摘要
试验构建丝氨酸蛋白酶抑制剂亚家族成员6b(Serine peptidase inhibitor,clade B,member 6b,SerpinB6b)基因过表达载体,研究其对小鼠子宫基质细胞蜕膜化的影响。根据Gen Bank中已发表的SerpinB6b基因序列,设计含酶切位点的特异性引物,PCR扩增并与p GEM-T载体连接,经限制性内切酶EcoRⅠ和HindⅢ双酶切后,与pc DNA3. 1(+)载体融合得到SerpinB6b过表达载体。将其转染到体外分离培养的小鼠子宫基质细胞,利用荧光定量PCR法检测子宫基质细胞中SerpinB6b mRNA表达的变化,以及SerpinB6b过表达后蜕膜化标志分子Prl8a2和Prl3c1表达的变化,对照组转染pc DNA3. 1(+)空载体。结果表明:SerpinB6b过表达载体经双酶切鉴定后,结果与目的基因条带吻合,测序结果与Gen Bank中小鼠SerpinB6b基因序列同源性为100%。与对照组相比,转染SerpinB6b过表达载体后,SerpinB6b在小鼠子宫基质细胞中的表达显著升高。同时,过表达SerpinB6b可明显促进蜕膜化标志分子Prl8a2和Prl3c1 mRNA的表达。提示SerpinB6b可能在小鼠子宫基质细胞蜕膜化过程中起重要的调节作用。
In this study,an overexpression vector of SerpinB6 b(serine peptidase inhibitor,clade B,member 6 b)was constructed to explore its effects on mouse uterine stromal cell decidualization.According to the published SerpinB6 b gene sequence in GenBank,specific primers containing restriction enzyme sites were designed and amplified by PCR.The amplified products were cloned into pGEM-T vector.After being digested with restriction endonucleases EcoRⅠand HindⅢ,the fragments were ligated into pcDNA3.1(+)to construct the SerpinB6 b overexpression vector.The expression of SerpinB6 b mRNA and the level of decidualization markers Prl8 a2 and Prl3 c1 mRNA in mouse uterine stromal cell tranfected with SerpinB6 b overexpression plasmid were detected by realtime PCR.An empty pcDNA3.1(+)expression vector was served as control.The results of SerpinB6 b overexpression vector identification by double enzyme digestion were proved to be consistent with the target gene band,and the sequencing result was 100%homologous to mouse SerpinB6 b gene sequence in GenBank.Compared with the control group,SerpinB6 b expression was significantly enhanced in mouse uterine stromal cells transfected with SerpinB6 b overexpression plasmid.Compared with the control group,SerpinB6 b expression was significantly enhanced in mouse uterine stromal cells transfected with SerpinB6 b overexpression plasmid.Simultaneously,overexpression of SerpinB6 b could significantly promote the expression of decidualized marker molecules Prl8 a2 and Prl3 c1 mRNA,suggesting that SerpinB6 b may play an important regulatory role in mouse uterine stromal cell decidualization.
作者
王雨思
于海帆
郭斌
杨占清
岳占碰
WANG Yusi;YU Haifan;GUO Bin;YANG Zhanqing;YUE Zhanpeng(College of Veterinary Medicine,Jilin University,Changchun 130062,China)
出处
《吉林农业大学学报》
CAS
CSCD
北大核心
2019年第2期248-252,共5页
Journal of Jilin Agricultural University
基金
国家自然科学基金项目(31672503
31472158)
吉林大学研究生创新基金项目(2017085)
