PKC-NF-κB信号传导对子前期患者脐血清诱导脐动脉平滑肌细胞胶原mRNA表达影响的体外研究

In vitro study on the effect of PKC-NF-κB signal transduction pathway on preeclamptic umbilical sera-induced collagen expression in HUASMC

目的 探讨蛋白激酶C-核因子-kappa β（protein kinase C-nuclearfactor-kappaB，PKC-NF-κB）信号传导通路对子瘌前期患者脐静脉血清诱导脐动脉平滑肌细胞（human umbilical artery smooth muscle cell，HUASMC）增殖及前胶原Ⅰ、ⅢmRNA的表达的影响。方法 采用组织块培养法培养正常妊娠HUASMC，传代，待细胞长满至70％～80％，加或不加PKC的抑制剂多黏菌素B（ploymyxin B，PMB）作用30min后分别加入子瘌前期患者脐静脉血清，培养2h，Western印迹法测定细胞胞浆Ⅰ-κB、胞核NF-κB活性；细胞培养48h，MTT测定细胞活力，流式细胞学测定细胞周期，逆转录-聚合酶链反应（reverse transcription-polymerase chain reaction，RT-PCR）测定HUA-SMC的bcl-3及前胶原Ⅰ、ⅢmRNA的表达。结果 子瘌前期患者血清组HUSMC的胞浆Ⅰ-κB含量（0．3765±0．0321）明显低于对照组（0．7857±0．0296），胞核NF-κB含量（0．8549±0．0395）、bcl-3表达（0．6782±0．213）、细胞活力（0．249±0．0748）、前胶原Ⅰ的表达（0．7387±0．208）、S期细胞百分比（20．13±1．21）％及G2／M期细胞百分比（16．45±0．89）％明显高于对照组（P〈0．01），G0＋G1期细胞百分比（62．53±2．85）％明显低于对照组（P〈0．01）。PMB预处理组Ⅰ-κB含量（0．8120±0．0352）明显高于子瘌前期组（P〈0．01）；NF-κB核转位（0．4528±0．0315）、bcl-3表达（0．216±0．193）、S期细胞百分比（8．21±0．56）％及G2／M期细胞百分比（5．38±0．52）％、细胞活力（0．1525±0．0454）及前胶原Ⅰ（0．3084±0．022）的表达低于子瘌前期组（P〈0．01），G0＋G1期细胞百分比（89．61±3．18）％明显高于子痴前期组（P〈0．01）。结论 PKC-NF-κB信号传导途径的激活在子痴前期患者胎盘血管病变过程中可能起重要作用。

Objective To study the effect of protein kinase C-nuclear factor-kappa B （PKC-NF-κB） signal transduction pathway on the umbilical sera-induced human umbilical artery smooth muscle cell （HUASMC） proliferation and the expression of precollagen Ⅰ , and Ⅲ mRNA in preeclamptic women. Methods HUASMC from normal pregnancy were cultured and generated. After 30 minutes of incubation with or without Polymyxin B （PMB）, the umbilical serum from the preeclamptic women was added. Two hours later, the expression of Ⅰ-κB and NF-κB were detected by Western blot. After 48 h, the activity of HUASMC was evaluated by MTT, the cell cycle by flow cytomerty and the expression of precollagen Ⅰ , Ⅲ Ⅲ and bcl-3 mRNA by reverse transcription-polymerase chain reaction（RT-PCR）. Results Ⅰ-κB level （0. 3765±0. 0321） and the percentage of Go ＋G1 phase cells （62.53±2. 85）% in preeclamptic group were significantly lower than those in control group [（0. 7857±0. 0296）,（78.66±3.25）%,P〈0.01] and PMB group [（0. 8120±0. 0352）%, （89.61±3.18）%, P〈0.01]. The level of NF-κB （0. 8549±0. 0395）, precollagen mRNA （0. 7387±0. 208） and bcl-3 mRNA （0. 6782±0. 213） of HUASMC, and percentage of cells in S [（20. 13±1.21）%] and G2 ＋M phase [（16. 45±0. 89）%] were significantly higher comparing with the normal group [（0.4832±0.0273）,（0.1084±0.177）,（0.1529±0.125）,（12.31±0.75）%,（9.7±0.95）%,P〈0.05]and PMB group[（90.4528±0.0315）,（0.3084±0.022）,（0.216±0.193）,（8.21±0.56）%,（5.38±0.52）%,P〈0.05].Conclusions PKC=NF-κB signal transduction pathway might play a key role in SMC phenotype modulation,which is important in the pathogenesis of placental blood vessel changes in preeclampsia.