CM-Dil标记大鼠骨髓间充质干细胞的生物学特性探讨
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摘要
背景:目前已有部分实验采用CM-Dil标记间充质干细胞开展体内外实验,取得良好成果。目的:观察CM-Dil标记大鼠骨髓间充质干细胞的生物学特性及其定向迁移能力。方法:分离培养大鼠骨髓间充质干细胞,通过流式细胞技术分析其特异性细胞表面标记抗原(CD29,CD44,CD11b,CD45)的表达。用CM-Dil标记大鼠骨髓间充质干细胞,继续培养细胞,观察细胞的形态及增殖情况,及传代后荧光的强度与持久性。采用Transwell小室进行标记后大鼠骨髓间充质干细胞向mdx鼠腓肠肌匀浆液的迁移实验。结果与结论:所培养细胞均一性好,均表达CD29和CD44,不表达CD11b和CD45,符合骨髓间充质干细胞的特点。经CM-Dil标记后对大鼠骨髓间充质干细胞的形态、增殖无明显影响,传代后仍可保持荧光强度,但随传代有所减弱。标记前后大鼠骨髓间充质干细胞的迁移能力无显著性差异(P>0.05)。结果说明,CM-Dil标记过程安全、简单、高效,对大鼠骨髓间充质干细胞的形态、增殖及迁移能力无明显影响,体外细胞传代后仍可保持荧光,是大鼠骨髓间充质干细胞标记与示踪的理想试剂。
BACKGROUND: Existing evidence has shown that CM-Dil-labeled mesenchymal stem cells have delivered better results in experiments in vivo and in vitro. OBJECTIVE: To investigate the effect of in vitro CM-Dil labeling on biological characteristics and directed migration of rat bone marrow mesenchymal stem cells. METHODS: Mesenchymal stem cells were isolated and cultured from the bone marrow of Sprague-Dawley rats. The specific surface antigens, CD29, CD44, CD11 b, CD45, were detected by flow cytometry. The rat bone marrow mesenchymal stem cells were labeled with CM-Dil, and then were cultured and passed continually. Thereafter the morphology and proliferation ability of labeled cells were assessed. The strength and duration of fluorescence after CM-Dil labeling were detected. Transwell chambers were used to investigate the migration ability of labeled cells towards the homogenate of the gastrocnemius muscles of mdx mice. RESULTS AND CONCLUSION: The cultured cells were markedly positive for CD29 and CD44, but negative for CD11 b and CD45, which were in accordance with the features of bone marrow mesenchymal stem cells. CM-Dil-labeled cells were not different from unlabeled cells in the aspect of morphology and proliferation. Fluorescence was still visible after passage; however, a reduction in fluorescent intensity was observed under an inverted fluorescent contrast phase microscope after labeling. The labeled cells showed no different migration ability from the unlabeled cells(P > 0.05). In conclusion, the procedure of CM-Dil labeling is safe, simple and effective. CM-Dil could be a good choice for labeling and tracing rat bone marrow mesenchymal stem cells.
BACKGROUND: Existing evidence has shown that CM-Dil-labeled mesenchymal stem cells have delivered better results in experiments in vivo and in vitro. OBJECTIVE: To investigate the effect of in vitro CM-Dil labeling on biological characteristics and directed migration of rat bone marrow mesenchymal stem cells. METHODS: Mesenchymal stem cells were isolated and cultured from the bone marrow of Sprague-Dawley rats. The specific surface antigens, CD29, CD44, CD11 b, CD45, were detected by flow cytometry. The rat bone marrow mesenchymal stem cells were labeled with CM-Dil, and then were cultured and passed continually. Thereafter the morphology and proliferation ability of labeled cells were assessed. The strength and duration of fluorescence after CM-Dil labeling were detected. Transwell chambers were used to investigate the migration ability of labeled cells towards the homogenate of the gastrocnemius muscles of mdx mice. RESULTS AND CONCLUSION: The cultured cells were markedly positive for CD29 and CD44, but negative for CD11 b and CD45, which were in accordance with the features of bone marrow mesenchymal stem cells. CM-Dil-labeled cells were not different from unlabeled cells in the aspect of morphology and proliferation. Fluorescence was still visible after passage; however, a reduction in fluorescent intensity was observed under an inverted fluorescent contrast phase microscope after labeling. The labeled cells showed no different migration ability from the unlabeled cells(P > 0.05). In conclusion, the procedure of CM-Dil labeling is safe, simple and effective. CM-Dil could be a good choice for labeling and tracing rat bone marrow mesenchymal stem cells.
