目的研究早发型重度子痫前期(ESPE)和晚发型重度子痫前期(LSPE)患者胎盘组织中Staufen1(STAU1)的表达情况及其临床意义。方法选取2016年5月至2018年4月于我院剖宫产分娩的重度子痫前期(SPE)孕妇30例作为SPE组,其中ESPE15例,LSPE15例;另...目的研究早发型重度子痫前期(ESPE)和晚发型重度子痫前期(LSPE)患者胎盘组织中Staufen1(STAU1)的表达情况及其临床意义。方法选取2016年5月至2018年4月于我院剖宫产分娩的重度子痫前期(SPE)孕妇30例作为SPE组,其中ESPE15例,LSPE15例;另选择同期正常妊娠孕妇30例作为对照组(N组),其中ESPE对照(EN)组10例,LSPE对照(LN)组20例。采用实时PCR(RT-PCR)方法检测孕妇胎盘组织STAU1 mRNA的相对表达量,采用Western blotting方法及免疫组织化学方法检测胎盘组织中STAU1蛋白的表达及定位情况。结果实时PCR结果显示,SPE组胎盘组织中STAU1 mRNA表达水平显著低于N组,差异有统计学意义(0.844±0.074 vs 1.096±0.057,P<0.01);其中,ESPE组胎盘组织中STAU1 mRNA表达水平显著低于EN组,差异有统计学意义(0.683±0.061 vs 1.015±0.060,P<0.01),而LSPE组胎盘组织中STAU1 mRNA表达水平与LN组无统计学差异(1.005±0.124 vs 1.136±0.080,P>0.05)。Western blotting结果显示,STAU1蛋白表达于胎盘组织中,SPE组胎盘组织中STAU1蛋白的表达水平显著低于N组,差异有统计学意义(0.916±0.102 vs 1.573±0.135,P<0.001);其中ESPE组胎盘组织中STAU1蛋白表达水平显著低于EN组,差异有统计学意义(0.719±0.101 vs 2.025±0.155,P<0.001),而LSPE组胎盘组织STAU1蛋白表达水平与LN组无统计学差异(1.112±0.165 vs 1.337±0.165,P>0.05)。免疫组织化学结果显示,胎盘组织的细胞滋养细胞和合体滋养细胞中均有STAU1蛋白表达,且主要在合体滋养细胞的细胞质中表达。结论STAU1低表达可能与SPE中ESPE的发病密切相关。展开更多
The proteins Inscuteable and Staufen are key components during asymmetric cell division of neuroblasts for the development of Drosophila melanogaster. Expression and purification of both proteins has been a difficult ...The proteins Inscuteable and Staufen are key components during asymmetric cell division of neuroblasts for the development of Drosophila melanogaster. Expression and purification of both proteins has been a difficult task for structure-function studies. Based on codon optimization for protein expression in Escherichia coli, we have been able to produce, in soluble form, the C-terminal domains of Inscuteable and Staufen as chimeras with N-terminal maltose binding protein tag that contains a rigid linker between them for feasible crystallization. In addition, using an optimized synthetic gene, corresponding to the amino acid region 250 - 623 of Inscuteable fused to glutathione-S-transferase, low-scale expression experiments showed production of soluble protein. Finally, eukaryotic expression of Inscuteable in the methylothropic yeast Pichia pastoris failed to produce the Drosophila protein at detectable amounts, reinforcing the fact that E. coli still was the microorganism of choice for high-yield protein expression.展开更多
文摘目的研究早发型重度子痫前期(ESPE)和晚发型重度子痫前期(LSPE)患者胎盘组织中Staufen1(STAU1)的表达情况及其临床意义。方法选取2016年5月至2018年4月于我院剖宫产分娩的重度子痫前期(SPE)孕妇30例作为SPE组,其中ESPE15例,LSPE15例;另选择同期正常妊娠孕妇30例作为对照组(N组),其中ESPE对照(EN)组10例,LSPE对照(LN)组20例。采用实时PCR(RT-PCR)方法检测孕妇胎盘组织STAU1 mRNA的相对表达量,采用Western blotting方法及免疫组织化学方法检测胎盘组织中STAU1蛋白的表达及定位情况。结果实时PCR结果显示,SPE组胎盘组织中STAU1 mRNA表达水平显著低于N组,差异有统计学意义(0.844±0.074 vs 1.096±0.057,P<0.01);其中,ESPE组胎盘组织中STAU1 mRNA表达水平显著低于EN组,差异有统计学意义(0.683±0.061 vs 1.015±0.060,P<0.01),而LSPE组胎盘组织中STAU1 mRNA表达水平与LN组无统计学差异(1.005±0.124 vs 1.136±0.080,P>0.05)。Western blotting结果显示,STAU1蛋白表达于胎盘组织中,SPE组胎盘组织中STAU1蛋白的表达水平显著低于N组,差异有统计学意义(0.916±0.102 vs 1.573±0.135,P<0.001);其中ESPE组胎盘组织中STAU1蛋白表达水平显著低于EN组,差异有统计学意义(0.719±0.101 vs 2.025±0.155,P<0.001),而LSPE组胎盘组织STAU1蛋白表达水平与LN组无统计学差异(1.112±0.165 vs 1.337±0.165,P>0.05)。免疫组织化学结果显示,胎盘组织的细胞滋养细胞和合体滋养细胞中均有STAU1蛋白表达,且主要在合体滋养细胞的细胞质中表达。结论STAU1低表达可能与SPE中ESPE的发病密切相关。
文摘The proteins Inscuteable and Staufen are key components during asymmetric cell division of neuroblasts for the development of Drosophila melanogaster. Expression and purification of both proteins has been a difficult task for structure-function studies. Based on codon optimization for protein expression in Escherichia coli, we have been able to produce, in soluble form, the C-terminal domains of Inscuteable and Staufen as chimeras with N-terminal maltose binding protein tag that contains a rigid linker between them for feasible crystallization. In addition, using an optimized synthetic gene, corresponding to the amino acid region 250 - 623 of Inscuteable fused to glutathione-S-transferase, low-scale expression experiments showed production of soluble protein. Finally, eukaryotic expression of Inscuteable in the methylothropic yeast Pichia pastoris failed to produce the Drosophila protein at detectable amounts, reinforcing the fact that E. coli still was the microorganism of choice for high-yield protein expression.
