低氧诱导c-Kit~+骨髓间充质干细胞增殖及旁分泌中ROCK信号的作用
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摘要
背景:研究显示低氧能刺激骨髓间充质干细胞增殖,但具体机制尚不清楚。ROCK信号对细胞增殖、凋亡等具有重要影响,当前对ROCK信号与骨髓间充质干细胞的生物学功能研究较少。目的:分析ROCK信号在低氧诱导c-Kit+骨髓间充质干细胞增殖及旁分泌中的作用。方法:贴壁法获得大鼠股骨骨髓间充质干细胞,应用磁激活细胞分选法获得c-Kit+亚群,细胞培养于常氧(体积分数21%O_2)及低氧(体积分数2%O_2)条件下,并用ROCK信号抑制剂Fasudil (10μmol/L)处理细胞。观察:①Westernblot检测p-MLC2(T18/S12)和MLC2蛋白表达,以p-MLC2(T18/S12)/MLC2比值变化反映ROCK信号的活化情况;②细胞计数分析细胞增殖,Westernblot检测细胞增殖标记物PCNA和细胞周期相关蛋白的表达;③流式细胞术检测细胞周期的变化;④ELISA检测细胞培养上清中血管内皮生长因子、转化生长因子β1和碱性成纤维细胞生长因子水平,了解细胞的旁分泌情况。结果与结论:成功分选出大鼠c-Kit+骨髓间充质干细胞。①相对于常氧而言,低氧显著下调MLC表达和上调p-MLC(T18/S19)表达进而增加p-MLC(T18/S19)/MLC2比值;②低氧促进c-Kit+骨髓间充质干细胞的增殖,表现为细胞数量增多和PCNA蛋白表达上调,Fasudil减弱了低氧诱导的c-Kit+骨髓间充质干细胞增殖,使细胞周期阻滞于S期并抑制CDK2、CDK4和上调p16的表达;③低氧显著促进c-Kit+骨髓间充质干细胞分泌上述3种细胞因子,Fasudil减弱低氧诱导的血管内皮生长因子和转化生长因子β1但上调碱性成纤维细胞生长因子的分泌。实验结果提示低氧刺激促进c-Kit+骨髓间充质干细胞增殖和旁分泌并活化ROCK信号;Fasudil抑制低氧诱导的细胞增殖和旁分泌作用,提示ROCK信号对c-Kit+骨髓间充质干细胞的增殖和旁分泌具有重要的正性调节作用。
BACKGROUND: Hypoxia stimulates the proliferation of bone marrow mesenchymal stem cells, but the specific mechanism is still unclear.The Rho-associated coiled-coil containing kinases(ROCK) signaling has an important influence on cell proliferation, migration and apoptosis.Few studies have been reported on the effect of ROCK signaling on the biological functions of bone marrow mesenchymal stem cells.OBJECTIVE: To analyze the effect of ROCK signaling on the hypoxia-induced proliferation and paracrine of c-Kit+ bone marrow mesenchymal stem cells.METHODS: Rat bone marrow mesenchymal stem cells derived from the rat femur were obtained by adherent method, and the c-Kit+subpopulation was sorted by magnetic activated cell sorting. Cells were cultured under normoxia(21% O_2) and hypoxia(2% O_2). After treatment with an ROCK signal inhibitor Fasudil(10 μmol/L), western blot was used to detect the expression of p-MLC(T18/S19), MLC,p-MLC2(T18/S12)/MLC2, PCNA and cell cycle related proteins. The cell proliferation was analyzed by cell counting, the cell cycle was detected using flow cytometry, and the changes of vascular endothelial growth factor, transforming growth factor β1 and basic fibroblast growth factor in the cell supernatant were detected by ELISA.RESULTS AND CONCLUSION: c-Kit+ subpopulation were successfully isolated from rat bone marrow mesenchymal stem cells. Compared with normoxia, hypoxia treatment significantly down-regulated MLC expression, up-regulated p-MLC(T18/S19) expression and therefore increased the ratio of p-MLC(T18/S19)/MLC2. Meanwhile, hypoxia promoted the proliferation of c-Kit+ bone marrow mesenchymal stem cells,as evidenced by the increase in cell number and PCNA protein expression. Fasudil inhibited the hypoxia-induced the proliferation of c-Kit+bone marrow mesenchymal stem cells, arrested the cell cycle at S phase, inhibited CDK2 and CDK4 and increased p16 expression. Hypoxia significantly promoted the secretion of vascular endothelial growth factor, transforming growth factor β1 and basic fibroblast growth factor by c-Kit+ bone marrow mesenchymal stem cells, and Fasudil attenuated hypoxia-induced vascular endothelial growth factor and transforming growth factor β1 expression but astonishingly upregulated basic fibroblast growth factor level. These results indicate that hypoxia promotes cell proliferation and paracrine of angiogenic factors and activates ROCK signaling in c-Kit+ bone marrow mesenchymal stem cells; Fasudil inhibits hypoxia-induced proliferation and expression of angiogenic factors in c-Kit+ bone marrow mesenchymal stem cells, suggesting that ROCK signaling plays a positive role in the proliferation and paracrine of c-Kit+ bone marrow mesenchymal stem cells.
