成纤维细胞生长因子2和丹参酮ⅡA在大鼠骨髓间充质干细胞向心肌样细胞分化中的作用

李娇; 吕洋; 刘洋; 王浩宇; 赵亚如; 王雪姣; 王海萍

doi:10.16098/j.issn.0529-1356.2020.04.008

PDF(10011 KB)

解剖学报 ›› 2020, Vol. 51 ›› Issue (4) : 520-527. DOI: 10.16098/j.issn.0529-1356.2020.04.008

细胞和分子生物学

成纤维细胞生长因子2和丹参酮ⅡA在大鼠骨髓间充质干细胞向心肌样细胞分化中的作用

李娇吕洋刘洋王浩宇赵亚如王雪娇王海萍*

作者信息 +

Role of fibroblast growth factor 2 and tanshinoneⅡA in the differentiation of rat bone marrow mesenchymal stem cells into cardiomyocyte-like cells

LI Jiao Lü Yang LIU Yang WANG Hao-yu ZHAO Ya-ru WANG Xue-jiao WANG Hai-ping*

Author information +

文章历史 +

摘要

目的探讨成纤维细胞生长因子2（FGF-2）和丹参酮ⅡA（tanshinoneⅡA）在大鼠骨髓间充质干细胞（BMSCs）向心肌样细胞分化中的作用。方法体外分离、培养BMSCs，利用流式细胞术进行鉴定。实验分组：对照组（不加任何诱导剂）、FGF-2组、丹参酮ⅡA组及两者联合诱导组。MTT检测诱导后的活性及增殖情况；Real-time PCR检测早期心肌转录因子GATA-4、Nkx2.5的表达；免疫细胞化学染色法检测缝隙连接蛋白43(Cx43)以及心肌肌钙蛋白Ⅰ（cTnI）的表达；免疫荧光化学染色法检测结蛋白(desmin)、原肌球蛋白（Tm）的表达；Western blotting检测结蛋白、Tm的表达。结果各诱导组较对照组增殖明显。与对照组相比，诱导组GATA-4和Nkx2.5基因的表达增强，联合组表达量最高，差异具有统计学意义（P＜0.05）。联合诱导组各标记物Cx43、cTnI、结蛋白、Tm的阳性表达率高于FGF-2及丹参酮ⅡA单独诱导组，差异具有统计学意义（P＜0.05）。Western blotting结果显示，联合诱导组结蛋白、Tm的表达量明显高于其他实验组，差异具有统计学意义（P＜0.05）。透射电子显微镜结果显示，诱导后细胞的细胞核居中，细胞质中可见肌丝、粗面内质网、线粒体和核糖体。结论 FGF-2和丹参酮ⅡA均能促进BMSCs增殖，诱导BMSCs分化为心肌样细胞，两者联合诱导的效果较其他组更佳。

Abstract

Objective To investigate the effects of fibroblast growth factor 2 (FGF-2) combined with tanshinoneⅡA on differentiation of rat bone marrow mesenchymal stem cells (BMSCs）into cardiomyocyte-like cells. Methods BMSCs were isolated and cultured. The cultured cells were identified by flow cytometry, BMSCs were divided into experimental control group, FGF-2 group, tanshinoneⅡA group and the combined induction group. The activity and value of BMSCs were detected by MTT. The Real-time PCR method was used to detect BMSCs. Expression of early myocardial transcription factors GATA-4 and Nkx2.5; Immunocytochemical staining for detection of connexin43（Cx43）and cardiac troponin-Ⅰ(cTnI);Immunofluorescence staining for detection of desmin and Tm; The expression of desmin and tropomyosin（Tm）was detected by Western blotting. Results The cell activity and proliferation after induction were good. The expression of GATA-4 and Nkx2.5 in the induction group were stronger than that in the experimental control group, and the difference was statistically significant (P<0.05). The positive expression of Cx43 and cTnI in the induction group increased significantly. The markers of the combined group were the most obvious, and the difference was statistically significant (P<0.05). The expression of Myo-specific protein desmin and Tm in the combined induction group increased significantly, and the difference was statistically significant (P<0.05). Conclusion Both FGF-2 and tanshinoneⅡA can promote the proliferation of BMSCs and induce the differentiation of BMSCs into cardiomyocyte-like cells. The synergistic effect of the two is better than other groups.

