VCAM-1抗体包被促进骨髓间充质干细胞迁移的体外实验研究
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摘要
目的观察包被VCAM-1抗体(Ab-VCAM)能否改变骨髓间充质干细胞(BMSCs)的特性及增加迁移率。方法①利用骨片法贴壁培养小鼠BMSCs;②利用抗体包被技术分两步将Ab-VCAM附着于BMSCs,第一步将PPG包被于BMSCs,即PPG-MSC,第二步将Ab-VCAM包被于PPG-MSC,即V-MSC;③利用免疫荧光技术证实V-MSC的成功构建;④观察未包被BMSCs (即MSC)及V-MSC组BMSCs的形态、生长曲线及多向分化潜能;⑤利用Transwell迁移实验比较体外迁移率。结果①选择浓度为100μg/mL的PPG以及1∶200的VCAM-1抗体浓度进行包被的效果最佳;②MSC组、PPG-MSC组、V-MSC组中绿色荧光数目的数值分别为(2.25±1.71)个/视野、(24.75±7.63)个/视野、(66±5.72)个/视野,差异具有显著统计学意义(P<0.01);③第四代BMSCs均可见正常细胞形态、生长情况一致、均可被诱导成脂肪细胞及成骨细胞;④两组细胞均具有迁移特性,V-MSC的迁移数量[(34.33±9.48)细胞/视野]多于MSC[(23±7.16)细胞/视野],但差异无统计学意义(P>0.05)。结论利用抗体包被技术成功地构建了Ab-VCAM-1靶向性干细胞,证实了抗体包被不影响BMSCs的生物学特性,并强化了BMSCs的迁移特性,可用于下一步移植治疗相关疾病。
Objective To observe whether coating VCAM-1 antibody(Ab-VCAM) can alter the characteristics of bone marrow mesenchymal stem cells(BMSCs) and increase its migration rate. Methods ① Mice BMSCs were cultivated from bone-adherent cells. ② Ab-VCAM1 was attached to BMSCs in two steps using antibody coating technique.The first step was to coat PPG in BMSCs, namely PPG-MSC. The second step was to package Ab-VCAM in PPG-MSC,namely V-MSC. ③Successful construction of V-MSC was confirmed by immunofluorescence technique. ④The morphology, growth curve and multi-directional differentiation potential of BMSCs were observed in uncoated BMSCs(MSC group) and V-MSC group. ⑤ In vitro mobility was compared using Transwell migration assay. Results ① PPG with a concentration of 100 μg/mL and a VCAM-1 antibody of 1∶200 were selected for optimal coating. ② The number of green fluorescence in MSC group, PPG-MSC group and V-MSC group were respectively(2.25 ± 1.71) cells/view,(24.75±7.63) cells/view and(66±5.72) cells/view(P<0.01). ③ The fourth generation of BMSCs showed normal cell morphology, uniform growth, and could be induced into adipocytes and osteoblasts. ④ Both groups of cells had migration characteristics, and the number of migration of V-MSC group was(34.33±9.48) cells/view, which was more than(23 ± 7.16) cells/view of MSC group(P>0.05). Conclusion Ab-VCAM-1 targeting stem cells were successfully constructed by antibody coating technique, which confirmed that antibody coating does not affect the biological characteristics of BMSCs and enhanced the migration characteristics of BMSCs. This can be used for the next step of transplantation to treat related diseases.
Objective To observe whether coating VCAM-1 antibody(Ab-VCAM) can alter the characteristics of bone marrow mesenchymal stem cells(BMSCs) and increase its migration rate. Methods ① Mice BMSCs were cultivated from bone-adherent cells. ② Ab-VCAM1 was attached to BMSCs in two steps using antibody coating technique.The first step was to coat PPG in BMSCs, namely PPG-MSC. The second step was to package Ab-VCAM in PPG-MSC,namely V-MSC. ③Successful construction of V-MSC was confirmed by immunofluorescence technique. ④The morphology, growth curve and multi-directional differentiation potential of BMSCs were observed in uncoated BMSCs(MSC group) and V-MSC group. ⑤ In vitro mobility was compared using Transwell migration assay. Results ① PPG with a concentration of 100 μg/mL and a VCAM-1 antibody of 1∶200 were selected for optimal coating. ② The number of green fluorescence in MSC group, PPG-MSC group and V-MSC group were respectively(2.25 ± 1.71) cells/view,(24.75±7.63) cells/view and(66±5.72) cells/view(P<0.01). ③ The fourth generation of BMSCs showed normal cell morphology, uniform growth, and could be induced into adipocytes and osteoblasts. ④ Both groups of cells had migration characteristics, and the number of migration of V-MSC group was(34.33±9.48) cells/view, which was more than(23 ± 7.16) cells/view of MSC group(P>0.05). Conclusion Ab-VCAM-1 targeting stem cells were successfully constructed by antibody coating technique, which confirmed that antibody coating does not affect the biological characteristics of BMSCs and enhanced the migration characteristics of BMSCs. This can be used for the next step of transplantation to treat related diseases.
