浓缩生长因子促人脐静脉血管内皮细胞成血管化作用研究
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摘要
目的研究浓缩生长因子(CGF)对人脐静脉血管内皮细胞(HUVECs)的增殖、迁移和分化的作用。方法取健康志愿者静脉血制成CGF,再用CGF制备浓缩生长因子提取液(CGFe)。体外培养细胞分为2%CGFe组、5%CGFe组、10%CGFe组和对照组。CCK-8和细胞周期实验检测各组细胞增殖活性;划痕实验检测内皮细胞迁移;实时荧光定量聚合酶链反应(q RT-PCR)检测各组细胞血管内皮生长因子(VEGF)、趋化因子受体4(CXCR4)、血小板衍生因子(PDGF)m RNA表达量。结果 CCK-8法和细胞周期结果显示CGFe明显促进细胞增殖(P<0.05),且增殖效应呈CGFe浓度依赖性,各组间均有统计学差异(P<0.05);划痕实验中12 h时实验组划痕愈合效率明显高于对照组,且愈合效率与CGFe浓度呈正比(P<0.05);CGFe明显促进VEGF、CXCR4、PDGF的m RNA表达量,促进效应与浓度呈正比(P<0.05)。结论 CGFe能有效促进HUVECs的增殖、迁移和成血管分化。
Objective This study aimed to explore the effects of concentrate growth factor extracts(CGFe) on human umbilical vein endothelial cells(HUVECs) in vitro. Methods Concentrate growth factor(CGF) were prepared from the peripheral blood of healthy donors, followed by CGFe. Four groups were designed based on cell culture medium, as follows: 2%CGFe, 5%CGFe, 10%CGFe, and control. The proliferation activity of HUVECs was detected by cell cycle and CCK-8 assays. The migration of HUVECs was detected by scratch assay. The m RNA expression levels of vascular endothelial growth factor(VEGF), chemokine receptor 4(CXCR4), and platelet derived growth factor(PDGF) were examined by quantitative real time polymerase chain reaction(q RT-PCR). Results Results of CCK-8 and cell cycle assays showed that CGFe promoted the proliferation capability of HUVECs in a dose-dependent manner, and the data had statistical significance among four groups(P<0.05). The cell migration assay indicated that CGF accelerated wound closure in a dose-dependent manner after 12 h of culture(P<0.05). The results of q RT-PCR showed that CGF upregulated the expression levels of VEGF, CXCR4, and PDGF in HUVECs. Conclusion CGFe can promote the proliferation, migration, and angiogenic differentiation of HUVECs. Thus, CGF might be an appropriate cure for dental pulp revascularization.
Objective This study aimed to explore the effects of concentrate growth factor extracts(CGFe) on human umbilical vein endothelial cells(HUVECs) in vitro. Methods Concentrate growth factor(CGF) were prepared from the peripheral blood of healthy donors, followed by CGFe. Four groups were designed based on cell culture medium, as follows: 2%CGFe, 5%CGFe, 10%CGFe, and control. The proliferation activity of HUVECs was detected by cell cycle and CCK-8 assays. The migration of HUVECs was detected by scratch assay. The m RNA expression levels of vascular endothelial growth factor(VEGF), chemokine receptor 4(CXCR4), and platelet derived growth factor(PDGF) were examined by quantitative real time polymerase chain reaction(q RT-PCR). Results Results of CCK-8 and cell cycle assays showed that CGFe promoted the proliferation capability of HUVECs in a dose-dependent manner, and the data had statistical significance among four groups(P<0.05). The cell migration assay indicated that CGF accelerated wound closure in a dose-dependent manner after 12 h of culture(P<0.05). The results of q RT-PCR showed that CGF upregulated the expression levels of VEGF, CXCR4, and PDGF in HUVECs. Conclusion CGFe can promote the proliferation, migration, and angiogenic differentiation of HUVECs. Thus, CGF might be an appropriate cure for dental pulp revascularization.
引文
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[15]Jin DK,Shido K,Kopp HG,et al.Cytokine-mediated deployment of SDF-1 induces revascularization through recruitment of CXCR4+hemangiocytes[J].Nat Med,2006,12(5):557-567.
