鞘氨醇激酶1修饰的脂肪来源间充质干细胞对组织工程化骨成骨效果的影响
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摘要
目的研究鞘氨醇激酶1(sphingosine kinase 1,SPK1)修饰的脂肪来源间充质干细胞(adipose tissue-derived stromal cells,ADSC)对组织工程化骨成骨能力的影响。方法将从SD大鼠脂肪细胞中分离提取的ADSC分为对照组(CON组)与实验组(SPK1组),分别感染10 MOI的CON和SPK1慢病毒,在感染病毒14 d后,分别使用茜素红和油红O染色,检测A_(595 nm)和A_(490 nm)以判定成骨、成脂能力,检测两组成骨细胞碱性磷酸酶(alkaline phosphatase,ALP)活性变化。构建SD大鼠股骨缺损模型,在CON与SPK1两组ADSC与β磷酸三钙陶瓷(β-TCP)结合后,将组织工程骨填压至缺损处,4、6周后X线检测大鼠骨缺损修复情况,Western blot检测SPK1、骨形态发生蛋白7(bone morphogenetic protein,BMP7)表达变化。结果流式细胞仪检测CON和SPK1慢病毒感染效率分别为94.4%、94.9%;CON和SPK1组SPK1蛋白相对表达量分别为(0.73±0.10)vs.(1.29±0.17),P<0.05;CON和SPK1组A_(595 nm)分别为(0.20±0.02)vs.(0.41±0.01),A_(490 nm)分别为(0.72±0.01)vs.(0.51±0.02),均P<0.05。CON与SPK1的ALP活性分别为(1.42±0.09)vs.(2.68±0.09),P<0.01。在骨缺损修复中,第4周SPK1组大鼠骨缺损处形成的高密度组织面积显著大于CON组,在第6周时SPK1组大鼠骨缺损治愈,而CON组则尚有缺损。CON和SPK1组在感染病毒48 h后BMP7相对表达量分别为(1.13±0.16)vs.(4.46±0.23),P<0.05。结论 SPK1修饰的ADSC体外、体内成骨能力增强,其机制可能与BMP7激活相关。
Objective To investigate the impact of sphmgosme kinase 1(SPKl) modified adipose tissue-derived stromal cells(ADSC)on tissue engineered bone osteogenesis.Methods ADSC cells isolated from SD rat fat cells were divided into CON group and SPK1 group,and then the cells were respectively infected with 10 MOI CON and SPK1 lentivirus for 48 h.The infection efficiency was confirmed by using flow cytometry.Alizarin red and oil red O was used to stain the cells 14 days after ADSC infection,and osteogenic and adipogenic ability was evaluated by detecting A_(595 nm) and A_(490 nm).In the meantime,the activity change of alkaline phosphatase(ALP)was detected.The SD rat femoral defect model was created,and then after combining ADSC with β-TCP,the tissue engineered bone was pressed to the defect site.The repairment of bone defect was detected by X-ray in 4,6 weeks.After infection of CON and SPK1 virus,bone morphogenetic protein(BMP7) expression in ADSC of these two groups was detected.Results The infection efficiency of CON and SPK1 lentivirus was 94.4%vs.94.9%respectively by flow cytometry.The SPK1 protein expression level in CON group and SPK1 group was(0.73±0.10) vs.(1.29±0.17)(P<0.05).The A value of CON and SPK1 group at 595 nm was(0.20±0.02) vs.(0.41±0.01)(P<0.05),respectively.The A value of CON and SPK1 group at 490 nm was(0.72±0.01) vs.(0.51±0.02)(P<0.05),respectively.The expression level of ALP in CON and SPK1 group was(1.42±0.09) vs.(2.68±0.09)(P<0.01),respectively.In the repairment of bone defect,high density tissue at rat bone defect was significantly larger in SPK1 group than in CON group in 4 weeks,and in 6 weeks,bone defect of SPK1 group was healed,but CON group still had defect The expression level of BMP7 in CON and SPK1 group was(1.13±0.16) vs.(4.46±0.23)(P<0.05) respectively,48 h after infection.Conclusion SPK1 modified ADSC has an enhanced osteogenesis ability in vitro and in vivo,which was related to the activation of BMP7.
