CAJ | 학술논문

目的建立以胰岛新生相关蛋白启动子为靶点的高通量筛选模型，用于胰岛新生相关蛋白基因表达上调剂的筛选。方法以叙利亚金黄地鼠基因组 DNA为模板扩增胰岛新生相关蛋白核心启动子序列，克隆至 pGL4.17质粒载体构建重组质粒。采用脂质体介导的方法将重组质粒和内参质粒 pRL-TK共转染金黄地鼠胰岛β细胞 HIT-T15，双荧光素酶报告基因系统检测荧光素酶的表达活性。对瞬时转染条件进行优化，以豆蔻酸-佛波醇-乙酸酯作为阳性对照来评价模型的有效性，并利用该细胞模型对本研究所的化合物库进行筛选。结果构建了重组荧光素酶报告基因质粒 pGL4.17-INGAP，并成功转染 HIT-T15细胞株。对模型进行优化后，应用阳性对照对其进行评价，Z'因子为0.57，适用于进行高通量筛选。用该模型筛选了本所化合物库，其中白藜芦醇显示出较好的上调作用，EC50为1.6μg/ml。结论成功建立了以胰岛新生相关蛋白启动子为靶点的基因表达上调剂筛选模型。
목적건립이이도신생상관단백계동자위파점적고통량사선모형，용우이도신생상관단백기인표체상조제적사선。방법이서리아금황지서기인조 DNA위모판확증이도신생상관단백핵심계동자서렬，극륭지 pGL4.17질립재체구건중조질립。채용지질체개도적방법장중조질립화내삼질립 pRL-TK공전염금황지서이도β세포 HIT-T15，쌍형광소매보고기인계통검측형광소매적표체활성。대순시전염조건진행우화，이두구산-불파순-을산지작위양성대조래평개모형적유효성，병이용해세포모형대본연구소적화합물고진행사선。결과구건료중조형광소매보고기인질립 pGL4.17-INGAP，병성공전염 HIT-T15세포주。대모형진행우화후，응용양성대조대기진행평개，Z'인자위0.57，괄용우진행고통량사선。용해모형사선료본소화합물고，기중백려호순현시출교호적상조작용，EC50위1.6μg/ml。결론성공건립료이이도신생상관단백계동자위파점적기인표체상조제사선모형。
Objective To establish a screening model for identifying up-regulators of INGAP expression and screen active compounds. Methods Syrian golden hamster genome was used as template to amplify the core promoter sequence of INGAP. Then the obtained promoter fragment was cloned into pGL4.17 vector to construct the recombinant plasmid which subsequently was co-transfected into HIT-T15 cell line together with the internal reference plasmid pRL-TK using Lipofectamine 2000. The luciferase activity was detected using dual-luciferase reporter assay system in 96-well format. After optimizing the conditions of transient transfection, PMA was used as positive control to assess the validation of the model. The compounds in the library of our institute were then screened. Results The recombinant plasmid named pGL4.17-INGAP was constructed and successfully transfected into HIT-T15 cell strain. The model was evaluated by positive control after optimization, and Z' factor is 0.57, showing that the model was able to use for screening up-regulators. Among the screened compounds, resveratrol showed up-regulated activity with EC50 value of 1.6μg/ml. Conclusion The cellular screening model for identifying up-regulators of INGAP expression was established.