NK细胞、HLA-G、和HLA-DRB1等位基因在子痫前期发病机制中的作用研究

目的:研究子痫前期患者发病机制中相关的免疫学变化及基因背景。方法:SAP法检测血液中NK细胞数量。MTT珐检测NK细胞增殖能力。51Cr释放法测定NK细胞杀伤活性。免疫组化法检测胎盘蜕膜浸润的NK细胞数量及HLA-G蛋白表达水平。RT-PCR和real time PCR法检测胎盘蜕膜组织HLA-G mRNA。PCR-SSP法检测两组HLA-DRB1等位基因频率,并分析母/儿间所携带某些HLA-DRB1等位基因配伍的频率。结果:①子痫前期患者外周血及其新生儿脐血NK细胞数均显著高于正常晚孕者及其新生儿。②子痫前期患者外周血及其新生儿脐血NK细胞增殖活性及杀伤活性均明显高于正常晚孕组(P<0.05)。③轻度与重度子痫前期组胎盘蜕膜NK细胞均多于正常晚孕组,但仅重度子痫前期组蜕膜NK细胞数量增加有统计学意义(P<0.05)。④轻度子痫前期组、重度子痫前期组和正常晚孕组之间HLA-G mRNA水平两两比较差异均无统计学意义(P>0.05)。⑤轻度子痫前期组HLA-G蛋白表达水平与正常晚孕组无显著性差异,重度子痫前期组胎盘HLA-G蛋白表达水平较正常晚孕组显著降低(P<0.05)。⑥两组孕妇和新生儿均检出13个HLA-DRB1等位基因,各等位基因频率在两组间均无明显差异。子痫前期组母不携带HLA-DRB1*04基因/儿携带DRB1*04基因、均不携带HLA-DRB1*14基因的频率显著高于正常晚孕组(P<0.05);母/儿均不携带HLA-DRB1*10基因、均携带HLA-DRB1*14的频率显著低于正常晚孕组(P<0.05)。结论:①先兆子痫患者外周血、脐血及蜕膜的NK细胞数量增多,活性增强,在妊娠过程中影响母胎免疫耐受。②母婴所携带的某些HLA-DRB1基因配伍与子痫前期易感性或抗性相关;父源性HLA-DRB1*04基因与子痫前期易感性相关。

Effects of NK cells, HLA-G and HLA-DRB1 on the etiology of pre-eclampsia

Objective: To investigate the immune and genetic aspects of pre-eclamptic patients.Methods: By SAP method NK cells were detected in pre-eclampsia group and control group.Detect NK cells proliferating ability by MTT and killing power by51Cr releasing test.Use the immunohistochemistry method to determine the quantity of NK cells and the expression of HLA-G on decidua.Use RT-PCR and real time PCR to quantify the HLA-G mRNA on deciduas.By PCR-SSP the polymorphism of HLA-DRB1 was detected in pre-eclamptic group and control group,and some frequence of common genes and specific bingding were analysed.Results: ①Both in peripheral maternal blood(PMB) and in umbilical blood(UB) the quantity of NK cells was significantly higher in pre-eclampsia group than in control group(P<0.05).②Proliferating ability of NK cells from pre-eclampsia group PMB and UB was increased than that from control group PMB and UB respectively(P<0.05).③Compared with that in control group,the counts of NK cells on decidua of mild and severe pre-eclampsia group were higher,while only the increasement of severe preeclampsia group had statistical difference(P<0.05).④The quantity of HLA-G mRNA on deciduas of mild and severe pre-eclampsia group had no statistical difference with that of control group.⑤The quantity of HLA-G protein on deciduas of mild and severe pre-eclampsia group was lower than control group,while only it had qualitative difference between severe pre-eclampsia group and normal group(P<0.05).⑥In PMB and UB,13 HLA-DRB1 allele genes were detected and there was no significant difference between pre-eclampsia group and control group.Compared with control group,in pre-eclampsia group the frequencies of HLA-DRB1*04 maternal/fetus(-)/(+)and HLA-DRB1*14 maternal/fetus(-)/(-)were increased while the frequencies of HLA-DRB1*10 maternal/fetus(-)/(-)and HLA-DRB1*14 maternal/fetus(+)/(+)were decreased(P<0.05).Conclusion: ①Both quantity and function of the NK cells yn preeclampsia patients became much more and stronger,respectively.That the NK cells may have relationship with the etiology of preeclampsia.②HLA-DRB1 maternal/fetus special bindings may be associated with the susceptibility or protective of pre-eclampsia.Paternal HLA-DRB1*04 may be a susceptible gene.