Objective To investigate the impact of sphmgosme kinase 1(SPKl) modified adipose tissue-derived stromal cells(ADSC)on tissue engineered bone osteogenesis.Methods ADSC cells isolated from SD rat fat cells were divided into CON group and SPK1 group,and then the cells were respectively infected with 10 MOI CON and SPK1 lentivirus for 48 h.The infection efficiency was confirmed by using flow cytometry.Alizarin red and oil red O was used to stain the cells 14 days after ADSC infection,and osteogenic and adipogenic ability was evaluated by detecting A_(595 nm) and A_(490 nm).In the meantime,the activity change of alkaline phosphatase(ALP)was detected.The SD rat femoral defect model was created,and then after combining ADSC with β-TCP,the tissue engineered bone was pressed to the defect site.The repairment of bone defect was detected by X-ray in 4,6 weeks.After infection of CON and SPK1 virus,bone morphogenetic protein(BMP7) expression in ADSC of these two groups was detected.Results The infection efficiency of CON and SPK1 lentivirus was 94.4%vs.94.9%respectively by flow cytometry.The SPK1 protein expression level in CON group and SPK1 group was(0.73±0.10) vs.(1.29±0.17)(P<0.05).The A value of CON and SPK1 group at 595 nm was(0.20±0.02) vs.(0.41±0.01)(P<0.05),respectively.The A value of CON and SPK1 group at 490 nm was(0.72±0.01) vs.(0.51±0.02)(P<0.05),respectively.The expression level of ALP in CON and SPK1 group was(1.42±0.09) vs.(2.68±0.09)(P<0.01),respectively.In the repairment of bone defect,high density tissue at rat bone defect was significantly larger in SPK1 group than in CON group in 4 weeks,and in 6 weeks,bone defect of SPK1 group was healed,but CON group still had defect The expression level of BMP7 in CON and SPK1 group was(1.13±0.16) vs.(4.46±0.23)(P<0.05) respectively,48 h after infection.Conclusion SPK1 modified ADSC has an enhanced osteogenesis ability in vitro and in vivo,which was related to the activation of BMP7.
引文
[1]Yang Z,Jin L,Tao H,et al.Reconstruction of large tibial bone defects following osteosarcoma resection using bone transport distraction:A report of two cases[J].Oncol Lett,2016,12(2):1445-1447.
[2]Iviglia G,Cassinelli C,Torre E,et al.Novel bioceramic-reinforced hydrogel for alveolar bone regeneration[J].Acta Biomater,2016,44:97-109.
[3]Badieyan Z S,Berezhanskyy T,Utzinger M,et al.Transcriptactivated collagen matrix as sustained mRNA delivery system for bone regeneration[J].J Control Release,2016,239:137-148.
[4]Li S,Hu C,Li J,et al.Effect of miR-26a-5p on the Wnt/Ca2+pathway and osteogenic differentiation of mouse adipose-derived mesenchymal stem cells[J].Calcif Tissue Int,2016,99(2):174-186.
[5]王之发,翁雁鸣,刘彦普,等.兔骨髓干细胞和脂肪干细胞体外成骨能力的比较[J].口腔颌面外科杂志,2013,23(1):8-13.
[6]Yu H,Yuan L,Xu M,et al.Sphingosine kinase 1improves cutaneous wound healing in diabetic rats[J].Injury,2014,45(7):1054-1058.
[7]Yester J W,Bryan L,Waters M R,et al.Sphingosine-1-phosphate inhibits IL-1-induced expression of C-C motif ligand 5via c-Fos-dependent suppression of IFN-βamplification loop[J].Faseb J,2015,29(12):4853-4865.
[8]Park E S,Choi S,Shin B,et al.Tumor necrosis factor(TNF)receptor-associated factor(TRAF)-interacting protein(TRIP)negatively regulates the TRAF2ubiquitin-dependent pathway by suppressing the TRAF2-sphingosine 1-phosphate(S1P)interaction[J].J Biol Chem,2015,290(15):9660-9673.
[9]赛岩,代学强,白纪民,等.鞘氨醇激酶通过调控血管发生促进肝癌转移[J].中国医药生物技术,2012,7(3):171-176.
[10]Rose F R,Oreffo R O.Bone tissue engineering:hope vs hype[J].Biochem Biophys Res Commun,2002,292(1):1-7.
[11]Ho S S,Vollmer N L,Refaat M I,et al.Bone morphogenetic protein-2promotes human mesenchymal stem cell survival and resultant bone formation when entrapped in photocrosslinked alginate hydrogels[J].Adv Healthc Mater,2016,5(19):2501-2509.
[12]Lee D E,Kim J H,Choi S H,et al.The sphingosine-1-phosphate receptor 1binding molecule FTY720inhibits osteoclast formation in rats with ligature-induced periodontitis[J].J Periodontal Res,2016,doi:10.111/jre.12366.