引文
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[16]Chen B,Bo CJ,Jia RP,et al.The renoprotective effect of bone marrow-derived endothelial progenitor cell transplantation on acute ischemia-reperfusion injury in rats.Transplant Proc.2013;45(5):2034-2039.
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[18]Qiao PF,Yao L,Zhang XC,et al.Heat shock pretreatment improves stem cell repair following ischemia-reperfusion injury via autophagy.World JGastroenterol.2015;21(45):12822-12834.
[19]Omidi A,Kashani IR,Akbari M,et al.Homing of allogeneic nestin-positive hair follicle-associated pluripotent stem cells after maternal transplantation in experimental model of cortical dysplasia.Biochem Cell Biol.2015;93(6):619-625.
[20]Cai J,Yu X,Zhang B,et al.Atorvastatin improves survival of implanted stem cells in a rat model of renal ischemia-reperfusion injury.Am J Nephrol.2014;39(6):466-475.
[21]Edalatmanesh MA,Bahrami AR,Hosseini E,et al.Neuroprotective effects of mesenchymal stem cell transplantation in animal model of cerebellar degeneration.Neurol Res.2011;33(9):913-920.
[22]Sun S,Chen G,Xu M,et al.Differentiation and migration of bone marrow mesenchymal stem cells transplanted through the spleen in rats with portal hypertension.PLoS One.2013;8(12):e83523.
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[24]陈朝,黎奔,郭建文,等.大鼠骨髓间充质干细胞的分离培养及CM-Dil标记的脑内示踪[J].解放军医学杂志,2010,35(8):946-953.
[25]李朝中,肖践明,陈丽星,等.大鼠骨髓间充质干细胞体外分离培养与CM-Di I荧光标记[J].中国组织工程研究,2014,18(1):39-44.
[26]陈丽,吴本清,程涵蓉,等.CM-Dil体外标记人脐带间充质干细胞传代示踪的可行性[J].中国组织工程研究与临床康复,2010,14(40):7435-7438.
[27]Ji F,Duan HG,Zheng CQ,et al.Comparison of chloromethyl-dialkylcarbocyanine and green fluorescent protein for labeling human umbilical mesenchymal stem cells.Biotechnol Lett.2015;37(2):437-447.
[28]Naaldijk Y,Johnson AA,Ishak S,et al.Migrational changes of mesenchymal stem cells in response to cytokines,growth factors,hypoxia,and aging.Exp Cell Res.2015;338(1):97-104.
[29]Zhou SB,Wang J,Chiang CA,et al.Mechanical stretch upregulates SDF-1αin skin tissue and induces migration of circulating bone marrow-derived stem cells into the expanded skin.Stem Cells.2013;31(12):2703-2713.
[30]Consentius C,Akyüz L,Schmidt-Lucke JA,et al.Mesenchymal stromal cells prevent allostimulation in vivo and control checkpoints of Th1 priming:migration of human dc to lymph nodes and nk cell activation.Stem Cells.2015;33(10):3087-3099.
[31]Lee SH,Jin KS,Bang OY,et al.Differential migration of mesenchymal stem cells to ischemic regions after middle cerebral artery occlusion in rats.PLo S One.2015;10(8):e0134920.