导出引用

李娇吕洋刘洋王浩宇赵亚如王雪娇王海萍. 成纤维细胞生长因子2和丹参酮ⅡA在大鼠骨髓间充质干细胞向心肌样细胞分化中的作用[J]. 解剖学报. 2020, 51(4): 520-527 https://doi.org/10.16098/j.issn.0529-1356.2020.04.008

LI Jiao Lü Yang LIU Yang WANG Hao-yu ZHAO Ya-ru WANG Xue-jiao WANG Hai-ping. Role of fibroblast growth factor 2 and tanshinoneⅡA in the differentiation of rat bone marrow mesenchymal stem cells into cardiomyocyte-like cells[J]. Acta Anatomica Sinica. 2020, 51(4): 520-527 https://doi.org/10.16098/j.issn.0529-1356.2020.04.008

中图分类号： R329.2

参考文献

［1］ Zhang XF, Hu DY, Ding RJ, et al. Current status and prevalence of cardiovascular and cerebrovascular diseases in China［J］. Chinese Journal of Hypertension, 2012,4(3):179-187.（in Chinese）

张啸飞, 胡大一, 丁荣晶, 等. 中国心脑血管疾病死亡现况及流行趋势［J］. 中国高血压杂志, 2012,4(3):179-187.

［2］ Zhang XQ, Ling B, Sun J. Study on inducers for differentiation of bone marrow mesenchymal stem cells into cardiomyoid cells ［J］. Journal of Cardiovascular Rehabilitation Medicine, 2014, 23(6):683-685.（in Chinese）

张晓琼,凌斌,孙洁.关于骨髓间充质干细胞向心肌样细胞分化的诱导剂研究概况［J］.心血管康复医学杂志,2014,23(6):683-685.

［3］ Sluijter JP, Condorelli G, Davidson SM, et al. Novel therapeutic strategies for cardioprotection［J］. Pharmacol Ther, 2014, 144(1):60-70.

［4］ Beltrami AP, Urbanek K, Kajstura J, et al. Evidence that human cardiac myocytes divide after myocardial infarction［J］. N Engl J Med, 2001, 344(23):1750-1757.

［5］ Tang GM, Qian G, Zhai ChL, et al. Tanshinone Ⅱ A effect on ischemic heart failure rat myocardial fibrosis ［J］. Journal of Medical Research, 2018, 47 (9):105-108.（in Chinese）

唐关敏,钱钢,翟昌林, 等. 丹参酮ⅡA对缺血性心力衰竭大鼠心肌纤维化的影响［J］. 医学研究杂志, 2018, 47(9):105-108.

［6］ Wang XY, Fan ChX, Zhao Q, et al. TanshinoneⅡA ectomesenchymal stem cells A placenta to the person to the entrainment of myocardial cell differentiation and its mechanism［J］. Journal of Jilin University (Medical Edition), 2019, 45(1):33-38, 218.（in Chinese）

王秀艳，樊晨星，赵千，等.丹参酮ⅡA对人胎盘间充质干细胞向心肌细胞分化的诱导作用及其机制［J］.吉林大学学报(医学版),2019,45(1):33-38,218.

［7］ Ornitz DM, Marie PJ. Fibroblast growth factor signaling in skeletal development and disease［J］. Genes Dev, 2015, 29(14):1463-1486.

［8］ Wang X, Zhen L, Miao H, et al. Concomitant retrograde cor-onary venous infusion of basic fibroblast growth factor enhances engraftment and differentation of bone marrow mesenchymal stem cells for cardiac repair after myocarction infarction［J］. Theranostics, 2015, 5(9):995-1006.

［9］ Xu J, Lu H, Miao ZN, et al. Immunoregulatory effect of neuronallike cells in inducting differentiation of bone marrow mesenchymal stem cells［J］. Eur Rev Med Pharmacol Sci, 2016, 20(24):5041-5048.

［10］ Lü Y, Liu B, Wang HP, et al. Transforming growth factor beta 1 and tanshinone ⅡA joint between induced bone marrow mesenchymal stem cells to myocardial cells［J］. Acta Anatomica Sinica, 2016, 47 (5):620-627.（in Chinese）

吕洋, 刘博, 王海萍, 等. 转化生长因子-β1和丹参酮ⅡA联合诱导骨髓间充质干细胞向心肌样细胞的分化［J］. 解剖学报, 2016, 47(5):620-627.

