。结论氟诱导大鼠肾脏后caspase-3和caspase-9基因表达增高,为氟中毒发病机制的研究提供一定的理论依据。

Objective To investigate the caspase-3 and caspsase-9 mRNA expression of kidney in rats induced by fluoride.Methods Male Sprague-Dawley rats were divided randomly into four groups with the administrations via drinking water of 0,50,100 and200 mg/L of sodium fluoride（control,low-fluoride,medium-fluoride and high-fluoride concentration） for 120 days,respectively.Total RNA of kidney was extracted,the mRNA expression levels of caspase-9 and caspase-3 were analyzed using real-time PCR.The relative expression was determined using the comparative 2-△△CTmethod,with the housekeeping gene β-actin used as a calibrator.Results The control,low-,medium-and high-fluoride concentration group displayed mRNA relative expression levels of caspase-3 are 1.01 ± 0.01,1.41 ± 0.68,2.31 ±0.95 and 2.69 ± 0.49 respectively,the mRNA expression levels of caspase-3 were higher in the fluoride exposed medium-and high-fluoride concentration group than the control significantly（P 〈 0.05）.In the control,low-,medium-and high-fluoride concentration group the mRNA relative expression levels of caspase-9 were 1.01 ± 0.01,1.30 ± 0.26,2.63 ± 0.56 and 3.97 ± 0.60,respectively,the mRNA expression levels of caspase-9 were higher in the fluoride exposed medium-and high-fluoride concentration group than the control significantly（P 〈0.01）.Conclusion Fluoride treatment increased mRNA expression levels of caspase-9 and caspase-3,and to provide theoretical basis and new clues for the research of Fluorosis pathogenesis.