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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。
Objective To investigate the effect of ODN on the expression of collagen Ⅰ, Ⅲ and tumor necrosis factor α (TNF-α) in human umbilical artery smooth muscle cells (HUASMC) induced by cord blood serum of preeclampsia patients . Methods Primary cultured HUASMC was divided into 4 groups: group A (cultured in normal neonatal umbilical serum), group B (cultured umbilical serum in neonates with preeclampsia), group C (in neonates with preeclampsia Umbilical cord serum and nuclear factor κB cis decoy ODN culture) and D (neonatal cord serum supplemented with preeclampsia and disordered ODN culture). The viability of HUASMC was measured by MTT assay and flow cytometry [absorbance (A)] and apoptosis rate, and the mRNA of procollagen ¢ ó and ¢ ó of HUASMC in each group was detected by RT-PCR The expression of TNF-α protein was detected by Western blot [all expressed as A value]. Results: (1) Proliferation activity in group A was 0.11 ± 0.02, in group B was 0.19 ± 0.02, in group C was 0.14 ± 0.02 and in group D was 0.18 ± 0.03. The proliferative activity of group B, C and D was significantly higher than that of group A <0.05), while those in group B and D were significantly higher than those in group C (P <0.05). (2) The apoptosis rate was (14.3 ± 1.2)% in group A, (7.8 ± 1.3)% in group B, (10.1 ± 1.2)% in group C and (8.1 ± 1.3)% in group D , The apoptotic rate in group D was significantly lower than that in group A (P <0.05), and the difference between group B and group C was also statistically significant (P <0.05). (3) The expression of procollagen Ⅰ mRNA in group A was 0.16 ± 0.02, in group B was 0.31 ± 0.04, in group C was 0.23 ± 0.04, in group D was 0.30 ± 0.03, and in group B, C and D In group A (P <0.05), the difference between group B and group C was also statistically significant (P <0.05). (4) Collagen ⅢmRNA: The mRNA expression levels of procollagen Ⅲ in the four groups were not significantly different from each other (P> 0.05). (5) The level of TNF-αprotein in group A was 0.15 ± 0.03, in group B was 0.74 ± 0.11, in group C was 0.36 ± 0.09, in group D was 0.79 ± 0.12, and in group B, C and D There was no significant difference between group B and group D (P <0.05); group B and D were significantly higher than group C (P <0.05). Conclusions ODN can decrease the proliferation, collagen synthesis and inflammatory cytokines synthesis of HUASMC in cord blood of patients with preeclampsia. Nuclear factor κB may play an important role in the pathogenesis of placental vascular lesions in preeclampsia.