多效生长因子对小鼠子宫基质细胞蜕膜化的影响

Effect of pleiotrophin on decidualization of uterine stromal cells in mice

目的:构建多效生长因子(PTN)基因过表达载体,研究PTN对小鼠子宫基质细胞蜕膜化的影响。方法:根据GenBank中已发表的PTN基因序列,设计含酶切位点的特异性引物,进行PCR扩增及胶回收后,与pGEM-T载体连接,经限制性内切酶EcoRⅠ和XhoⅠ双酶切后与pcDNA 3.1(+)载体融合得到PTN过表达质粒,将其转染至体外分离培养的小鼠子宫基质细胞中,利用实时荧光定量PCR(qRT-PCR)法检测子宫基质细胞中PTN mRNA表达水平及PTN过表达作用下蜕膜化标志性分子Prl8a2和Prl3c1表达水平。对照组为转染pcDNA 3.1(+)空载体的子宫基质细胞。结果:PTN过表达质粒经双酶切鉴定后,结果与目的基因条带吻合,测序结果与GenBank中小鼠PTN基因序列同源性为100%。与对照组比较,PTN过表达载体组小鼠子宫基质细胞中PTN、Prl8a2和Prl3c1mRNA表达水平明显升高(P<0.05)。结论:PTN过表达能够使小鼠子宫内膜基质细胞中蜕膜化标志分子的表达水平升高,提示PTN可能在小鼠子宫内膜蜕膜化过程中起促进作用。

Objective：To construct the pleiotrophin （PTN ）overexpression vector,and to explore the effect of PTN on the decidualization of uterine stromal cells in the mice.Methods：The specific primers containing restriction enzyme cutting sites were designed according to the PTN gene sequences published in GenBank for PCR amplification.The amplified fragment of PTN was recovered from the agarose gel and cloned into the pGEM-T vector.The pGEMT-PTN was cut by double enzyme digestion and ligated into pcDNA3.1 （＋）to construct the PTN overexpression plasmid.After transfection with PTN overexpression plasmid,the expression levels of PTN mRNA in the uterine stromal cells and the expression levels of decidualization markers Prl8a2 and Prl3c1 were detected by qRT-PCR method.The uterine stromal cells transfected with pcDNA3.1 （＋）empty vector were used as control group. Results：The results of identification by double enzyme digestion indicated that the bands of PTN overexpression plasmid were consistent with those of the target gene,and the clone sequencing results suggested that it had 100% homology with mouse PTN gene sequence published in GenBank.Compared with control group, the expression levels of PTN,Prl8a2 and Prl3c1 mRNA in mouse uterine stromal cells in PTN overexpression group were significantly increased （P 〈 0.05）.Conclusion：PTN overexpression could increase the expression levels of decidualization markers in mouse uterine stromal cells,indicating that PTN might play an enhancement effect during＆amp;nbsp;uterine decidualization in the mice.