摘要
目的:观察P300/CBP相关因子(P300/CBP associated factor,PCAF)对人子宫内膜间质细胞增殖的调控作用。方法:通过Western blotting、实时定量PCR及Cell Counting Kit-8(CCK-8)细胞增殖实验,观察分析Ad-FLAG-PCAF重组腺病毒介导的PCAF过表达及PCAF-siRNA介导的PCAF低表达对体外培养的人子宫内膜间质细胞增殖的影响。结果:获得滴度为8×1010ifu/ml的重组Ad-FLAG-PCAF腺病毒,转染体外培养的人子宫内膜间质细胞后,可有效介导细胞内FLAG-PCAF融合蛋白的高表达;而转染PCAF特异的siRNA后,可有效抑制人子宫内膜间质细胞中PCAF的表达。PCAF过表达的人子宫内膜间质细胞中,其细胞周期标志蛋白cyclinD1和cyclinD3的表达水平显著降低(P<0.05),而PCAF低表达的人子宫内膜间质细胞中,其cyclinD1和cyclinD3的表达水平显著升高(P<0.05);此外,CCK-8实验结果显示,PCAF过表达能显著抑制由雌、孕激素共同刺激所引起的人子宫内膜间质细胞的增殖,而基因沉默人子宫内膜间质细胞中内源性PCAF的表达后,间质细胞的增殖增加>20%(P<0.05)。结论:PCAF可能通过蛋白乙酰化修饰作用,参与调控人子宫内膜间质细胞的增殖过程。
Objective： To detect the effects of P300/CBP associated factor （PCAF） expression level on the proliferation of human endometrial stromal cell （hESCs） in vitro. Methods： Western blotting, real-time PCR, cell counting Kit-8 （CCK-8）, adenovirus-mediated over-expression of PCAF and PCAF-siRNA mediated down-regulation of PCAF expression were performed to study the effects of PCAF in regulating the proliferation of hESCs. Results： A total of 8 X 101 ifu/ml adenovirus Ad-FLAG-PCAF was obtained, which can over-express FLAG-PCAF fusion protein in hESCs. On the other hand, PCAF siRNA could effectively inhibit PCAF protein expression level in hESCs. PCAF over-expression resulted in a decreased both of cyclinD 1 and cyclinD3 protein and mRNA expression in hESCs （P〈0.05）. Down-regulation of PCAF in hESCs significantly increased cyclinD 1 and cyclinD3 protein and mRNA expression （P〈0.05）. Moreover, adenovirus-mediated over-expression of PCAF markedly impaired cell proliferation by 33% induced by estrogen and progesterone and down-regulation of endogenous PCAF increased cell proliferation over 20% （P〈0.05）. Conclusion： PCAF might play critical roles in regulation of hESCs proliferation.
出处
《生殖与避孕》
CAS
CSCD
2013年第3期159-166,共8页
Reproduction and Contraception
基金
国家自然科学基金(No.81170570
No.30900727
No.81070492)
南京市卫生局资金(QYK09172)
留学回国人员科研启动基金(No.2011-1139)资助项目