引文
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[10]GHOBADI A,RETTIG MP,COOPER ML,et al.Bortezomib is a rapid mobilizer of hematopoietic stem cells in mice via modulation of the VCAM-1/VLA-4 axis[J].Blood,2014,124(17):2752-2754.
[11]KAUNDAL U,BAGAI U,RAKHA A.Immunomodulatory plasticity of mesenchymal stem cells:a potential key to successful solid organ transplantation[J].J Transl Med,2018,16(1):31.
[12]SHERIFF L,ALANAZI A,WARD LSC,et al.Origin-specific adhesive interactions of mesenchymal stem cells with platelets influence their behavior after infusion[J].Stem Cells,2018,36(7):1062-1074.
[13]CORRADETTI B,TARABALLI F,MARTINEZ JO,et al.Hyaluronic acid coatings as a simple and efficient approach to improve MSChoming toward the site of inflammation[J].Sci Rep,2017,7(1):7991.
[2]陈倩倩,万军,阎丽,等.小鼠骨髓间充质干细胞移植后在炎症性肠病模型的定位[J].军医进修学院学报,2012,33(6):638-641.
[3]陈倩倩.CXCR-4基因转染联合VCAM-1抗体包被促MSCs归巢及其对实验性IBD小鼠肠黏膜修复机制研究[D].北京:中国人民解放军总医院·解放军医学院,2015.
[4]陈峥.CXCR-4基因转染的BMMSCs对实验性IBD小鼠肠黏膜修复机制研究[D].北京:中国人民解放军总医院·解放军医学院,2015.
[5]CHEN QQ,WAN J,YAN L,et al.Mesenchymal stem cells alleviate TNBS-induced colitis by modulating inflammatory and autoimmune responses[J].World J Gastroenterol,2013,19(29):4702-4717.
[6]ANSBORO S,GREISER U,BARRY F,et al.Strategies for improved targeting of therapeutic cells:implications for tissue repair[J].Eur Cell Mater,2012,23:310-318.
[7]XINARIS C,MORIGI M,BENEDETTI V,et al.A novel strategy to enhance mesenchymal stem cell migration capacity and promote tissue repair in an injury specific fashion[J].Cell Transplant,2013,22(3):423-436.
[8]LI Q,YU P,WANG W,et al.Gene expression profiles of various cytokines in mesenchymal stem cells derived from umbilical cord tissue and bone marrow following infection with human cytomegalovirus[J].Cell Mol Biol Lett,2014;19(1):140-157.
[9]WANG CM,GUO Z,XIE YJ,et al.Co-treating mesenchymal stem cells with IL-1βand TNF-αincreases VCAM-1 expression and improves post-ischemic myocardial function[J].Mol Med Rep,2014,10(2):792-798.
[10]GHOBADI A,RETTIG MP,COOPER ML,et al.Bortezomib is a rapid mobilizer of hematopoietic stem cells in mice via modulation of the VCAM-1/VLA-4 axis[J].Blood,2014,124(17):2752-2754.
[11]KAUNDAL U,BAGAI U,RAKHA A.Immunomodulatory plasticity of mesenchymal stem cells:a potential key to successful solid organ transplantation[J].J Transl Med,2018,16(1):31.
[12]SHERIFF L,ALANAZI A,WARD LSC,et al.Origin-specific adhesive interactions of mesenchymal stem cells with platelets influence their behavior after infusion[J].Stem Cells,2018,36(7):1062-1074.
[13]CORRADETTI B,TARABALLI F,MARTINEZ JO,et al.Hyaluronic acid coatings as a simple and efficient approach to improve MSChoming toward the site of inflammation[J].Sci Rep,2017,7(1):7991.
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