Different concentrations of angiotensin II induce directional differentiation of bone marrow mesenchymal stem cells into cardiomyocytes

doi:10.3969/j.issn.2095-4344.2014.50.007

Abstract

Abstract:

BACKGROUND: There are less experimental studies addressing angiotensin II-induced differentiation of bone marrow mesenchymal stem cells into cardiomyocytes, and there is no uniform standard for this new inducer used in the experiments, so the results of the induced experimental studies are certainly different.
OBJECTIVE: To discuss the optimal concentration of angiotensin II to induce the differentiation of bone marrow mesenchymal stem cells into cardiomyocytes.
METHODS: Bone marrow mesenchymal stem cells from rats were isolated, cultured and purified by the density gradient centrifugation combined with adherence method. Passage 3 cells were divided into four induction groups (0.05, 0.1, 0.2, 0.5 μmol/L angiotensin II) and a control group. In the four induction groups, the corresponding inducer was added 24 hours after cells were seeded, and after 24 hours of induction, the cells were cultured in complete medium for 4 weeks. Cells in the control group were only cultured in the complete medium. MTT assay was used to detect the absorbance values at days 1, 3, 5, 7 to calculate the cell proliferation inhibition rate in each group. Expression of cardiac-specific cTnT and β-MHC in bone marrow mesenchymal stem cells was detected by immuno?uorescence staining at 1 and 4 weeks after induction. mRNA expression of cardiac-transcription-factor GATA4 and NKx2.5 in bone marrow mesenchymal stem cells was measured by real-time PCR at 4 weeks after induction.
RESULTS AND CONCLUSION: The cell proliferation curves were similar among different groups. The cell proliferation inhibition rate in the 0.05 μmol/L group began to appear negative, which was significantly different from the other groups at the 5th day (P < 0.05). The cell proliferation inhibition rate of 0.1 μmol/L group was significantly lower than that of 0.2 or 0.5 μmol/L groups at the 5th and 7th days. cTnT and β-MHC were detected in the bone marrow mesenchymal stem cells of four induction groups, but the control group was negative for cTnT and β-MHC. After 1 week of induction, the positive rates of cTnT and β-MHC in the 0.5 μmol/L group was significantly higher than that in the 0.05 μmol/L group (P < 0.05). After 4 weeks of induction, the positive rates of cTnT and β-MHC had no difference between the 0.1 μmol/L and
0.2 μmol/L groups (P > 0.05). The early cardiac transcription factor GATA-4 and NKx2.5 were detected by RT-PCR in the cells of four induction groups after 4 weeks of induction, while their expression was negative in the control group. These findings suggest that after angiotensin II induction, bone marrow mesenchymal stem cells can express the cardiac-specific protein cTnT, β-MHC and transcription factor GATA-4 and NKx2.5. As mentioned above, the suitable concentration of angiotensin II is 0.1 μmol/L.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: bone marrow, mesenchymal stem cells, myocytes, cardiac, angiotensin II

CLC Number:

R394.2

Shi Jing-bao, Xing Wan-hong. Different concentrations of angiotensin II induce directional differentiation of bone marrow mesenchymal stem cells into cardiomyocytes[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(50): 8072-8079.

