VEGF基因转染脂肪干细胞后上清液对皮肤成纤维细胞及脐静脉血管内皮细胞的影响
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摘要
目的:通过慢病毒将血管内皮生长因子(VEGF)基因转染于人脂肪干细胞(HADSCs),检测其上清液(CM)中生长因子的表达,并用其上清液作用于人皮肤成纤维细胞(HDFs)及人脐静脉内皮细胞(HUVECs),观察对这2种细胞活力和迁移的影响。方法:准备HADSCs、HDFs及HUVECs 3种细胞并鉴定;将携带VEGF165基因的慢病毒转染于HADSCs,定时收集上清液;ELISA法检验上清中生长因子分泌情况;将VEGF-CM与完全培养液以一定比例混合分为5组,分别培养HDFs及HUVECs,CCK-8法检验对2种细胞活力的影响;将最佳比例的VEGF-CM、正常CM(Nor-CM)和完全培养液分别作用于HDFs及HUVECs,划痕法测试出对2种细胞迁移的影响。结果:ELISA结果表明VEGF-CM中VEGF及碱性成纤维细胞生长因子(bFGF)的表达均较Nor-CM组提高;相对于其它比例,当完全培养液与VEGF-CM以1∶2的比例混合时,HDFs和HUVECs的活力显著增强(P<0.05);VEGF-CM与其它培养液相比可显著提高HDFs和HUVECs的迁移能力(P<0.05)。结论:转染VEGF165基因后的HADSCs可同时增强VEGF及bFGF的分泌,其上清液可提高成纤维细胞及血管内皮细胞的活力和迁移能力。
AIM: To investigate the influence of conditioned medium(CM) of human adipose-derived stem cells(HADSCs) transfected with vascular endothelial growth factor(VEGF) gene on human dermal fibroblasts(HDFs) and human umbilical vein endothelial cells(HUVECs) in vitro. METHODS: The HADSCs, HDFs and HUVECs were prepared and identified. The HADSCs were transfected with lentivirus carrying VEGF165 gene and the CM was collected regularly. ELISA method was used to detect the growth factor secretion in the CM. The VEGF-CM mixed with complete medium were divided into 5 groups for culturing with HDFs or HUVECs. The cell viability was measured by CCK-8 assay. The optimal ratio of VEGF-CM, normal CM(Nor-CM) and complete medium were applied to HDFs or HUVECs. The migration ability was detected by wound-healing assay. RESULTS: The results of ELISA showed that the expression levels of VEGF and basic fibroblast growth factor(bFGF) in VEGF-CM group were higher those in Nor-CM group(P<0.05). Compared with other groups, the cell viability and migration abilities of HDFs and HUVECs were obviously enhanced in VEGF-CM group(P<0.05). CONCLUSION: The HADSCs transfected with VEGF165 gene greatly enhances the expression of VEGF and bFGF. The CM of HADSCs promotes the viability and migration abilities of HDFs and HUVECs in vitro.
AIM: To investigate the influence of conditioned medium(CM) of human adipose-derived stem cells(HADSCs) transfected with vascular endothelial growth factor(VEGF) gene on human dermal fibroblasts(HDFs) and human umbilical vein endothelial cells(HUVECs) in vitro. METHODS: The HADSCs, HDFs and HUVECs were prepared and identified. The HADSCs were transfected with lentivirus carrying VEGF165 gene and the CM was collected regularly. ELISA method was used to detect the growth factor secretion in the CM. The VEGF-CM mixed with complete medium were divided into 5 groups for culturing with HDFs or HUVECs. The cell viability was measured by CCK-8 assay. The optimal ratio of VEGF-CM, normal CM(Nor-CM) and complete medium were applied to HDFs or HUVECs. The migration ability was detected by wound-healing assay. RESULTS: The results of ELISA showed that the expression levels of VEGF and basic fibroblast growth factor(bFGF) in VEGF-CM group were higher those in Nor-CM group(P<0.05). Compared with other groups, the cell viability and migration abilities of HDFs and HUVECs were obviously enhanced in VEGF-CM group(P<0.05). CONCLUSION: The HADSCs transfected with VEGF165 gene greatly enhances the expression of VEGF and bFGF. The CM of HADSCs promotes the viability and migration abilities of HDFs and HUVECs in vitro.
引文
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[2] 王于, 连帆, 谭国胜, 等. 同种异体脂肪干细胞门脉移植对大鼠纤维化肝脏的影响[J]. 中国病理生理杂志, 2010, 26(11):2197-2201.
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[5] Rehman J, Traktuev D, Li J, et al. Secretion of angioge-nic and antiapoptotic factors by human adipose stromal cells[J]. Circulation, 2004, 109(10):1292-1298.
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[7] Gaur M, Dobke M, Lunyak VV. Mesenchymal stem cells from adipose tissue in clinical applications for dermatological indications and skin aging[J]. Int J Mol Sci, 2017, 18(1):E208.
