人大隐静脉干细胞原代培养方法的改良
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摘要
旨在探讨如何高效获取优质的人大隐静脉(Great saphenous vein)原代干细胞。大隐静脉剪碎后分别采用组织块贴壁法和Ⅱ型胶原酶消化法获取血管壁细胞。倒置显微镜下观察不同时间段两组血管壁细胞形态学变化,台盼蓝染色测定血管壁细胞存活率,流式分选CD34和CD117双阳性干细胞,免疫荧光进一步证实。细胞培养至第三代(P3)时组织块贴壁法提取的细胞出现纤维化老化,而酶消化法提取的细胞仍有集落样生长,细胞存活率分别为(91.7±1.2)%和(97.2±0.7)%(P=0.005)。流式分选结果显示:组织块法和酶消化法获得CD34和CD117双阳性细胞所占比例分别为(0.16±0.05)%和(0.44±0.07)%,差异有统计学意义(P=0.005)。免疫荧光染色显示,分选出的干细胞培养1周后组织块法获得双阳性干细胞的阳性率(89.41±2.06)%,酶消化法为(94.03±1.83)%,P<0.05。流式细胞仪检测分选出的干细胞中CD31、VEGF2和SMA阳性率分别为(0.12±0.01)%、(0.19±0.02)%和(0.45±0.01)%,与阴性对照组差异无统计学意义(P>0.05),排除成熟内皮细胞和平滑肌细胞存在的可能性。分选出的干细胞进行管腔形成实验进一步证实其具有向内皮分化的潜能。上述结果显示,采用酶消化法可以获得形态更好、数量更多、存活率相对更高的干细胞,可广泛用于临床和基础研究。
The objectiue was to explore how to improve stem cell derivation from human great saphenous vein. After the saphenous vein was cut into small pieces, the cells of the vessel wall were obtained by tissue adherent method and digestion with type Ⅱ collagenase. The morphological changes of blood vessel wall were observed under inverted microscope. The survival of vascular wall cells was assessed by trypan blue staining. Stem cells doubly positive for CD34 and CD117 were sorted out by immunofluorescent staining and flow cytometry. The cells obtained by tissue adherence method exhibited signs of fibrotic changes and aging at the third passage(P3), while the cells extracted by enzymatic digestion still showed colony-like growth. Survival rates of these two groups of cells were(91.7±1.2)% and(97.2±0.7)%,(P=0.005). The results of flow cytometry showed that the positive rates of CD34 and CD117 double positive cells in these two groups were(0.16± 0.05)% and(0.44±0.07)%, respectively, with statistical significance(P=0.005). Immunofluorescent staining showed that the positive rates of double positive stem cells in the two groups were(89.41±2.06)% and(94.03±1.83)%, P<0.05 one week after the sorted stem cells were cultured. The positive rates of CD31, VEGF2 and SMA in the stem cells determined by flow cytometry were(0.12±0.01)%,(0.19±0.02)% and(0.45±0.01)%, respectively, which were not statistically different from those of the control groups. This could rule out substantial inclusion of mature endothelial cells and smooth muscle cells. Tube forming experiment confirmed that these vascular stem cells had developmental plasticity. More viable and morphologically healthy vascular stem cells can be derived by enzymatic digestion. These cells can be widely used in clinical and basic research.
The objectiue was to explore how to improve stem cell derivation from human great saphenous vein. After the saphenous vein was cut into small pieces, the cells of the vessel wall were obtained by tissue adherent method and digestion with type Ⅱ collagenase. The morphological changes of blood vessel wall were observed under inverted microscope. The survival of vascular wall cells was assessed by trypan blue staining. Stem cells doubly positive for CD34 and CD117 were sorted out by immunofluorescent staining and flow cytometry. The cells obtained by tissue adherence method exhibited signs of fibrotic changes and aging at the third passage(P3), while the cells extracted by enzymatic digestion still showed colony-like growth. Survival rates of these two groups of cells were(91.7±1.2)% and(97.2±0.7)%,(P=0.005). The results of flow cytometry showed that the positive rates of CD34 and CD117 double positive cells in these two groups were(0.16± 0.05)% and(0.44±0.07)%, respectively, with statistical significance(P=0.005). Immunofluorescent staining showed that the positive rates of double positive stem cells in the two groups were(89.41±2.06)% and(94.03±1.83)%, P<0.05 one week after the sorted stem cells were cultured. The positive rates of CD31, VEGF2 and SMA in the stem cells determined by flow cytometry were(0.12±0.01)%,(0.19±0.02)% and(0.45±0.01)%, respectively, which were not statistically different from those of the control groups. This could rule out substantial inclusion of mature endothelial cells and smooth muscle cells. Tube forming experiment confirmed that these vascular stem cells had developmental plasticity. More viable and morphologically healthy vascular stem cells can be derived by enzymatic digestion. These cells can be widely used in clinical and basic research.
引文
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[24]Ma Z,Chen YK.Establishment of inferior vena cava thrombosis model in nude mice.Med J Chin People’s Lib Army,2016,41(11):883-886(in Chinese).马振,陈以宽.裸鼠下腔静脉血栓模型的构建.解放军医学杂志,2016,41(11):883-886.
[25]Jonlin EC.Differing standards for the NIH stem cell registry and FDA approval render most federally funded h ESC lines unsuitable for clinical use.Cell Stem Cell,2014,14(2):139-140.
