国家自然科学基金(30770816, 30270510)和重庆市自然科学基金项目(CSTC2009BB5409)资助项目
This work was supported by grants from The National Natural Science Foundation of China(30770816, 30270510) and Chongqing Natural Science Foundation (CSTC2009BB5409)
为了观察肿瘤坏死因子相关凋亡诱导配体(TRAIL)基因对体外培养的小鼠蜕膜基质细胞增殖及凋亡的作用,探讨TRAIL对小鼠子宫蜕膜化进程的影响,构建TRAIL过表达及干扰质粒,转染小鼠基质细胞后诱导蜕膜化发生.转染72 h后,应用半定量RT-PCR和Western blotting检测蜕膜基质细胞中TRAIL mRNA和蛋白质的表达情况、 MTT法观察蜕膜基质细胞的生长和增殖能力、流式细胞术检测蜕膜基质细胞的细胞周期分布情况和凋亡率.经酶切和核苷酸测序证实,TRAIL基因正确克隆入真核表达载体且能够上调TRAIL的表达,干扰质粒能有效地抑制TRAIL基因的表达.TRAIL过表达和RNA干扰的结果表明:TRAIL具有将蜕膜基质细胞阻滞在G0/G1期、抑制蜕膜基质细胞增殖并促使其凋亡的功效,提示TRAIL可能参与调节胚胎植入后基质细胞的有序蜕膜化进程.
To explore the functional role of TRAIL on the proliferation,apoptosis of mouse decidual stromal cells (DSC) artificially induced in vitro, a TRAIL gene expressing eukaryotic vector and a short interfering RNA(siRNA) expressing vector were constructed. 72h after transfecting with each vector, semi-quantitative reverse transcription polymerase chain reaction (semi-qRT-PCR) and Western blotting were applied to detect the expression levels of TRAIL mRNA and protein, respectively in DSC. Besides, MTT assay was performed to investigate the cell growth activity and proliferation capacity, and flow cytometry was used to examine cell cycle distribution and apoptosis rate of DSC. Restriction endonuclease digestion and sequencing of nucleotide acid confirmed the correct construction of both over-expression and interfering vectors. The combined results from TRAIL overexpression and interfering in DSC demonstrated that TRAIL is capable of markedly inhibiting DSC proliferation by blocking most of cells at G0/G1 phase, meanwhile the apoptosis rate of DSC was notably increased.
张晋平,罗文萍,张 倩,彭洪英,谭冬梅,王应雄,谭 毅.肿瘤坏死因子相关的凋亡诱导配体(TRAIL)基因抑制小鼠子宫基质细胞的蜕膜化[J].生物化学与生物物理进展,2010,37(3):288-296
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