[8] Efimenko A, Dzhoyashvili N, Kalinina N, et al. Adipose-derived mesenchymal stromal cells from aged patients with coronary artery disease keep mesenchymal stromal cell properties but exhibit characteristics of aging and have impaired angiogenic potential[J]. Stem Cells Transl Med, 2014, 3(1):32-41.
[9] Kober J, Gugerell A, Schmid M, et al. Wound healing effect of conditioned media obtained from adipose tissue on human skin cells: a comparative in vitro study[J]. Ann Plast Surg, 2016, 77(2):156-163.
[10] 李江璇, 肖丽玲. 脂肪干细胞分泌功能的研究进展及应用前景[J]. 广东医学, 2014, 35(2):293-296.
[11] Shevchenko EK, Makarevich PI, Tsokolaeva ZI, et al. Transplantation of modified human adipose derived stromal cells expressing VEGF165 results in more efficient angiogenic response in ischemic skeletal muscle[J]. J Transl Med, 2013, 11:138.
[12] Sun XZ, Liu GH, Wang ZQ, et al. Over-expression of VEGF165 in the adipose tissue-derived stem cells via the lentiviral vector[J]. Chin Med J (Engl), 2011, 124(19):3093-3097.
[13] Roy H, Bhardwaj S, Yla-Herttuala S. Biology of vascular endothelial growth factors[J]. FEBS Lett, 2006, 580(12):2879-2887.
[14] Barrientos S, Stojadinovic O, Golinko MS, et al. Growth factors and cytokines in wound healing[J]. Wound Repair Regen, 2008, 16(5):585-601.
[15] 黄芳, 杨晶金, 王雪玲, 等. VEGF和bFGF在大鼠压疮模型中的表达及其意义[J]. 中国病理生理杂志, 2012, 28(8):1504-1506.
[2] 王于, 连帆, 谭国胜, 等. 同种异体脂肪干细胞门脉移植对大鼠纤维化肝脏的影响[J]. 中国病理生理杂志, 2010, 26(11):2197-2201.
[3] Hsiao ST, Lokmic Z, Peshavariya H, et al. Hypoxic conditioning enhances the angiogenic paracrine activity of human adipose-derived stem cells[J]. Stem Cells Dev, 2013, 22(10):1614-1623.
[4] Lee EY, Xia Y, Kim WS, et al. Hypoxia-enhanced wound-healing function of adipose-derived stem cells: increase in stem cell proliferation and up-regulation of VEGF and bFGF[J]. Wound Repair Regen, 2009, 17(4):540-547.
[5] Rehman J, Traktuev D, Li J, et al. Secretion of angioge-nic and antiapoptotic factors by human adipose stromal cells[J]. Circulation, 2004, 109(10):1292-1298.
[6] Kim WS, Park BS, Sung JH, et al. Wound healing effect of adipose-derived stem cells: a critical role of secretory factors on human dermal fibroblasts[J]. J Dermatol Sci, 2007, 48(1):15-24.
[7] Gaur M, Dobke M, Lunyak VV. Mesenchymal stem cells from adipose tissue in clinical applications for dermatological indications and skin aging[J]. Int J Mol Sci, 2017, 18(1):E208.
[8] Efimenko A, Dzhoyashvili N, Kalinina N, et al. Adipose-derived mesenchymal stromal cells from aged patients with coronary artery disease keep mesenchymal stromal cell properties but exhibit characteristics of aging and have impaired angiogenic potential[J]. Stem Cells Transl Med, 2014, 3(1):32-41.
[9] Kober J, Gugerell A, Schmid M, et al. Wound healing effect of conditioned media obtained from adipose tissue on human skin cells: a comparative in vitro study[J]. Ann Plast Surg, 2016, 77(2):156-163.
[10] 李江璇, 肖丽玲. 脂肪干细胞分泌功能的研究进展及应用前景[J]. 广东医学, 2014, 35(2):293-296.
[11] Shevchenko EK, Makarevich PI, Tsokolaeva ZI, et al. Transplantation of modified human adipose derived stromal cells expressing VEGF165 results in more efficient angiogenic response in ischemic skeletal muscle[J]. J Transl Med, 2013, 11:138.
[12] Sun XZ, Liu GH, Wang ZQ, et al. Over-expression of VEGF165 in the adipose tissue-derived stem cells via the lentiviral vector[J]. Chin Med J (Engl), 2011, 124(19):3093-3097.
[13] Roy H, Bhardwaj S, Yla-Herttuala S. Biology of vascular endothelial growth factors[J]. FEBS Lett, 2006, 580(12):2879-2887.
[14] Barrientos S, Stojadinovic O, Golinko MS, et al. Growth factors and cytokines in wound healing[J]. Wound Repair Regen, 2008, 16(5):585-601.
[15] 黄芳, 杨晶金, 王雪玲, 等. VEGF和bFGF在大鼠压疮模型中的表达及其意义[J]. 中国病理生理杂志, 2012, 28(8):1504-1506.