[13]Zhang J N,Zhao Y,Liu C,et al.The role of the sphingosine-1-phosphate signaling pathway in osteocyte mechanotransduction[J].Bone,2015,79:71-78.
[14]Wong R W K,Rabie A B M.Early healing pattern of statininduced osteogenesis[J].Br J Oral Maxillofac Surg,2005,43(1):46-50.
[15]Lieberman J R,Daluiski A,Stevenson S,et al.The effect of regional gene therapy with bone morphogenetic protein-2-producing bone-marrow cells on the repair of segmental femoral defects in rats[J].J Bone Joint Surg Am,1999,81(7):905-917.
[16]Ma P X.Biomimetic materials for tissue engineering[J].Adv Drug Deliver Rev,2008,60(2):184-198.
[17]Sun P,Wang J,Zheng Y,et al.BMP2/7heterodimer is a stronger inducer of bone regeneration in peri-implant bone defects model than BMP2or BMP7homodimer[J].Dent Mater J,2012,31(2):239-248.
[2]Iviglia G,Cassinelli C,Torre E,et al.Novel bioceramic-reinforced hydrogel for alveolar bone regeneration[J].Acta Biomater,2016,44:97-109.
[3]Badieyan Z S,Berezhanskyy T,Utzinger M,et al.Transcriptactivated collagen matrix as sustained mRNA delivery system for bone regeneration[J].J Control Release,2016,239:137-148.
[4]Li S,Hu C,Li J,et al.Effect of miR-26a-5p on the Wnt/Ca2+pathway and osteogenic differentiation of mouse adipose-derived mesenchymal stem cells[J].Calcif Tissue Int,2016,99(2):174-186.
[5]王之发,翁雁鸣,刘彦普,等.兔骨髓干细胞和脂肪干细胞体外成骨能力的比较[J].口腔颌面外科杂志,2013,23(1):8-13.
[6]Yu H,Yuan L,Xu M,et al.Sphingosine kinase 1improves cutaneous wound healing in diabetic rats[J].Injury,2014,45(7):1054-1058.
[7]Yester J W,Bryan L,Waters M R,et al.Sphingosine-1-phosphate inhibits IL-1-induced expression of C-C motif ligand 5via c-Fos-dependent suppression of IFN-βamplification loop[J].Faseb J,2015,29(12):4853-4865.
[8]Park E S,Choi S,Shin B,et al.Tumor necrosis factor(TNF)receptor-associated factor(TRAF)-interacting protein(TRIP)negatively regulates the TRAF2ubiquitin-dependent pathway by suppressing the TRAF2-sphingosine 1-phosphate(S1P)interaction[J].J Biol Chem,2015,290(15):9660-9673.
[9]赛岩,代学强,白纪民,等.鞘氨醇激酶通过调控血管发生促进肝癌转移[J].中国医药生物技术,2012,7(3):171-176.
[10]Rose F R,Oreffo R O.Bone tissue engineering:hope vs hype[J].Biochem Biophys Res Commun,2002,292(1):1-7.
[11]Ho S S,Vollmer N L,Refaat M I,et al.Bone morphogenetic protein-2promotes human mesenchymal stem cell survival and resultant bone formation when entrapped in photocrosslinked alginate hydrogels[J].Adv Healthc Mater,2016,5(19):2501-2509.
[12]Lee D E,Kim J H,Choi S H,et al.The sphingosine-1-phosphate receptor 1binding molecule FTY720inhibits osteoclast formation in rats with ligature-induced periodontitis[J].J Periodontal Res,2016,doi:10.111/jre.12366.
[13]Zhang J N,Zhao Y,Liu C,et al.The role of the sphingosine-1-phosphate signaling pathway in osteocyte mechanotransduction[J].Bone,2015,79:71-78.
[14]Wong R W K,Rabie A B M.Early healing pattern of statininduced osteogenesis[J].Br J Oral Maxillofac Surg,2005,43(1):46-50.
[15]Lieberman J R,Daluiski A,Stevenson S,et al.The effect of regional gene therapy with bone morphogenetic protein-2-producing bone-marrow cells on the repair of segmental femoral defects in rats[J].J Bone Joint Surg Am,1999,81(7):905-917.
[16]Ma P X.Biomimetic materials for tissue engineering[J].Adv Drug Deliver Rev,2008,60(2):184-198.
[17]Sun P,Wang J,Zheng Y,et al.BMP2/7heterodimer is a stronger inducer of bone regeneration in peri-implant bone defects model than BMP2or BMP7homodimer[J].Dent Mater J,2012,31(2):239-248.