[32]Motaln H,Turnsek TL.Cytokines play a key role in communication between mesenchymal stem cells and brain cancer cells.Protein Pept Lett.2015;22(4):322-331.
[33]Hengartner NE,Fiedler J,Schrezenmeier H,et al.Crucial role of IL1beta and C3a in the in vitro-response of multipotent mesenchymal stromal cells to inflammatory mediators of polytrauma.PLo S One.2015;10(1):e0116772.
[34]Marquez-Curtis LA,Janowska-Wieczorek A.Enhancing the migration ability of mesenchymal stromal cells by targeting the SDF-1/CXCR4 axis.Biomed Res Int.2013;2013:561098.
[35]Joos H,Wildner A,Hogrefe C,et al.Interleukin-1 beta and tumor necrosis factor alpha inhibit migration activity of chondrogenic progenitor cells from non-fibrillated osteoarthritic cartilage.Arthritis Res Ther.2013;15(5):R119.
[36]Sl?rdahl TS,Denayer T,Moen SH,et al.Anti-c-METNanobody-a new potential drug in multiple myeloma treatment.Eur J Haematol.2013;91(5):399-410.
[37]Hengartner NE,Fiedler J,Ignatius A,et al.IL-1βinhibits human osteoblast migration.Mol Med.2013;19:36-42.
[38]Zou C,Luo Q,Qin J,et al.Osteopontin promotes mesenchymal stem cell migration and lessens cell stiffness via integrinβ1,FAK,and ERK pathways.Cell Biochem Biophys.2013;65(3):455-462.
[39]Dittmar T,Entschladen F.Migratory properties of mesenchymal stem cells.Adv Biochem Eng Biotechnol.2013;129:117-136.
[40]Wobus M,Benath G,Ferrer RA,et al.Impact of lenalidomide on the functional properties of human mesenchymal stromal cells.Exp Hematol.2012;40(10):867-876.
[41]Hurst NJ Jr,Najy AJ,Ustach CV,Movilla L,Kim HR,et al.Platelet-derived growth factor-C(PDGF-C)activation by serine proteases:implications for breast cancer progression.Biochem J.2012;441(3):909-918.
[42]Hu J,Qin K,Zhang Y,et al.Downregulation of transcription factor Oct4 induces an epithelial-tomesenchymal transition via enhancement of Ca2+influx in breast cancer cells.Biochem Biophys Res Commun.2011;411(4):786-791.
[43]Zou C,Song G,Luo Q,et al.Mesenchymal stem cells require integrinβ1 for directed migration induced by osteopontin in vitro.In Vitro Cell Dev Biol Anim.2011;47(3):241-250.
[2]Ji AT,Chang YC,Fu YJ,et al.Niche-dependent regulations of metabolic balance in high-fat diet-induced diabetic mice by mesenchymal stromal cells.Diabetes.2015;64(3):926-936.
[3]Shang YC,Wang SH,Xiong F,et al.Wnt3a signaling promotes proliferation,myogenic differentiation,and migration of rat bone marrow mesenchymal stem cells.Acta Pharmacol Sin.2007;28(11):1761-1774.
[4]Liu X,Wang X,Li A,et al.Effect of mesenchymal stem cell transplantation on brain-derived neurotrophic factor expression in rats with Tourette syndrome.Exp Ther Med.2016;11(4):1211-1216.
[5]Li M,Zhang YX,Zhang Z,et al.Endomicroscopy Will Track Injected Mesenchymal Stem Cells in Rat Colitis Models.Inflamm Bowel Dis.2015;21(9):2068-2077.
[6]Hendijani F,Javanmard SH,Sadeghi-aliabadi H.Human Wharton's jelly mesenchymal stem cell secretome display antiproliferative effect on leukemia cell line and produce additive cytotoxic effect in combination with doxorubicin.Tissue Cell.2015;47(3):229-234.
[7]Lin CS,Xin ZC,Dai J,et al.Commonly used mesenchymal stem cell markers and tracking labels:limitations and challenges.Histol Histopathol.2013;28(9):1109-1116.