［11］ Sun W, Wang HP, Lü Y, et al. Differentiation of rat bone marrow mesenchymal stem cells into cardiomyoid cells induced by fibroblast growth factor-2 combined with bone morphogenetic protein-2 in vitro［J］. Journal of Zhengzhou University (Medical Edition), 2018, 53(1):47-52.（in Chinese）

孙微, 王海萍, 吕洋, 等. 成纤维生长因子-2联合骨形态发生蛋白-2体外诱导大鼠骨髓间充质干细胞向心肌样细胞的分化［J］. 郑州大学学报(医学版), 2018, 53(1):47-52.

［12］ Li R, Wang HP, Lü Y, et al. Differentiation of bone marrow mesenchymal stem cells into cardiomyoid cells induced by FGF-2 and TGF-1［J］. Journal of Practical Medicine, 2017, 33(24):4019-4023.（in Chinese）

李柔, 王海萍, 吕洋,等. FGF-2与TGF-β1联合诱导骨髓间充质干细胞向心肌样细胞的分化［J］. 实用医学杂志, 2017, 33(24):4019-4023.

［13］ Khezri S, Valojerdi MR, Sepehri H, et al. Effect of basic fibroblast growth factor on cardiomyocyte differentiation from mouse embryonic stem cells［J］. Saudi Med J, 2007, 28(2):181-186.

［14］ Hafez P, Jose S, Chowdhury SR, et al. Cardiomyogenic differentiation of human sternal bone marrow mesenchymal stem cells using a combination of basic fibroblast growth factor and hydrocortisone［J］. Cell Biol Int, 2016, 40(1):55-64.

［15］ Ruscic KJ, Francesco M, VillalbaGalea CA, et al. IKs channels open slowly because KCNE1 accessory subunits slow the movement of S4 voltage sensors in KCNQ1 pore-forming subunits［J］. Proc Natl Acad Sci USA, 2013, 110(7):559-566.

［16］ Apple FS. Tissue specificity of cardiac troponin Ⅰ, cardiac troponin T and creatine kinase-MB［J］. Clin Chim Acta, 1999, 284(2):151-159.

［17］ Liang W, Guo J, Li J, et al. Downregulation of miR-122 attenuates hypoxia/reoxygenation (H/R)-induced myocardial cell apoptosis by upregulating GATA-4 ［J］. Biochem Biophys Res Commun, 2016, 478(3): 1416-1422.

［18］ Yu B, Yang Y, Liu H, et al. Clusterin/Akt up-regulation is critical for GATA-4 mediated cytoprotection of mesenchymal stem cells against ischemiainjury［J］. PLoS One, 2016, 11(3):e0151542.

［19］ Zhang Y, Chen RK, Cai ZhJ. The role of Nkx2.5 and GATA-4 transcription factors in heart development［J］. Journal of Medical Molecular Biology,2004, 1(4):257-260.（in Chinese）

张勇, 陈如坤, 蔡振杰. Nkx2-5、GATA-4转录因子在心脏发育中的作用［J］.医学分子生物学杂志, 2004, 1(4): 257-260.

［20］ Ruan Z, Zhu L, Yin Y, et al. Overexpressing NKx2.5 increases the differentiation of human umbilical cord drived mesenchymal stem cells into cardiomyocyte-like cells［J］. Biomed Pharmacother, 2016, 78:110-115.

［21］ Li P, Zhang L. Exogenous Nkx2.5 or GATA-4 transfected rabbit bone marrow mesenchymal stem cells and myocardial cell culture on the treatment of myocardial infarction in rabbits［J］. Mol Med Rep, 2015, 12(2):2607-2621.

［22］ Gao XR, Tan YZ, Wang HJ. Overexpression of Csx/Nkx2.5 and GATA-4 enhances the efficacy of mesenchymal stem cell transplantation after myocardial infarction［J］. Circ J, 2011, 75(11):2683-2691.

［23］ Kishi A, Yamamoto M, Kikuchi A, et al. Gene and protein expressions of vimentin and desmin during embryonic development of the mylohyoid muscle［J］. Anat Sci Int, 2012, 87(3):126-131.

［24］ Hnia K, Ramspacher C, Vermot J, et al. Desmin in muscle and associated diseases: beyond the structural function［J］. Cell Tissue Res, 2015, 360(3):591-608.

［25］ El-Mezgueldi M. Tropomyosin dynamics ［J］. J Muscle Res Cell Motil, 2014, 35(3-4): 203-210.

［26］ Gunning PW, Hardeman EC, Lappalainen P, et al. Tropomyosin master regulator of actin filament function in the cytoskeleton［J］. J Cell Sci, 2015, 128(16):2965-2974.