BACKGROUND: Hypoxia stimulates the proliferation of bone marrow mesenchymal stem cells, but the specific mechanism is still unclear.The Rho-associated coiled-coil containing kinases(ROCK) signaling has an important influence on cell proliferation, migration and apoptosis.Few studies have been reported on the effect of ROCK signaling on the biological functions of bone marrow mesenchymal stem cells.OBJECTIVE: To analyze the effect of ROCK signaling on the hypoxia-induced proliferation and paracrine of c-Kit+ bone marrow mesenchymal stem cells.METHODS: Rat bone marrow mesenchymal stem cells derived from the rat femur were obtained by adherent method, and the c-Kit+subpopulation was sorted by magnetic activated cell sorting. Cells were cultured under normoxia(21% O_2) and hypoxia(2% O_2). After treatment with an ROCK signal inhibitor Fasudil(10 μmol/L), western blot was used to detect the expression of p-MLC(T18/S19), MLC,p-MLC2(T18/S12)/MLC2, PCNA and cell cycle related proteins. The cell proliferation was analyzed by cell counting, the cell cycle was detected using flow cytometry, and the changes of vascular endothelial growth factor, transforming growth factor β1 and basic fibroblast growth factor in the cell supernatant were detected by ELISA.RESULTS AND CONCLUSION: c-Kit+ subpopulation were successfully isolated from rat bone marrow mesenchymal stem cells. Compared with normoxia, hypoxia treatment significantly down-regulated MLC expression, up-regulated p-MLC(T18/S19) expression and therefore increased the ratio of p-MLC(T18/S19)/MLC2. Meanwhile, hypoxia promoted the proliferation of c-Kit+ bone marrow mesenchymal stem cells,as evidenced by the increase in cell number and PCNA protein expression. Fasudil inhibited the hypoxia-induced the proliferation of c-Kit+bone marrow mesenchymal stem cells, arrested the cell cycle at S phase, inhibited CDK2 and CDK4 and increased p16 expression. Hypoxia significantly promoted the secretion of vascular endothelial growth factor, transforming growth factor β1 and basic fibroblast growth factor by c-Kit+ bone marrow mesenchymal stem cells, and Fasudil attenuated hypoxia-induced vascular endothelial growth factor and transforming growth factor β1 expression but astonishingly upregulated basic fibroblast growth factor level. These results indicate that hypoxia promotes cell proliferation and paracrine of angiogenic factors and activates ROCK signaling in c-Kit+ bone marrow mesenchymal stem cells; Fasudil inhibits hypoxia-induced proliferation and expression of angiogenic factors in c-Kit+ bone marrow mesenchymal stem cells, suggesting that ROCK signaling plays a positive role in the proliferation and paracrine of c-Kit+ bone marrow mesenchymal stem cells.
引文
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[29]Wang X,Tang P,Guo F,et al.RhoA regulates Activin B-induced stress fiber formation and migration of bone marrow-derived mesenchymal stromal cell through distinct signaling.Biochim Biophys Acta Gen Subj.2017;1861(1 Pt A):3011-3018.
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[32]Patel RA,Liu Y,Wang B,et al.Identification of novel ROCKinhibitors with anti-migratory and anti-invasive activities.Oncogene.2014;33(5):550-555.
