| 倪 菁,白 丹,雷 飞,马建梅,周 韵,王春阳.促甲状腺激素对大鼠心肌成纤维细胞影响的研究[J].现代生物医学进展英文版,2018,(15):2855-2860. |
| 促甲状腺激素对大鼠心肌成纤维细胞影响的研究 |
| The Effect of Thyroid-Stimulating Hormone on Rat Cardiac Fibroblasts |
| Received:December 07, 2017 Revised:December 31, 2017 |
| DOI：10.13241/j.cnki.pmb.2018.15.011 |
| 中文关键词: 促甲状腺激素 心肌成纤维细胞 胶原 金属蛋白酶 |
| 英文关键词: TSH Cardiac fibroblasts Collagen MMPs |
| 基金项目:陕西省教育厅科技计划项目(17JK0667)；西安医学院校级课题(2016QN14) |
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| 中文摘要: |
| 摘要 目的：探讨促甲状腺激素（TSH）对SD乳鼠心肌成纤维细胞（CFs）的影响及机制。方法：新生1-3 d的SD乳鼠15只，处死后进行CFs的分离与培养，倒置显微镜对原代培养细胞形态进行鉴定，免疫组织化学染色法对CFs胞浆内波动蛋白进行染色并鉴定。后续实验过程中，按照使用bTSH工作液的稀释浓度分为A组（1 μmol?L-1）、B组（2 μmol?L-1）、C组（4 μmol?L-1）。MTT法检测心肌成纤维细胞增殖率。酶联免疫吸附法（ELISA）检测培养细胞的上清液中Ⅰ、Ⅲ型胶原蛋白含量。荧光定量PCR反应测定培养细胞上清液中基质金属蛋白酶-2（MMP-2）、基质金属蛋白酶-9（MMP-9）mRNA的相对表达量。Western-blot法检测培养细胞上清液中MMP-2、MMP-9的蛋白表达水平。结果：CFs的形态学变化及纯度鉴定结果显示乳鼠CFs培养成功。MTT试验结果显示，24 h时后，A、B、C组CFs的增殖率分别为112.91±10.23、123.22±9.34、132.56±9.36；48 h时后，A、B、C组CFs的增殖率分别为124.4±8.34、133.5±9.02、139.6±11.36。随着bTSH浓度的增加，CFs增殖率呈逐渐增加趋势。ELISA检测结果显示，A、B、C组I 型胶原蛋白表达水平分别为（2.61±0.31）μg?L-1、（5.53±0.66）μg?L-1、（7.91±0.74）μg?L-1；A、B、C组III 型胶原蛋白表达水平分别为（3.96±0.25）μg?L-1、（6.72±0.52）μg?L-1、（8.11±0.65）μg?L-1。随着bTSH浓度的升高，I 型和III 型胶原蛋白表达水平呈升高趋势。荧光定量PCR法检测结果显示，A、B、C组MMP-2的mRNA表达量分别为9.124±1.021、15.223±1.536、18.678±1.742；A、B、C组MMP-9的mRNA表达量分别为16.447±1.664、17.402±1.881、24.247±2.364。随着bTSH浓度的增加，MMP-2、MMP-9的mRNA相对表达量呈增加趋势。Western-blot法检测结果显示，A、B、C组MMP-2灰度值分别为165.41±16.57、198.25±21.34、223.41±19.08；A、B、C组MMP-9灰度值分别为140.30±15.09、190.47±20.06、230.14±21.45。随着bTSH浓度的增加，MMP-2、MMP-9的蛋白表达水平呈增加趋势。结论：TSH能够促进乳鼠CFs细胞增殖及I、III型胶原蛋白表达增加，导致细胞外基质成分降解及合成过程失调。 |
| 英文摘要: |
| ABSTRACT Objective: To explore the effect and mechanism of TSH on cardiac fibroblast (CFs) in rat. Methods: 1-3 d SD rats(n=15) were put death, to separate and culture CFs. The cell morphology of the original culture was identified by the inverted microscope and the immunohistochemical staining method was used to dye and identify the volatile protein in CFs cytoplasm. According concentra- tion of the working fluid of bTSH, which were divided into group A (1 μmol?L-1), group B (2 μmol?L-1), and group C (4 μmol?L-1). My- ocardial fibroblast proliferation rate was detected by MTT method. The supernatant of cultured cells Ⅰ, Ⅲ collagen content was detected by ELISA method. Fluorescence quantitative PCR was used to determine the relative expression of MMP-2 and MMP-9 mRNA in cul- tured cells. The protein expression levels of MMP-2 and MMP-9 were cultured in cultured cells by Western- blot. Results: CFs morpho- logical changes and purity identification results showed that CFs culture was successful. The results of MTT test showed, after 24 h, the proliferation rate of CFs of group A, B and C were 112.91±10.23, 123.22±9.34, 132.56±9.36; after 48 h, the proliferation rate of CFs of group A, B and C were 124.4±8.34, 133.5±9.02, 139.6±11.36. With the increase of bTSH concentration, the proliferation of CFs increased. ELISA test results showed, expression of type I collagen of group A, B and C were（2.61±0.31）μg?L-1,（5.53±0.66）μg?L-1,（7.91±0.74）μg?L-1; type III collagen of group A, B and C were （3.96±0.25）μg?L-1,（6.72±0.52）μg?L-1,（8.11±0.65）μg?L-1. With the increase of bTSH concentration, the expression of type I and type III collagen increased. The PCR results showed, mRNA relative expres- sion in group A, B and C of MMP-2 were 9.124±1.021, 15.223±1.536, 18.678±1.742; MMP-9 were 16.447±1.664, 17.402±1.881, 24.247±2.364. With the increase of bTSH concentration, the mRNA relative expression of MMP-2 and MMP-9 increased. The results of Western-blot test showed, grey value of MMP-2 in group A, B and C were 165.41±16.57, 198.25±21.34, 223.41±19.08; grey value of MMP-9 were 140.30±15.09, 190.47±20.06, 230.14±21.45. With the increase of bTSH concentration, the expression level of MMP-2 and MMP-9 increased. Conclusion: TSH can promote CFs cell proliferation and the expression of I, III collagen, resulting in the degra- dation of extracellular matrix components and the dysregulation of the synthesis process. |
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