反义寡核苷酸对精氨酸升压素诱导心脏成纤维细胞β_1整合素表达的抑制作用

Antisense oligonucleotides inhibit the expression of β_1 integrin induced by arginine vasopressin in cardiac fibroblasts

目的 探讨反义寡核苷酸对精氨酸升压素 (AVP)诱导心脏成纤维细胞 (CFs) β1整合素表达及细胞增殖的影响 ,为防治AVP诱导的心肌纤维化提供理论依据。方法 设计合成反义、正义及错配 β1整合素寡核苷酸片段 ,转入体外培养的CFs中 ,采用原位ELISA技术检测不同状态下 β1整合素表达水平 ,应用四氮唑盐 (MTT)比色法检测细胞增殖。结果 (1)随着AVP浓度的增加 ,CFs表面β1整合素表达水平呈上升趋势 ,各实验组之间吸光度比较差异有显著意义 (F =4 93;P <0 0 1)。其中 1× 10 -7mol/LAVP和 1× 10 -6mol/LAVP组的CFsβ1整合素表达水平分别为 (0 2 5± 0 0 1)A值和(0 30± 0 0 4 )A值 ,均明显高于对照组的 (0 2 1± 0 0 2 )A值 ,差异有非常显著意义 (P <0 0 1) ;(2 )反义链 +AVP组的CFsβ1整合素表达水平为 (0 19± 0 0 2 )A值 ,反义链与AVP共同作用组明显低于AVP组的 (0 2 5± 0 0 1)A值 ,并有非常显著性差异 (P <0 0 1)。正义链 +AVP组和错配链 +AVP组的CFsβ1整合素表达水平分别为 (0 2 5± 0 0 1)和 (0 2 5± 0 0 2 )A值 ,均明显高于反义链与AVP共同作用组 (P <0 0 1)。 (3)空白对照组、AVP组、反义链 +AVP组、正义链 +AVP组和错配链 +AVP组CFsMTT法分别为 (0 36± 0 0 1)OD值。

Objective To develop a new way for preventing cardiac fibrosis, antisense oligonucleotides were applied to modulate the expression of β 1 integrin and cell proliferation induced by argipressin(AVP) in cardiac fibroblasts.Methods ELISA technique was used to detect the expression of β 1 integrin.Phosphorothioate modified antisense, sense and mismatched oligonucleotides were used to interfere with the expression of β 1 integrin.MTT assay was used to measure cell proliferation.Results (1) AVP prominently induced the expression of β 1 integrin on cardiac fibroblasts in a dose dependent manner.After 1×10 -7 mol/L AVP and 1×10 -6 mol/L AVP were added, the absorption value by ELISA were (0.25±0.01)and (0.30±0.04),respectively.Both were significantly higher than that of control (P<0.01).The expression of β 1 integrin in 1×10 -6mol/L AVP group was also higher than those in 1×10 -9mol/L AVP group(0.24±0.03)and 1×10 -8mol/L AVP group(0.24±0.03),respectively.(2) The absorption value of AVP group, sense oligonucleotides+AVP group and mismatched oligonucleotides+AVP group were (0.25±0.01),(0.25±0.01)and (0.25±0.02), respectively.They all had significantly higher level of β 1 integrin compared with that of cardiac fibroblasts treated with antisense oligonucleotides(0.19±0.02,P<0.01).(3)OD value of control, AVP group, antisense oligonucleotides+AVP group, sense oligonucleotides+AVP group and mismatched oligonucleotides+AVP group measured with MTT assay were (0.36±0.01), (0.38±0.02), (0.35±0.03), (0.38±0.02) and (0.38±0.01), respectively.OD value of antisense oligonucleotides+AVP group was significantly lower fthose of AVP, sense oligonucleotides+AVP and mismatched oligonucleotides+AVP groups(P<0.05). Conclusions Antisense oligonucleotides may reverse the expression of β 1 integrin and cell proliferation promoted by AVP in cardiac fibroblasts, whereas those treated with sense and mismatched oligonucleotides may not.Antisense technology may be useful for dealing with matrix remodeling.