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目的探讨在高静水压条件下心肌营养素1(CT-1)对成纤维细胞增殖作用机制。方法3～4代心肌成纤维细胞用于实验,分为对照组、助溶剂(二甲亚砜,DMSO)组、CT-1反义寡核苷酸组(ASODN)、正义寡核苷酸组(SODN)、MEK-EPK阻断剂PD98059组、JAK-STAT3阻断剂AG490组、PI3K阻断剂LY294002。利用自制压力培养装置,将各组细胞置于160mmHg压力下培养8h。STAT3、ERK1/2和PI3-K的活性通过Westernblot分析测定;MTT测定心肌成纤维细胞增殖。结果高静水压明显促进心肌成纤维细胞增殖,CT-1表达上调,CT-1ASODN干预后,CT-1ASODN能抑制高静水压所致的细胞增殖,吸光度值(A值)为(0.132±0.013vs0.154±0.011,P<0.05),STAT3(2.09±0.25vs2.47±0.28,P<0.05)、ERK1/2(1.13±0.19vs1.61±0.22,P<0.05)和PI3-K(1.25±0.23vs1.71±0.25,P<0.05),蛋白表达水平明显低于对照组。AG490组明显减弱高静水压的促增殖作用(0.118±0.018vs0.155±0.010,P<0.05);PD98059增强高静水压的促增殖(0.185±0.011vs0.155±0.010,P<0.05),PI3-K阻断剂LY294002干预后对高静水压的促增殖作用无影响(0.157±0.015vs0.155±0.010,P>0.05);SODN与对照组相比对心肌成纤维细胞增殖无明显影响。结论高静水压下,可以激活STAT3、ERK1/2、PI3-K信号通路,心肌成纤维细胞增殖主要通过STAT3通路;ERK1/2通路起负向调节作用;PI3-K与细胞增殖关系不是很密切。
Objective To investigate the mechanism of fibroblast proliferation by cardiac troponin-1 (CT-1) under high hydrostatic pressure. Method 3 ~ 4 on behalf of myocardial fibroblasts for the experiment, divided into control group, cosolvent (dimethyl sulfoxide, DMSO) group, CT-1 antisense oligonucleotide group (ASODN), sense oligonucleotide group (SODN), MEK-EPK blocker PD98059 group, JAK-STAT3 blocker AG490 group and PI3K blocker LY294002. Using self-made pressure culture device, each group of cells was placed under 160mmHg pressure for 8h. The activities of STAT3, ERK1 / 2 and PI3-K were determined by Western blot analysis; myocardial fibroblast proliferation was measured by MTT. Results High hydrostatic pressure significantly enhanced the proliferation of cardiac fibroblasts, and the expression of CT-1 was up-regulated. CT-1 ASODN could inhibit the cell proliferation induced by high hydrostatic pressure after CT-1 ASODN treatment. The absorbance value (A value) was (0.132 ± 0.013 vs 0.165 ± 0.011, P <0.05), STAT3 (2.09 ± 0.25vs2.47 ± 0.28, P <0.05), ERK1 / 2 (1.13 ± 0.19vs1.61 ± 0.22, P <0.05) and PI3-K .71 ± 0.25, P <0.05), the protein expression level was significantly lower than the control group. AG490 group significantly attenuated the effects of high hydrostatic pressure on proliferation (0.118 ± 0.018vs0.155 ± 0.010, P <0.05); PD98059 enhanced the hypertrophic proliferation (0.185 ± 0.011vs0.155 ± 0.010, P <0.05) The blocker LY294002 had no effect on the proliferation of cardiac fibroblasts (0.157 ± 0.015 vs 0.155 ± 0.010, P> 0.05) after intervention with LY294002. SODN had no significant effect on the proliferation of cardiac fibroblasts compared with the control group. Conclusion Under high hydrostatic pressure, STAT3, ERK1 / 2 and PI3-K signaling pathways can be activated. The proliferation of cardiac fibroblasts mainly through STAT3 pathway. ERK1 / 2 pathway plays a negative regulatory role. The relationship between PI3-K and cell proliferation is not very close.