[33]Das R,Jahr H,van Osch GJ,et al.The role of hypoxia in bone marrow-derived mesenchymal stem cells:considerations for regenerative medicine approaches.Tissue Eng Part B Rev.2010;16(2):159-168.
[34]Johnson C,Huynh V,Hargrove L,et al.Inhibition of Mast Cell-Derived Histamine Decreases Human Cholangiocarcinoma Growth and Differentiation via c-Kit/Stem Cell Factor-Dependent Signaling.Am J Pathol.2016;186(1):123-133.
[35]Gude NA,Firouzi F,Broughton KM,et al.Cardiac c-Kit Biology Revealed by Inducible Transgenesis.Circ Res.2018;123(1):57-72.
[36]Ashman LK,Griffith R.Therapeutic targeting of c-KIT in cancer.Expert Opin Investig Drugs.2013;22(1):103-115.
[37]Lennartsson J,R?nnstrand L.Stem cell factor receptor/c-Kit:from basic science to clinical implications.Physiol Rev.2012;92(4):1619-1649.
[38]Sheng L,Mao X,Yu Q,et al.Effect of the PI3K/AKT signaling pathway on hypoxia-induced proliferation and differentiation of bone marrow-derived mesenchymal stem cells.Exp Ther Med,2017;13(1):55-62.
[39]Wang ZH,Zhu D,Xie S,et al.Inhibition of Rho-kinase Attenuates Left Ventricular Remodeling Caused by Chronic Intermittent Hypoxia in Rats via Suppressing Myocardial Inflammation and Apoptosis.J Cardiovasc Pharmacol.2017;70(2):102-109.
[40]Qiao F,Zou Z,Liu C,et al.ROCK2 mediates the proliferation of pulmonary arterial endothelial cells induced by hypoxia in the development of pulmonary arterial hypertension.Exp Ther Med.2016;11(6):2567-2572.
[41]曹美霞,屈长青.Rho-ROCK信号通路功能及其活性调节[J].生物学杂志,2015,32(6):81-85.
[42]Xu N,Chen SH,Qu GY,et al.Fasudil inhibits proliferation and collagen synthesis and induces apoptosis of human fibroblasts derived from urethral scar via the Rho/ROCKsignaling pathway.Am J Transl Res.2017;9(3):1317-1325.
[43]Golias CH,Charalabopoulos A,Charalabopoulos K.Cell proliferation and cell cycle control:a mini review.Int J Clin Pract.2004;58(12):1134-1141.
[44]Tang L,Dai F,Liu Y,et al.RhoA/ROCK signaling regulates smooth muscle phenotypic modulation and vascular remodeling via the JNK pathway and vimentin cytoskeleton.Pharmacol Res.2018;133:201-212.
[45]Polymeri A,Giannobile WV,Kaigler D.Bone Marrow Stromal Stem Cells in Tissue Engineering and Regenerative Medicine.Horm Metab Res.2016;48(11):700-713.
[46]陈珂玲,周总光,周斌,等.骨髓间充质干细胞旁分泌因子治疗重症急性胰腺炎的潜能[J].生物医学工程学杂志,2015,32(1):245-248.
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[2]Sipp D,Robey PG,Turner L.Clear up this stem-cell mess.Nature.2018;561(7724):455-457.
[3]Mushahary D,Spittler A,Kasper C,et al.Isolation,cultivation,and characterization of human mesenchymal stem cells.Cytometry A.2018;93(1):19-31.
[4]Zhou B,Wu SM.Reassessment of c-Kit in cardiac cells:a complex interplay between expression,fate,and function.Circ Res.2018;123(1):9-11.
[5]Ding R,Jiang X,Ha Y,et al.Activation of Notch1 signalling promotes multi-lineage differentiation of c-Kit(POS)/NKX2.5(POS)bone marrow stem cells:implication in stem cell translational medicine.Stem Cell Res Ther.2015;6:91.
[6]Anam K,Davis TA.Comparative analysis of gene transcripts for cell signaling receptors in bone marrow-derived hematopoietic stem/progenitor cell and mesenchymal stromal cell populations.Stem Cell Res Ther.2013;4(5):112.
[7]哈艳平,王振良,雷洪,等.过表达Notch1胞内域对c-Kit+骨髓间充质干细胞分化的影响[J].中国组织工程研究,2016,20(6):785-792.