[8]Huntsman HD,Zachwieja N,Zou K,et al.Mesenchymal stem cells contribute to vascular growth in skeletal muscle in response to eccentric exercise.Am J Physiol Heart Circ Physiol.2013;304(1):H72-H81.
[9]Price MJ,Chou CC,Frantzen M,et al.Intravenous mesenchymal stem cell therapy early after reperfused acute myocardial infarction improves left ventricular function and alters electrophysiologic properties.Int JCardiol.2001;11(2):231-239.
[10]Dai W,Hale SL,Martin BJ,et al.Allogeneic mesenchymal stem cell transplantation in postinfarcted rat myocardium:short-and long-term effects.Circulation.2005;112(2):214-223.
[11]Feng SW,Chen F,Cao J,et al.Restoration of muscle fibers and satellite cells after isogenic MSCtransplantation with microdystrophin gene delivery.Biochem Biophys Res Commun.2012;419(1):1-6.
[12]Spitzer N,Sammons GS,Price EM,et al.Autofluorescent cells in rat brain can be convincing impostors in green fluorescent reporter studies.JNeurosci Methods.2011;197(1):48-55.
[13]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.
[14]Lin CS,Xin ZC,Dai J,et al.Commonly used mesenchymal stem cell markers and tracking labels:Limitations and challenges.Histol Histopathol.2013;28(9):1109-1116.
[15]Li N,Yang H,Lu L,et al.Comparison of the labeling efficiency of BrdU,DiI and FISH labeling techniques in bone marrow stromal cells.Brain Res.2008;1215:11-19.
[16]Chen B,Bo CJ,Jia RP,et al.The renoprotective effect of bone marrow-derived endothelial progenitor cell transplantation on acute ischemia-reperfusion injury in rats.Transplant Proc.2013;45(5):2034-2039.
[17]Gong J,Meng HB,Hua J,et al.The SDF-1/CXCR4axis regulates migration of transplanted bone marrow mesenchymal stem cells towards the pancreas in rats with acute pancreatitis.Mol Med Rep.2014;9(5):1575-1582.
[18]Qiao PF,Yao L,Zhang XC,et al.Heat shock pretreatment improves stem cell repair following ischemia-reperfusion injury via autophagy.World JGastroenterol.2015;21(45):12822-12834.
[19]Omidi A,Kashani IR,Akbari M,et al.Homing of allogeneic nestin-positive hair follicle-associated pluripotent stem cells after maternal transplantation in experimental model of cortical dysplasia.Biochem Cell Biol.2015;93(6):619-625.
[20]Cai J,Yu X,Zhang B,et al.Atorvastatin improves survival of implanted stem cells in a rat model of renal ischemia-reperfusion injury.Am J Nephrol.2014;39(6):466-475.
[21]Edalatmanesh MA,Bahrami AR,Hosseini E,et al.Neuroprotective effects of mesenchymal stem cell transplantation in animal model of cerebellar degeneration.Neurol Res.2011;33(9):913-920.
[22]Sun S,Chen G,Xu M,et al.Differentiation and migration of bone marrow mesenchymal stem cells transplanted through the spleen in rats with portal hypertension.PLoS One.2013;8(12):e83523.
[23]Schormann W,Hammersen FJ,Brulport M,et al.Tracking of human cells in mice.Histochem Cell Biol.2008;130(2):329-338.
[24]陈朝,黎奔,郭建文,等.大鼠骨髓间充质干细胞的分离培养及CM-Dil标记的脑内示踪[J].解放军医学杂志,2010,35(8):946-953.
[25]李朝中,肖践明,陈丽星,等.大鼠骨髓间充质干细胞体外分离培养与CM-Di I荧光标记[J].中国组织工程研究,2014,18(1):39-44.
[26]陈丽,吴本清,程涵蓉,等.CM-Dil体外标记人脐带间充质干细胞传代示踪的可行性[J].中国组织工程研究与临床康复,2010,14(40):7435-7438.
