β-肌动蛋白作为糖皮质激素作用后晶状体上皮细胞定量mRNA表达的内部参照标准

Study of β-actin as an internal standard for quantitative mRNA measurement in lens epithelial cells cultured in a dexamethasone medium

目的观察糖皮质激素作用体外培养的晶状体上皮细胞时,看家基因β肌动蛋白(βactin)mRNA表达的稳定性,建立该细胞定量mRNA研究的内部参照标准。方法采用含0、1×10-8、5×10-8、10×10-8、50×10-8mol/L不同浓度的地塞米松培养液体外培养晶状体上皮细胞,RTPCR测定β肌动蛋白(βactin)mRNA,并进行半定量灰度分析。结果各组均有明显表达,对照组晶状体上皮细胞β肌动蛋白表达(平均灰度值)为52.99±6.66,1×10-8、5×10-8、10×10-8、50×10-8mol/L地塞米松处理组分别为51.65±4.24、48.85±5.42、50.46±4.89和49.52±3.91,统计学分析各组间平均灰度值差异无显著性。结论体外培养的晶状体上皮细胞经0、1×10-8、5×10-8、10×10-8、50×10-8mol/L地塞米松作用后βactinmRNA表达稳定,是研究糖皮质激素作用于该细胞时可靠的内部参照标准。

Objective To establish an internal standard for quantitative mRNA measurement in cultured lens epithelial cells（LEGs）, and to observe the stability of the housekeeping gene beta-actin （β-actin）mRNA in normal and glucocorticoid treated cells in vitro. Methods LECs were cultured in vitro, then treated with a DMEM medium consisting of 0.1×10^-8、5×10^-8、10×10^-8、50×10^-8mol/L of dexmnethasone for 7 days. The expression of β-actin mRNA was measured by RT-PCR semi-quantitatively.Results The gnayscale in normal cultured LECs was 52.99±6.66 while it was 51.65±4.24,48.85±5.42,50.46±1.89,49.52±3.91 in the1×10^-8、5×10^-8、10×10^-8、50×10^-8mol/L dexamethasone-treated groups, respectively. There were no statistically significant differences between the control group and the dexamthasone-treated groups.Couclusion There is a stable β-actin mRNA expression both in dexamethasone-treated and untreated LECs. The results reveal that β-actin, is a preferable internal startdard when investigating the other effects of glucococoticoid on LE cells.