[8]Zhang GW,Gu TX,Guan XY,et al.Delayed enrichment for c-kit and inducing cardiac differentiation attenuated protective effects of BMSCs'transplantation in pig model of acute myocardial ischemia.Cardiovasc Ther.2015;33(4):184-92.
[9]Wang YL,Zhang G,Wang HJ,et al.Preinduction with bone morphogenetic protein-2 enhances cardiomyogenic differentiation of c-Kit+mesenchymal stem cells and repair of infarcted myocardium.Int J Cardiol.2018;265:173-180.
[10]Taghavi S,Sharp TE 3rd,Duran JM,et al.Autologous c-Kit+mesenchymal stem cell injections provide superior therapeutic benefit as compared to c-Kit+cardiac-derived stem cells in a feline model of isoproterenol-induced cardiomyopathy.Clin Transl Sci.2015;8(5):425-431.
[11]Hao ZC,Lu J,Wang SZ,et al.Stem cell-derived exosomes:Apromising strategy for fracture healing.Cell Prolif.2017;50(5).doi:10.1111/cpr.12359.
[12]Phinney DG,Pittenger MF.Concise Review:MSC-derived exosomes for cell-free therapy.Stem Cells.2017;35(4):851-858.
[13]Lazar E,Benedek T,Korodi S,et al.Stem cell-derived exosomes-an emerging tool for myocardial regeneration.World J Stem Cells.2018;10(8):106-115.
[14]Cunnane EM,Weinbaum JS,O'Brien FJ,et al.Future Perspectives on the Role of Stem Cells and Extracellular Vesicles in Vascular Tissue Regeneration.Front Cardiovasc Med.2018;5:86.
[15]Antebi B,Rodriguez LA 2nd,Walker KP 3rd,et al.Short-term physiological hypoxia potentiates the therapeutic function of mesenchymal stem cells.Stem Cell Res Ther.2018;9(1):265.
[16]Tong C,Hao H,Xia L,et al.Hypoxia pretreatment of bone marrow-derived mesenchymal stem cells seeded in a collagen-chitosan sponge scaffold promotes skin wound healing in diabetic rats with hindlimb ischemia.Wound Repair Regen.2016;24(1):45-56.
[17]Julian L,Olson MF.Rho-associated coiled-coil containing kinases(ROCK):structure,regulation,and functions.Small GTPases.2014;5:e29846.
[18]Schofield AV,Bernard O.Rho-associated coiled-coil kinase(ROCK)signaling and disease.Crit Rev Biochem Mol Biol.2013;48(4):301-316.
[19]Fu PC,Tang RH,Yu ZY,et al.The Rho-associated kinase inhibitors Y27632 and fasudil promote microglial migration in the spinal cord via the ERK signaling pathway.Neural Regen Res.2018;13(4):677-683.
[20]Zhang C,Wu JM,Liao M,et al.The ROCK/GGTase Pathway Are Essential to the Proliferation and Differentiation of Neural Stem Cells Mediated by Simvastatin.J Mol Neurosci.2016;60(4):474-485.
[21]Wang T,Kang W,Du L,et al.Rho-kinase inhibitor Y-27632facilitates the proliferation,migration and pluripotency of human periodontal ligament stem cells.J Cell Mol Med.2017;21(11):3100-3112.
[22]彭志明,赵晓阳,朱凯,等.Lingo-1介导Rho-ROCK通路调控神经干细胞的分化[J].中国组织工程研究,2018,22(29):4669-4674.
[23]徐亮,陶树清,文刚,等.RhoA/ROCK信号通路在骨质疏松大鼠BMSCs成骨分化中的研究[J].中国骨质疏松杂志,2017,23(11):1415-1419.
[24]Zhang L,Jiang G,Zhao X,et al.Dimethyloxalylglycine Promotes Bone Marrow Mesenchymal Stem Cell Osteogenesis via Rho/ROCK Signaling.Cell Physiol Biochem.2016;39(4):1391-403.
[25]Chen Z,Wang X,Shao Y,et al.Synthetic osteogenic growth peptide promotes differentiation of human bone marrow mesenchymal stem cells to osteoblasts via RhoA/ROCKpathway.Mol Cell Biochem.2011;358(1-2):221-227.
