显性负效应在不同类型胰岛素病发病机制中的作用

Action of the dominant-negative effect on the pathogenesis of insulinopathies

目的探讨显性负效应在不同类型胰岛素基因突变所致糖尿病发病机制中的作用。方法分别用含有突变型前胰岛素原互补DNA(complementary DNA,cDNA)的重组质粒和含有人野生型前胰岛素原cDNA的重组质粒共转染293T细胞,共有V(A3)L、C(A7)Y、R(SP6)H、G(B8)S和G(C28)R 5个突变组,将含有人野生型前胰岛素原cDNA的重组质粒与含有小鼠野生型前胰岛素原cDNA的重组质粒共转染作为正常对照组。转染48 h后收集细胞和培养液,放射免疫法检测细胞内和培养液人胰岛素原水平。结果与正常对照组[(135.84±1.89) pmol/L]相比,C(A7)Y组[(29.28±6.85) pmol/L]和G(B8)S组[(33.62±10.52) pmol/L]细胞培养液中人胰岛素原水平显著降低(P<0.01),其余各组培养液中人胰岛素原水平与正常对照组相比差异无统计学意义(P>0.05)。结论突变型胰岛素原C(A7)Y和G(B8)S对共存的野生型胰岛素原产生显性负效应。

ObjectiveTo explore the action of dominant-negative effect on mutant insulin gene-induced diabetes.Methods293T cells were transfected with a recombinant plasmid containing mutant preproinsulinogen complementary DNA （cDNA） and a recombinant plasmid containing human wild-type preproinsulinogen cDNA. There were 5 mutant groups which mutant preproinsulins respectively bear substitutions V（A3）L, C（A7）Y, R（SP6）H, G（B8）S or G（C28）R. Wild-type mouse preproinsulin and wild-type human preproinsulin were co-transfected as normal control group. After 48 hours, medium and cells were collected. Human proinsulin were detected by human-specific proinsulin radioimmunoassay.ResultsCompared with the control group [（135.84±1.89） pmol/L], human proinsulin levels in medium of C（A7）Y group [（29.28±6.85） pmol/L] and G（B8）S group[（33.62±10.52） pmol/L] decreased significantly （P〈0.01）. There was no significant difference in human proinsulin level between the other groups and the control group （P〉0.05）.ConclusionMutants C（A7）Y and G（B8）S induce the dominant-negative effect on co-existing wild-type proinsulin.