核因子κB顺式诱骗寡脱氧核苷酸对子痫前期患者脐血清培养的脐动脉平滑肌细胞功能的影响

目的 探讨核因子κB顺式诱骗寡脱氧核苷酸(ODN)对子痫前期患者脐血清诱导人脐动脉平滑肌细胞(HUASMC)前胶原Ⅰ、Ⅲ及肿瘤坏死因子α(TNF-α)表达的影响.方法 将原代培养的HUASMC分4组:A组(加入正常新生儿脐血清培养)、B组(加入子痫前期患者的新生儿脐血清培养)、C组(加入子痫前期患者的新生儿脐血清及核因子κB顺式诱骗ODN培养)和D组(加入子痫前期患者的新生儿脐血清及无序ODN培养).通过四甲基偶氮唑蓝比色法及流式细胞仪检测各组HUASMC的增殖活力[以吸光度(A)表示]和凋亡率;应用RT-PCR检测各组HUASMC的前胶原Ⅰ、ⅢmRNA的表达,蛋白印迹法检测TNF-α蛋白的表达[均以A值表示].结果 (1)增殖活力:A组为0.11±0.02,B组为0.19±0.02,C组为0.14±0.02,D组为0.18±0.03;B、C、D组增殖活力显著高于A组(P＜0.05),B、D组显著高于C组(P＜0.05).(2)凋亡率:A组为(14.3±1.2)%,B组为(7.8±1.3)%,C组为(10.1±1.2)%,D组为(8.1±1.3)%;B、C、D组凋亡率显著低于A组(P＜0.05),B组与C组比较,差异也有统计学意义(P＜0.05).(3)前胶原Ⅰ mRNA:A组为0.16±0.02,B组为0.31±0.04,C组为0.23±0.04,D组为0.30±0.03,B、C、D组前胶原Ⅰ mRNA表达水平显著高于A组(P＜0.05),B组与C组比较,差异也有统计学意义(P＜0.05).(4)前胶原ⅢmRNA:4组前胶原ⅢmRNA表达水平相互比较,差异均无统计学意义(P＞0.05).(5)TNF-α蛋白:A组为0.15±0.03,B组为0.74±0.11,C组为0.36±0.09,D组为0.79±0.12,B、C、D组TNF-α蛋白表达显著高于A组(P＜0.05);B、D组又显著高于C组(P＜0.05),但B组与D组比较,差异无统计学意义(P＞0.05).结论 核因子κB顺式诱骗ODN能降调子痫前期患者脐血清促HUASMC增殖、胶原合成及炎性因子合成的作用.核因子κB在子痫前期患者胎盘血管病变过程中可能起重要作用.

Effects of nuclear factor-kappaB decoy oligodeoxynucleotide on the function of human umbilical artery smooth muscle cells induced by umbilical sera in preeclampsia

OBJECTIVE: To study the effects of nuclear factor-kappaB (NF-kappaB) decoy oligodeoxynucleotide (ODN) on the preeclamptic umbilical serum induced expression of precollagen I, III mRNA and tumor necrosis factor-alpha (TNF-alpha) in cultured human umbilical artery smooth muscle cells (HUASMC). METHODS: Primary cultured HUASMC of normal pregnancy were divided into four groups: group A (HUASMC were incubated with umbilical serum of normal pregnancy); group B (HUASMC were incubated with umbilical serum of preeclampsia); group C (HUASMC were transfected with NF-kappaB cis decoy ODN 48 h before incubation with umbilical serum of preeclampsia); group D (HUASMC were transfected with NF-kappaB scramble ODN 24 h before incubation with umbilical serum of preeclampsia). NF-kappaB cis decoy ODN and NF-kappaB scramble ODN were transfected with cationic lipofectamine to the latter two groups, respectively. The proliferation of human umbilical artery smooth muscle cells was evaluated by methyl thiazolyl tetrazolium and the apoptosis was analyzed by flow cytometry. The expression levels of precollagen I, III mRNA were detected by RT-PCR, the expression levels of TNF-alpha were detected by western blot. RESULTS: (1) The proliferation of group B (0.19 +/- 0.02) and group D (0.18 +/- 0.03) was significantly increased as compared with those of group A (0.11 +/- 0.02) and group C (0.14 +/- 0.02) (P < 0.05). (2) The apoptosis rates of group B [(7.8 +/- 1.3)%], group C [(10.1 +/- 1.2)%] and group D [(8.1 +/- 1.3)%] were significantly reduced as compared with that of group A [(14.3 +/- 1.2)%] (P < 0.05), and there was a significant difference between groups B and C (P < 0.05). (3) The expression levels of precollagen I mRNA of group B (0.31 +/- 0.04), group C (0.23 +/- 0.04) and group D (0.30 +/- 0.03) were significantly increased as compared with that of group A (0.16 +/- 0.02) (P < 0.05), and there was a significant difference between groups B and C (P < 0.05). (4) There were no significant differences among the four groups in the expression level of precollagen III mRNA (P > 0.05). (5) The expression of TNF-alpha of group B (0.74 +/- 0.11), group C (0.36 +/- 0.09) and group D (0.79 +/- 0.12) were significantly higher than that of group A (0.15 +/- 0.03) (P < 0.05), and the expression of TNF-alpha of groups B and D were significantly higher than that of group C (P < 0.05), while there was no significant difference between groups B and D (P > 0.05). CONCLUSIONS: NF-kappaB cis decoy ODN could down-regulate the proliferation, as well as the expression levels of precollagen and TNF-alpha of HUASMC induced by umbilical serum of preeclampsia. NF-kappaB may play an important role in the pathogenesis of placental artery abnormalities in preeclampsia.