[26]Li Z,Han S,Wang X,et al.Rho kinase inhibitor Y-27632promotes the differentiation of human bone marrow mesenchymal stem cells into keratinocyte-like cells in xeno-free conditioned medium.Stem Cell Res Ther.2015;6:17.
[27]Liu X,Zhang Z,Yan X,et al.The Rho kinase inhibitor Y-27632facilitates the differentiation of bone marrow mesenchymal stem cells.J Mol Histol.2014;45(6):707-714.
[28]Chiba Y,Kuroda S,Shichinohe H,et al.Synergistic effects of bone marrow stromal cells and a Rho kinase(ROCK)inhibitor,fasudil on axon regeneration in rat spinal cord injury.Neuropathology.2010;30(3):241-50.
[29]Wang X,Tang P,Guo F,et al.RhoA regulates Activin B-induced stress fiber formation and migration of bone marrow-derived mesenchymal stromal cell through distinct signaling.Biochim Biophys Acta Gen Subj.2017;1861(1 Pt A):3011-3018.
[30]Li JR,Zhao YS,Chang Y,et al.Fasudil improves endothelial dysfunction in rats exposed to chronic intermittent hypoxia through RhoA/ROCK/NFATc3 pathway.PLoS One.2018;13(4):e0195604.
[31]高艳,谢敏,石俊青.Rho/Rho激酶信号通路在低氧致肺纤维化中的作用及法舒地尔的干预效应[J].第三军医大学学报,2010,32(22):2378-2382.
[32]Patel RA,Liu Y,Wang B,et al.Identification of novel ROCKinhibitors with anti-migratory and anti-invasive activities.Oncogene.2014;33(5):550-555.
[33]Das R,Jahr H,van Osch GJ,et al.The role of hypoxia in bone marrow-derived mesenchymal stem cells:considerations for regenerative medicine approaches.Tissue Eng Part B Rev.2010;16(2):159-168.
[34]Johnson C,Huynh V,Hargrove L,et al.Inhibition of Mast Cell-Derived Histamine Decreases Human Cholangiocarcinoma Growth and Differentiation via c-Kit/Stem Cell Factor-Dependent Signaling.Am J Pathol.2016;186(1):123-133.
[35]Gude NA,Firouzi F,Broughton KM,et al.Cardiac c-Kit Biology Revealed by Inducible Transgenesis.Circ Res.2018;123(1):57-72.
[36]Ashman LK,Griffith R.Therapeutic targeting of c-KIT in cancer.Expert Opin Investig Drugs.2013;22(1):103-115.
[37]Lennartsson J,R?nnstrand L.Stem cell factor receptor/c-Kit:from basic science to clinical implications.Physiol Rev.2012;92(4):1619-1649.
[38]Sheng L,Mao X,Yu Q,et al.Effect of the PI3K/AKT signaling pathway on hypoxia-induced proliferation and differentiation of bone marrow-derived mesenchymal stem cells.Exp Ther Med,2017;13(1):55-62.
[39]Wang ZH,Zhu D,Xie S,et al.Inhibition of Rho-kinase Attenuates Left Ventricular Remodeling Caused by Chronic Intermittent Hypoxia in Rats via Suppressing Myocardial Inflammation and Apoptosis.J Cardiovasc Pharmacol.2017;70(2):102-109.
[40]Qiao F,Zou Z,Liu C,et al.ROCK2 mediates the proliferation of pulmonary arterial endothelial cells induced by hypoxia in the development of pulmonary arterial hypertension.Exp Ther Med.2016;11(6):2567-2572.
[41]曹美霞,屈长青.Rho-ROCK信号通路功能及其活性调节[J].生物学杂志,2015,32(6):81-85.
[42]Xu N,Chen SH,Qu GY,et al.Fasudil inhibits proliferation and collagen synthesis and induces apoptosis of human fibroblasts derived from urethral scar via the Rho/ROCKsignaling pathway.Am J Transl Res.2017;9(3):1317-1325.
[43]Golias CH,Charalabopoulos A,Charalabopoulos K.Cell proliferation and cell cycle control:a mini review.Int J Clin Pract.2004;58(12):1134-1141.
[44]Tang L,Dai F,Liu Y,et al.RhoA/ROCK signaling regulates smooth muscle phenotypic modulation and vascular remodeling via the JNK pathway and vimentin cytoskeleton.Pharmacol Res.2018;133:201-212.
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