CAJ | 학술논문

目的:构建可随ATP浓度变化而在内质网与葡萄糖反应蛋白78(GRP78)可逆性结合与解聚的人胰岛索原基因表达载体,并研究其在HepG2细胞中的表达及葡萄糖对其分泌的影响.方法:人工合成含胰岛素信号肽(ss)及条件结合/解聚区域(CAD)的多聚核苷酸链,将其与人胰岛素原基因相连,构建SS-CAD-人胰岛素原表达载体(pSS-CAD-Proins),将其经脂质体转染HepG2细胞,于不同浓度葡萄糖的培养液中培养,检测培养液中的胰岛素浓度并提取细胞总RNA.结果:成功构建pSS-CAD-Proins表达质粒.转染HepG2细胞后48h.在葡萄糖浓度分别为5、15及25mmol/L的培养基中,胰岛素分泌量分别为(4.73±0.52)、(8.84土0.43)及(14.15±0.32)mU/L,不同葡萄糖浓度诱导的胰岛素mRNA表达量明显不同.结论:pSS-CAD-Proins载体能够在HepG2细胞中成功表达,并且胰岛素的分泌及转录受葡萄糖的调控.
목적:구건가수ATP농도변화이재내질망여포도당반응단백78(GRP78)가역성결합여해취적인이도색원기인표체재체,병연구기재HepG2세포중적표체급포도당대기분비적영향.방법:인공합성함이도소신호태(ss)급조건결합/해취구역(CAD)적다취핵감산련,장기여인이도소원기인상련,구건SS-CAD-인이도소원표체재체(pSS-CAD-Proins),장기경지질체전염HepG2세포,우불동농도포도당적배양액중배양,검측배양액중적이도소농도병제취세포총RNA.결과:성공구건pSS-CAD-Proins표체질립.전염HepG2세포후48h.재포도당농도분별위5、15급25mmol/L적배양기중,이도소분비량분별위(4.73±0.52)、(8.84토0.43)급(14.15±0.32)mU/L,불동포도당농도유도적이도소mRNA표체량명현불동.결론:pSS-CAD-Proins재체능구재HepG2세포중성공표체,병차이도소적분비급전록수포도당적조공.
Objective To construct and identify the pSS-CAD-Proins expression plasmid which can integrate and dissociate with GRP78 in the endoplasmic reticulum in ATP dependent way. Methods A fragment of oligonucleotide carrying insulin signal sequence (SS) and conditional aggregation domain (CAD) was synthesized and cloned into the pEGFP-N1 expression plasmid which was connected by human mutated proinsulin gene with furin site . The plasmid was transfected into HepG2 cells by lipofectamine. The insulin concentration in supernatant of cells cultured in different glucose medium was assayed by RIA. The expression levels of recombinant insulin mRNA at three glucose concentrations were determined by RT-PCR. Results The expression plasmid of pSS-CAD-Proins was successfully constructed. At the glucose concentration of 5.00 mmol/L, 15.00 mmol/L and 25.00 mmol/L, the amount of secreted insulin was (4.73±0.52) mU/L, (8.84±0.43) mU/L and (14.15±0.32) mU/L separately (n=3).The mRNA expression of insulin gene was positively correlated to glucose concentration. Conclusions The plasmid vector carrying glucose-regulating human proinsulin gene is successfully constructed and transfected in HepG2 cells, and the secretion and expression of insulin are regulated by glucose concentration.