CAJ | 학술논문

目的：构建几种与人类糖尿病相关的人突变型胰岛素原质粒，并在大鼠胰岛素瘤细胞（INS-1）中进行表达。方法以野生型人胰岛素原质粒基因PCMS-EGFP/HWT为模板，设计并合成引物，利用定点突变技术，PCR生成PCMS-EGFP/H-C(B19)G、H-L(B11)P、H-R(S6)C、H-F(B25)L 4种单点突变质粒，对每个质粒进行序列测定验证定点突变成功，用脂质体2000将上述野生型、4种突变型质粒及空质粒分别转染INS-1细胞，放射免疫法检测各细胞培养液中人胰岛素水平。结果序列测定证实上述质粒定点突变成功, H-C(B19)G、H-L(B11)P、H-R(S6)C 3组突变细胞培养液中胰岛素水平低于野生型和H-F(B25)L组（P＜0.05），与空质粒组差异无统计学意义，且3组彼此间差异无统计学意义；H-F(B25)L组与野生型差异无统计学意义（P＞0.05）。结论成功构建并表达4种突变型人胰岛素原质粒，不同突变型胰岛素原可能通过不同机制导致糖尿病。
목적：구건궤충여인류당뇨병상관적인돌변형이도소원질립，병재대서이도소류세포（INS-1）중진행표체。방법이야생형인이도소원질립기인PCMS-EGFP/HWT위모판，설계병합성인물，이용정점돌변기술，PCR생성PCMS-EGFP/H-C(B19)G、H-L(B11)P、H-R(S6)C、H-F(B25)L 4충단점돌변질립，대매개질립진행서렬측정험증정점돌변성공，용지질체2000장상술야생형、4충돌변형질립급공질립분별전염INS-1세포，방사면역법검측각세포배양액중인이도소수평。결과서렬측정증실상술질립정점돌변성공, H-C(B19)G、H-L(B11)P、H-R(S6)C 3조돌변세포배양액중이도소수평저우야생형화H-F(B25)L조（P＜0.05），여공질립조차이무통계학의의，차3조피차간차이무통계학의의；H-F(B25)L조여야생형차이무통계학의의（P＞0.05）。결론성공구건병표체4충돌변형인이도소원질립，불동돌변형이도소원가능통과불동궤제도치당뇨병。
Objective To construct several human proinsulin mutants plasmid related to diabetes and to express in INS-1 (Insulin secreting beta cell derived line) cell. Methods Human mild proinsulin gene was used as template , and site-directed mutagenesis PCR was employed to generate four human proinsulin plasmid mutants. Each mutant plasmid was sequenced then transfected with empty plasmid and mild plasmid into INS-1 cell by liposome 2000. Insulin value in each cell solution was determined by radioimmunoassay. Results Proinsulin mutants plasmid were confirmed by sequencing. In-sulin values in culture solution of H-C(B19)G、H-L(B11)P、H-R(S6)C mutants are less than those in wild type and H-F (B25)L(P<0.05). Comparison of insulin values between H-C(B19)G、H-L(B11)P、H-R(S6)C groups were not statistically significant(P>0.05), and all these three groups showed no significant differences with empty plasmid group statistically (P>0.05).Insulin value of H-F(B25)L was of no significant differences statistically with empty plasmid(P>0.05). Conclu-sion Four human proinsulin mutants plasmid were constructed and expressed successfully in INS-1 cell, and different mu-tants plasmid result in diabetes through different mechanism.