Figures/Tables 6

2.1 骨髓间充质干细胞的分离培养和形态学观察通过密度梯度离心+贴壁法获得骨髓间充质干细胞，培养48 h可见有纺锤形、三角形、短小梭形细胞，细胞折光性好，但可见细胞表面黏附有ficoll颗粒，并且培养基中有大量的ficoll分离液干扰，72 h成为可见细长纺锤、细长梭形细胞。细胞生长较零散，成簇的细胞生长较快，可形成折光双联体。第4天，细胞开始增殖，细胞密度较低，细胞排列不规则，可见三角形、梭形细胞及少量圆形细胞，第7天有70%-80%的细胞融合，细胞排列较规整，呈束状、旋涡状或鱼群状。多次换液并用PBS清洗后ficoll颗粒减少(图1A)。细胞传至第2，3代，细胞形态趋于一致，细胞呈梭形细胞样(图1B)。 2.2 诱导后的细胞形态观察第3代骨髓间充质干细胞经不同浓度血管紧张素Ⅱ诱导液分别诱导24 h，0.5 μmol/L组可见贴壁的部分细胞脱落，其余各组细胞均有死亡，且随药物浓度的升高细胞死亡率升高。剩余贴壁的细胞变扁平或呈短棒状(图2A)。2 d后存活细胞可见细胞核的分裂，开始增殖，细胞呈细长梭形，少数细胞呈三角形或多边形。培养2周后，80%-90%细胞融合，细胞生长呈蛇皮样、鱼群样，且细胞出现聚集生长(图2B)，3周时可见有克隆样聚集生长现象，细胞呈细长梭形，部分细胞有分支。不同浓度血管紧张素Ⅱ诱导4周后均未见自发性搏动。 2.3 大鼠骨髓间充质干细胞诱导后增殖能力及抑制率诱导后的前5 d诱导组与空白对照组细胞的增殖曲线无显著差异(图3)。第7天，0.05 μmol/L血管紧张素Ⅱ组的吸光度值与0.1，0.2，0.5 μmol/L血管紧张素Ⅱ组比较，差异有显著性意义(P < 0.05)，0.2，0.5 μmol/L血管紧张素Ⅱ组的吸光度值较空白对照组差异有显著性意义(P < 0.05)。通过对抑制率的计算，显示第3代骨髓间充质干细胞在诱导培养1 d内，各组间的增殖抑制能力大致相同，于第2天已开始增殖，随着诱导时间的延长，各诱导组相对空白对照组来说药物抑制率逐渐增强；第3天0.1 μmol/L组与0.2 μmol/L组的抑制率差异无显著性意义(P > 0.05)，0.05 μmol/L组于第5天开始出现负值，与其他组比较差异均有显著性意义(P < 0.05)。第5天0.2 μmol/L与0.5 μmol/L组的抑制率差异显著性无意义(P > 0.05)，第7天0.2 μmol/L与0.5 μmol/L组的抑制率差异显著性无意义(P > 0.05)，均与0.1 μmol/L组的抑制率差异有显著性意义(P < 0.05)，见表1。"

