β_1整合素反义寡核苷酸抑制精氨酸加压素诱导下心脏成纤维细胞DNA合成的作用

目的:探讨β1整合素反义寡核苷酸(ASODN)对精氨酸加压素(AVP)诱导大鼠心脏成纤维细胞(CFs)DNA合成功能的抑制作用。方法:以胰蛋白酶消化法分离、培养SD大鼠的CFs,采用MTT吸光度法及3H-胸腺嘧啶核苷(3H-TdR)掺入法,原位酶联免疫吸附测定(ELISA)等技术,观察基础状态下β1整合素ASODN对CFs细胞数目及DNA合成功能的影响;β1整合素ASODN对AVP诱导的β1整合素表达,CFs增殖及DNA合成功能的影响;基础状态下设空白对照组、反义链组和正义链组;AVP诱导下设空白对照组、1×10-7mol/L AVP组、反义链+1×10-7mol/L AVP组和正义链+1×10-7mol/L AVP组,每组均为8个复孔。结果:①反义链组的CFs细胞数目及3H-TdR掺入率明显低于空白对照组及正义链组,并且均有统计学意义(P0.01);正义链组与对照组比较无统计学意义。②1×10-7mol/LAVP组和正义链+1×10-7mol/LAVP组的β1整合素表达水平,CFs细胞数目均高于空白对照组(P0.05),β1整合素ASODN与AVP共同作用组的β1整合素表达水平,CFs细胞数目明显低于1×10-7mol/LAVP组和正义链+1×10-7mol/L AVP组,并且均有非常显著性意义(P0.01)。③1×10-7mol/L AVP组和正义链+1×10-7mol/L AVP组的CFs3H-TdR掺入率均高于空白对照组(P0.05),β1整合素ASODN与AVP共同作用组的CFs3H-TdR掺入率明显低于空白对照组、1×10-7mol/L AVP组和正义链+1×10-7mol/L AVP组,并且均有非常显著性意义(P0.01)。结论:β1整合素ASODN不但抑制基础状态下CFs生长及DNA合成代谢,而且抑制AVP刺激β1整合素表达、CFs增殖、DNA合成的作用,说明针对特异性靶基因片段合成的ASODN可能抑制β1整合素遗传信息传递的某个环节,干扰细胞内外信息的传递,影响β1整合素介导的CFs与细胞外基质的黏附,从而产生拮抗AVP刺激心脏间质重构形成的作用。进一步提示应用反义药物防治高血压心脏间质重构的可能性。

Inhibitory effects of antisense β1 integrin oligodeoxynucleotides on DNA synthesis in cardiac fibroblasts induced by arginine vasopressin

AIM: To explore the effects of β1 integrin antisense oligodeoxynucleotides (ASODN) on the DNA synthesis in cardiac fibroblasts (CFs) induced by arginine vasopressin (AVP). METHODS: CFs of Sprague Dawley neonatal rats were separated and cultured using trypsin. MTT techniques and enzyme-linked immunosorbent assay (ELISA) were adopted to evaluate the number and expression of β1 integrin in CFs. DNA synthesis was measured by incorporation of 3H-thymidine (3H-TdR) in blank control group (n=8), β1 integrin sense oligodeoxynucleotide group (n=8) and β1 integrin ASODN group (n=8). The effects of β1 integrin ASODN on the DNA synthesis in CFs pretreated with AVP were also observed. RESULTS: The number of CFs cultured and the 3H-TdR incorporation value in CFs by ASODN were significantly lower than in control and sense group (P<0.01). In the group pretreated with 1×10-7 mol/L AVP and group of sense+1×10-7 mol/L AVP, the expression of β1 integrin in CFs and the number of CFs were significantly higher than in control (P<0.05), whereas in the group of added ASODN+1×10-7 mol/L AVP, the expression of 1 integrin and the number of CFs was significantly lower than in 1×10-7 mol/L AVP group and sense oligos+1×10-7 mol/L AVP group (P<0.01). In the group pretreated with 1×10-7 mol/L AVP and group of sense+1×10-7 mol/L AVP, the 3H-TdR incorporation value in CFs was significantly higher than in control (P<0.05), whereas the 3H-TdR incorporation value in CFs with added ASODN+1×10-7 mol/L AVP was significantly lower than in blank control group, 1×10-7 mol/L AVP group and sense oligos+1×10-7 mol/L AVP group (P<0.01). CONCLUSION: CF proliferation and DNA synthesis can be effectively inhibited by β1 integrin ASODN. The effects of AVP on the expression of β1 integrin in CFs, CF proliferation and DNA synthesis can also be effectively inhibited by ASODN, but they are not weakened by sense oligodeoxynucleotides, indicating that restraining of a gene segment would limit one of the steps of information transduction on expression of integrin, inhibit the transduction of cell information, interfere with the inhesion of ECM and CFs, and then reverse cardiac remodeling by AVP. β1 integrin ASODN may be a new drug for modulating interstitial remodeling in essential hypertensive patients.