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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 observe the regulatory effect of P300 / CBP associated factor (PCAF) on the proliferation of human endometrial stromal cells. Methods: Ad-FLAG-PCAF recombinant adenovirus-mediated PCAF overexpression and PCAF-siRNA-mediated low expression of PCAF were detected by Western blotting, real-time quantitative PCR and Cell Counting Kit-8 On the proliferation of human endometrial stromal cells cultured in vitro. Results: The recombinant adenovirus Ad-FLAG-PCAF with a titer of 8 × 1010ifu / ml was obtained. The transfection of human endometrial stromal cells in vitro could effectively induce the high expression of FLAG-PCAF fusion protein. However, PCAF-specific siRNA transfection could effectively inhibit the expression of PCAF in human endometrial stromal cells. The expression of cyclinD1 and cyclinD3 in PCAF overexpressing human endometrial stromal cells was significantly decreased (P <0.05), whereas in PCAF low expression human endometrial stromal cells, cyclinD1 and cyclinD3 (P <0.05). In addition, CCK-8 results showed that PCAF overexpression significantly inhibited the proliferation of human endometrial stromal cells induced by estradiol and progesterone stimulation, while the gene silencing Proliferation of stromal cells increased by> 20% (P <0.05) after expression of endogenous PCAF in human endometrial stromal cells. Conclusion: PCAF may be involved in the regulation of the proliferation of human endometrial stromal cells through the modification of protein acetylation.