References

[1] Chen XQ, Chen LL, Fan L,et al.Stem cells with FGF4-bFGF fused gene enhances the expression of bFGF and improves myocardial repair in rats.Biochem Biophys Res Commun. 2014;447(1):145-151.
[2] Igura K, Okada M, Kim HW,et al.Identification of small juvenile stem cells in aged bone marrow and their therapeutic potential for repair of the ischemic heart.Am J Physiol Heart Circ Physiol. 2013;305(9):H1354-1362.
[3] Valarmathi MT, Goodwin RL, Fuseler JW,et al.A 3-D cardiac muscle construct for exploring adult marrow stem cell based myocardial regeneration.Biomaterials. 2010; 31(12): 3185-200.
[4] 徐信群,王泉兰,应国秋,等.骨髓间充质干细胞移植对心肌梗死大鼠损伤心肌的修复效果[J].南昌大学学报:医学版,2013,53(6): 1-4,21.
[5] 王海萍,张雷,赵静,等.大鼠骨髓间充质干细胞移植治疗心肌梗死的实验研究[J].中国临床解剖学杂志,2012,30(2):209-213.
[6] 田茂,朴海南,陈宇,等. 骨髓间充质干细胞体外构建工程化心肌组织的生存[J].中国组织工程研究,2014,18(20):3133-3138.
[7] 马红芬,张晓刚,史若飞,等.人工脑膜复合大鼠骨髓间充质干细胞修复心肌梗死[J].中国组织工程研究,2013,17(14): 2552-2557.
[8] 苑媛,吕安林,陈丹,等.血管紧张素诱导人骨髓间充质干细胞分化为心肌样细胞[J].心脏杂志,2006,18(3):258-261 .
[9] Xing Y, Lv A, Wang L,et al.The combination of angiotensin II and 5-azacytidine promotes cardiomyocyte differentiation of rat bone marrow mesenchymal stem cells.Mol Cell Biochem. 2012;360(1-2):279-287.
[10] 张卫泽,樊艳,陈永清,等.血管紧张素Ⅱ对成人脂肪间充质干细胞向心肌细胞分化的影响[J].第四军医大学学报, 2008,29(8): 692-695.
[11] 顾健,王爱玲,郝玉瑜,等.大鼠骨髓间充质干细胞体外诱导分化为心肌样细胞的研究[J].医学综述,2014,20(6):1109-1111,1116.
[12] 陈玲玲,尹立雪.超声辐照微泡介导5-氮杂胞苷诱导人骨髓间充质干细胞心肌样分化的实验研究[J].中华超声影像学杂志,2013, 22(11):991-996.
[13] 孙庆国,赵文静,王日中,等.体外定向诱导大鼠骨髓间充质干细胞分化为心肌样细胞的实验研究[J].中国实验诊断学,2012,16(12): 2202-2204.
[14] 邸军,李梓菲,陈艳,等.大鼠骨髓间充质干细胞体外向心肌细胞诱导分化的机制[J].中国老年学杂志,2013,33(12):2835-2836.
[15] 王宁,李新华,邢万红,等.细胞代数与5-氮胞苷浓度对体外诱导骨髓间充质干细胞向心肌样细胞定向分化的影响[J].中西医结合心脑血管病杂志,2010, 8(10):1220-1222.
[16] 王宁,邢万红,李新华,等.大鼠骨髓间充质干细胞体外心肌定向诱导及心肌组织构建研究[J].国际生物医学工程杂志,2010,33(3): 163-166.
[17] Makino S, Fukuda K, Miyoshi S,et al.Cardiomyocytes can be generated from marrow stromal cells in vitro.J Clin Invest. 1999;103(5):697-705.
[18] Fukuda K.Development of regenerative cardiomyocytes from mesenchymal stem cells for cardiovascular tissue engineering. Artif Organs. 2001;25(3):187-193.
[19] Liu Y, Song J, Liu W,et al.Growth and differentiation of rat bone marrow stromal cells: does 5-azacytidine trigger their cardiomyogenic differentiation?Cardiovasc Res. 2003;58(2): 460-468.
[20] Xu J, Lin SC, Chen J,et al.CCR2 mediates the uptake of bone marrow-derived fibroblast precursors in angiotensin II-induced cardiac fibrosis.Am J Physiol Heart Circ Physiol. 2011;301(2):H538-547.
[21] Sopel MJ, Rosin NL, Lee TD,et al.Myocardial fibrosis in response to Angiotensin II is preceded by the recruitment of mesenchymal progenitor cells.Lab Invest. 2011;91(4): 565-578.
[22] Qian C, Schoemaker RG, van Gilst WH,et al.The role of the renin-angiotensin-aldosterone system in cardiovascular progenitor cell function.Clin Sci (Lond). 2009;116(4):301-314.
[23] Hakuno D, Fukuda K, Makino S,et al.Bone marrow-derived regenerated cardiomyocytes (CMG Cells) express functional adrenergic and muscarinic receptors.Circulation. 2002;105(3): 380-386.
[24] Wang X, Phillips MI, Mehta JL.LOX-1 and angiotensin receptors, and their interplay.Cardiovasc Drugs Ther. 2011; 25(5):401-417.
[25] Wang X, Khaidakov M, Ding Z,et al.Cross-talk between inflammation and angiotensin II: studies based on direct transfection of cardiomyocytes with AT1R and AT2R cDNA.Exp Biol Med (Maywood). 2012;237(12):1394-1401.
[26] Coyne TM, Marcus AJ, Woodbury D,et al.Marrow stromal cells transplanted to the adult brain are rejected by an inflammatory response and transfer donor labels to host neurons and glia.Stem Cells. 2006;24(11):2483-2492.
[27] Heikinheimo M, Scandrett JM, Wilson DB.Localization of transcription factor GATA-4 to regions of the mouse embryo involved in cardiac development.Dev Biol. 1994;164(2): 361-373.
[28] Xing Y, Lv A, Wang L,et al.Engineered myocardial tissues constructed in vivo using cardiomyocyte-like cells derived from bone marrow mesenchymal stem cells in rats.J Biomed Sci. 2012;19:6.
[29] Liu BW, Lü AL, Hou J,et al.Electrophysiological characteristics of cardiomyocyte-like cells from rat bone marrow derived mesenchymal stem cells by four inductors.Chin Med J (Engl). 2013;126(18):3528-3533.
[30] Li L, Fan D, Wang C,et al.Angiotensin II increases periostin expression via Ras/p38 MAPK/CREB and ERK1/2/TGF-β1 pathways in cardiac fibroblasts.Cardiovasc Res. 2011;91(1): 80-89.
[31] He JG, Chen SL, Huang YY,et al.The nonpeptide AVE0991 attenuates myocardial hypertrophy as induced by angiotensin II through downregulation of transforming growth factor-beta1/Smad2 expression.Heart Vessels. 2010;25(5): 438-443.
[32] Wang N, Ren GD, Zhou Z,et al.Cooperation of myocardin and Smad2 in inducing differentiation of mesenchymal stem cells into smooth muscle cells.IUBMB Life. 2012;64(4):331-339.
[33] Haddad GE, Coleman BR, Zhao A,et al.Regulation of atrial contraction by PKA and PKC during development and regression of eccentric cardiac hypertrophy.Am J Physiol Heart Circ Physiol. 2005;288(2):H695-704.
[34] Li TF, O'Keefe RJ, Chen D.TGF-beta signaling in chondrocytes. Front Biosci. 2005;10:681-688.