[16]Huang Z,Miao X,Luan Y,et al.PAR1-stimulated platelet releasate promotes angiogenic activities of endothelial progenitor cells more potently than PAR4-stimulated platelet releasate[J].J Thromb Haemost,2015,13(3):465-476.
[17]Tran-Hung L,Mathieu S,About I.Role of human pulp fibroblasts in angiogenesis[J].J Dent Res,2006,85(9):819-823.
[2]Sakai VT,Cordeiro MM,Dong Z,et al.Tooth slice/scaffold model of dental pulp tissue engineering[J].Adv Dent Res,2011,23(3):325-332.
[3]Peng L,Ye L,Zhou XD.Mesenchymal stem cells and tooth engineering[J].Int J Oral Sci,2009,1(1):6-12.
[4]Torabinejad M,Faras H.A clinical and histological report of a tooth with an open apex treated with regenerative endodontics using platelet-rich plasma[J].J Endod,2012,38(6):864-868.
[5]Kakudo N,Morimoto N,Kushida S,et al.Platelet-rich plasma releasate promotes angiogenesis in vitro and in vivo[J].Med Mol Morphol,2014,47(2):83-89.
[6]Choukroun J,Diss A,Simonpieri A,et al.Platelet-rich fibrin(PRF):a second-generation platelet concentrate.Part V:histologic evaluations of PRF effects on bone allograft maturation in sinus lift[J].Oral Surg Oral Med Oral Pathol Oral Radiol Endod,2006,101(3):299-303.
[7]姜志红,李欣,柳忠豪.浓缩生长因子提取液对钛片表面MC3T3-E1细胞影响的实验研究[J].中国口腔种植学杂志,2016,21(2):63-66,93.Jiang ZH,Li X,Liu ZH.The effects of concentrate growth factors extract(CGFe)on osteoblast attached to the SLA titanium surfaces[J].Chin J Oral Implant,2016,21(2):63-66,93.
[8]Sohn DS,Heo JU,Kwak DH,et al.Bone regeneration in the maxillary sinus using an autologous fibrin-rich block with concentrated growth factors alone[J].Implant Dent,2011,20(5):389-395.
[9]Qin J,Wang L,Zheng L,et al.Concentrated growth factor promotes Schwann cell migration partly through the integrinβ1-mediated activation of the focal adhesion kinase pathway[J].Int J Mol Med,2016,37(5):1363-1370.
[10]Kobayashi M,Kawase T,Horimizu M,et al.A proposed protocol for the standardized preparation of PRF membranes for clinical use[J].Biologicals,2012,40(5):323-329.
[11]Clipet F,Tricot S,Alno N,et al.In vitro effects of Choukroun’s platelet-rich fibrin conditioned medium on 3 different cell lines implicated in dental implantology[J].Implant Dent,2012,21(1):51-56.
[12]Honda H,Tamai N,Naka N,et al.Bone tissue engineering with bone marrow-derived stromal cells integrated with concentrated growth factor in Rattus norvegicus calvaria defect model[J].J Artif Organs,2013,16(3):305-315.
[13]Masuki H,Okudera T,Watanebe T,et al.Growth factor and pro-inflammatory cytokine contents in platelet-rich plasma(PRP),plasma rich in growth factors(PRGF),advanced platelet-rich fibrin(A-PRF),and concentrated growth factors(CGF)[J].Int J Implant Dent,2016,2(1):19.
[14]Saghiri MA,Asatourian A,Sorenson CM,et al.Role of angiogenesis in endodontics:contributions of stem cells and proangiogenic and antiangiogenic factors to dental pulp regeneration[J].J Endod,2015,41(6):797-803.
[15]Jin DK,Shido K,Kopp HG,et al.Cytokine-mediated deployment of SDF-1 induces revascularization through recruitment of CXCR4+hemangiocytes[J].Nat Med,2006,12(5):557-567.
[16]Huang Z,Miao X,Luan Y,et al.PAR1-stimulated platelet releasate promotes angiogenic activities of endothelial progenitor cells more potently than PAR4-stimulated platelet releasate[J].J Thromb Haemost,2015,13(3):465-476.
[17]Tran-Hung L,Mathieu S,About I.Role of human pulp fibroblasts in angiogenesis[J].J Dent Res,2006,85(9):819-823.