[27]Ji F,Duan HG,Zheng CQ,et al.Comparison of chloromethyl-dialkylcarbocyanine and green fluorescent protein for labeling human umbilical mesenchymal stem cells.Biotechnol Lett.2015;37(2):437-447.
[28]Naaldijk Y,Johnson AA,Ishak S,et al.Migrational changes of mesenchymal stem cells in response to cytokines,growth factors,hypoxia,and aging.Exp Cell Res.2015;338(1):97-104.
[29]Zhou SB,Wang J,Chiang CA,et al.Mechanical stretch upregulates SDF-1αin skin tissue and induces migration of circulating bone marrow-derived stem cells into the expanded skin.Stem Cells.2013;31(12):2703-2713.
[30]Consentius C,Akyüz L,Schmidt-Lucke JA,et al.Mesenchymal stromal cells prevent allostimulation in vivo and control checkpoints of Th1 priming:migration of human dc to lymph nodes and nk cell activation.Stem Cells.2015;33(10):3087-3099.
[31]Lee SH,Jin KS,Bang OY,et al.Differential migration of mesenchymal stem cells to ischemic regions after middle cerebral artery occlusion in rats.PLo S One.2015;10(8):e0134920.
[32]Motaln H,Turnsek TL.Cytokines play a key role in communication between mesenchymal stem cells and brain cancer cells.Protein Pept Lett.2015;22(4):322-331.
[33]Hengartner NE,Fiedler J,Schrezenmeier H,et al.Crucial role of IL1beta and C3a in the in vitro-response of multipotent mesenchymal stromal cells to inflammatory mediators of polytrauma.PLo S One.2015;10(1):e0116772.
[34]Marquez-Curtis LA,Janowska-Wieczorek A.Enhancing the migration ability of mesenchymal stromal cells by targeting the SDF-1/CXCR4 axis.Biomed Res Int.2013;2013:561098.
[35]Joos H,Wildner A,Hogrefe C,et al.Interleukin-1 beta and tumor necrosis factor alpha inhibit migration activity of chondrogenic progenitor cells from non-fibrillated osteoarthritic cartilage.Arthritis Res Ther.2013;15(5):R119.
[36]Sl?rdahl TS,Denayer T,Moen SH,et al.Anti-c-METNanobody-a new potential drug in multiple myeloma treatment.Eur J Haematol.2013;91(5):399-410.
[37]Hengartner NE,Fiedler J,Ignatius A,et al.IL-1βinhibits human osteoblast migration.Mol Med.2013;19:36-42.
[38]Zou C,Luo Q,Qin J,et al.Osteopontin promotes mesenchymal stem cell migration and lessens cell stiffness via integrinβ1,FAK,and ERK pathways.Cell Biochem Biophys.2013;65(3):455-462.
[39]Dittmar T,Entschladen F.Migratory properties of mesenchymal stem cells.Adv Biochem Eng Biotechnol.2013;129:117-136.
[40]Wobus M,Benath G,Ferrer RA,et al.Impact of lenalidomide on the functional properties of human mesenchymal stromal cells.Exp Hematol.2012;40(10):867-876.
[41]Hurst NJ Jr,Najy AJ,Ustach CV,Movilla L,Kim HR,et al.Platelet-derived growth factor-C(PDGF-C)activation by serine proteases:implications for breast cancer progression.Biochem J.2012;441(3):909-918.
[42]Hu J,Qin K,Zhang Y,et al.Downregulation of transcription factor Oct4 induces an epithelial-tomesenchymal transition via enhancement of Ca2+influx in breast cancer cells.Biochem Biophys Res Commun.2011;411(4):786-791.
[43]Zou C,Song G,Luo Q,et al.Mesenchymal stem cells require integrinβ1 for directed migration induced by osteopontin in vitro.In Vitro Cell Dev Biol Anim.2011;47(3):241-250.