[2]Askarov MB,Onischenko NA.Multipotent mesenchymal stromal cells of autologous bone marrow stimulate neoangiogenesis,restore microcirculation,and promote healing of indolent ulcers of the stomach.Bull Experim Biol Med,2008,146(4):512-516.
[3]Invernici G,Emanueli C,Madeddu P,et al.Human fetal aorta contains vascular progenitor cells capable of inducing vasculogenesis,angiogenesis,and myogenesis in vitro and in a murine model of peripheral ischemia.Am J Pathol,2007,170(6):1879-1892.
[4]Li XQ,Meng QY,Wu HR.Effects of bone marrowderived endothelial progenitor cell transplantation on vein microenvironment in a rat model of chronic thrombosis.Chin Med J,2007,120(24):2245-2249.
[5]Chen YK,Jiang XM,Gong JP.Recombinant human granulocyte colony-stimulating factor enhanced the resolution of venous thrombi.J Vascul Surg,2008,47(5):1058-1065.
[6]Campagnolo P,Cesselli D,Al Haj Zen A,et al.Human adult vena saphena contains perivascular progenitor cells endowed with clonogenic and proangiogenic potential.Circulation,2010,121(15):1735-1745.
[7]Chen YK,Wong MM,Campagnolo P,et al.Adventitial stem cells in vein grafts display multilineage potential that contributes to neointimal formation.Arterioscl,Thromb,Vascul Biol,2013,33(8):1844-1851.
[8]Wong MM,Chen YK,Margariti A,et al.Macrophages control vascular stem/progenitor cell plasticity through tumor necrosis factor-alpha-mediated nuclear factorkappa B activation.Arterioscl,Thrombo,Vascul Biol,2014,34(3):635-643.
[9]Kirchhoffer DG,Dierickx K.Human dignity and human tissue:a meaningful ethical relationship?J Med Eth,2011,37(9):552-556.
[10]Hu YH,Zhang ZY,Torsney E,et al.Abundant progenitor cells in the adventitia contribute to atherosclerosis of vein grafts in Apo E-deficient mice.JClin Investigat,2004,113(9):1258-1265.
[11]Zengin E,Chalajour F,Gehling UM,et al.Vascular wall resident progenitor cells:a source for postnatal vasculogenesis.Development,2006,133(8):1543-1551.
[12]Lu WS,Li XR.Vascular stem/progenitor cells:functions and signaling pathways.Cellul Mol Life Sci,2018,75(5):859-869.
[13]Psaltis PJ,Simari RD.Vascular wall progenitor cells in health and disease.Circulat Res,2015,116(8):1392-1412.
[14]Bobryshev YV,Orekhov AN,Chistiakov DA.Vascular stem/progenitor cells:current status of the problem.Cell Tissue Res,2015,362(1):1-7.
[15]Havelka GE,Kibbe MR.The vascular adventitia:its role in the arterial injury response.Vasc Endovascr Surg,2011,45(5):381-390.
[16]Majesky MW,Dong XR,Hoglund V,et al.The adventitia:a dynamic interface containing resident progenitor cells.Arterioscler Thromb Vasc Biol,2011,31(7):1530-1539.
[17]Fadini GP,Madeddu P,Waltenberger J,et al.Vascular stem and progenitor cells in diabetic complications.Exp Diabetes Res,2012,2012:580343.
[18]Tabar V,Studer L.Pluripotent stem cells in regenerative medicine:challenges and recent progress.Nat Rev Genet,2014,15(2):82-92.
[19]Beltrami AP,Cesselli D,Bergamin N,et al.Multipotent cells can be generated in vitro from several adult human organs(heart,liver,and bone marrow).Blood,2007,110(9):3438-3446.
[20]Wang LB,Zhu H,Hao J,et al.Progress in stem cells and regenerative medicine.Chin J Biotech,2015,31(6):871-879(in Chinese).王立宾,祝贺,郝捷,等.干细胞与再生医学研究进展.生物工程学报,2015,31(6):871-879.
[21]Watt SM,Athanassopoulos A,Harris AL,et al.Human endothelial stem/progenitor cells,angiogenic factors and vascular repair.JR Soc Interface,2010,7(S6):S731-S751.
[22]Li L,Yang YH,Wang S,et al.Comparison of cellular activity detection methods.J Biol,2011,28(1):87-90,93(in Chinese).李磊,杨雨晗,王双,等.细胞活性检测方法之比较.生物学杂志,2011,28(1):87-90,93.
[23]Shi MX,Li WJ,Li BZ,et al.Comparison of cellular activity detection methods.Chin J Biotech,2009,25(5):754-760(in Chinese).史明霞,李维佳,李炳宗,等.人羊膜来源成体干细胞的多向分化潜能.生物工程学报,2009,25(5):754-760.
[24]Ma Z,Chen YK.Establishment of inferior vena cava thrombosis model in nude mice.Med J Chin People’s Lib Army,2016,41(11):883-886(in Chinese).马振,陈以宽.裸鼠下腔静脉血栓模型的构建.解放军医学杂志,2016,41(11):883-886.
[25]Jonlin EC.Differing standards for the NIH stem cell registry and FDA approval render most federally funded h ESC lines unsuitable for clinical use.Cell Stem Cell,2014,14(2):139-140.