无血清培养上调N2a细胞钙反应性反式激活因子的表达

Up-regulation of expression of calcium-responsive transactivator in N2a cells by serum-free culturing

目的探讨钙反应性反式激活因子(calcium-responsive transactivator,CREST)是否与神经细胞的分化有关。方法选用在促分化因素作用下具有分化为神经元能力的小鼠神经母细胞瘤(N2a)细胞,用无血清培养(血清撤除)诱导N2a细胞分化。接种和培养N2a细胞12～24h后,将其分为对照组和无血清诱导组,对照组继续在含5%胎牛血清(FBS)的培养基中培养,无血清诱导组在不含FBS的培养基中培养。采用免疫印迹技术、免疫荧光技术和RT-PCR检测无血清诱导分化对N2a细胞CREST蛋白和mRNA表达的影响。结果在含5%胎牛血清(FBS)培养基培养的对照N2a细胞中,CREST蛋白水平在各时间点均无明显变化,CREST mRNA水平仅在培养48h后略有升高。而在无血清诱导分化的N2a细胞中,CREST蛋白表达水平在无血清诱导12h时无明显变化,但在诱导24h后明显升高,诱导48h后进一步升高;RT-PCR检测显示,CREST mRNA在无血清诱导12h后即开始升高,诱导24h和48h则升高更为明显。结论无血清诱导分化的N2a细胞中CREST的表达上调,提示CREST可能参与神经细胞的分化。

Objective To investigate if the calcium-responsive transactivator （CREST） is involved in differentiation of neuronal cells. Methods Mouse neuroblastoma （N2a） cells capable of being differentiated into neurons by differential induction factors were used as a model. Serum-free culture was used to induce differentiation of N2a cells. The effect of serum-free culturing of different durations （0 h, 12 h, 24 h and 48 h） on the expression of CREST was detected by immunoblotting, immunofluorescence and RT-PCR. Results In control N2a cells cultured in a medium containing 5% fetal bovine serum （FBS）, immunoblot- ting and immunofluorescence showed that the protein level of CREST was not changed at all time points, and RT-PCR showed that the mRNA level of CREST was mildly elevated only after 48 h. In contrast, in N2a cells differentiated by serum-free culture, although CREST protein level did not change at 12 h, it was elevated at 24 h, with a more significant increase at 48 h. Moreover, the mRNA level in N2a cells treated with serum-free culture increased at as early as 12 h, and further increased with longer serum-free induc- tion. Conclusion The expression of CREST in N2a cells differentiated by serum-free culturing is up-regula- ted, which indicates that CREST may be involved in neuronal differentiation.