慢病毒介导TGF-β3转染兔脂肪间充质干细胞对其向成软骨细胞分化的影响
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摘要
目的探讨转化生长因子-β3((TGF-β)3)基因对脂肪间充质干细胞体向成软骨分化能力的影响。方法用含TGF-β3目的基因的慢病毒载体转染脂肪间充质干细胞,通过流式细胞术及共聚焦检测转染效率、细胞活性检测法(CCK-8)、荧光定量-PCR(RT-PCR)等实验方法进行成软骨分化相关内容的检测。结果转染96 h时感染复数MOI=50及MOI=100二者检测EGFP绿色荧光蛋白的表达无明显差异,TGF-β3慢病毒转染的脂肪间充质干细胞的长梭形细胞形态优于慢病毒空载体转染后脂肪间充质干细胞的形态。与空白组比较慢病毒转染后的细胞数量无明显减少。转染含有TGF-β3慢病毒的脂肪间充质干细胞从基因水平可合成更多的成软骨分化相关的聚蛋白多糖(Aggrecan)、Ⅱ型胶原(COL2A1)、性别决定区Y框蛋白(SOX-9)明显高于空白组(P<0.05),形成更多的细胞外基质。结论 TGF-β3目的基因由慢病毒载体导入细胞内,是诱导ADSCs成软骨分化的理想的途径,可作为软骨组织工程的种子细胞来源。
Objective To investigate the effect of transforming growth factor-β3 gene on the differentiation of adipose-derived mesenchymal stem cells into chondrocytes. Methods Adipose-derived mesenchymal stem cells were transfected with lentiviral vector containing transforming growth factor-β3 gene, and transfection efficiency was detected by Flow cytometry and Confocal microscopy, CCK-8, RT-PCR and other related indicators of chondrogenic differentiation. Results There was no significant difference in the expression of EGFP green fluorescent protein between the multiplicity of infections of MOI=50 and MOI=100 at 96 h after transfection. The morphology of long spindle cells of transfected growth factor-β3 lentivirus-transfected adipose-derived mesenchymal stem cells was superior. Morphology of adipose-derived mesenchymal stem cells after transfection of lentiviral vector. Compared with the blank group, the number of cells after lentivirus transfection was not significantly reduced. Transfected adipose-derived mesenchymal stem cells containing transforming growth factor-β3 lentivirus can synthesize more cartilage-differentiated Aggrecan, SOX-9 and COL2 at the gene levels, which was significantly higher than those of the blank group(P<0.05), and more extracellular matrix was formed. Conclusion The TGF-β3 gene is introduced into cells by lentiviral vector, which is an ideal pathway for inducing chondrogenic differentiation of ADSCs, and can be used as a seed cell source for cartilage tissue engineering.
Objective To investigate the effect of transforming growth factor-β3 gene on the differentiation of adipose-derived mesenchymal stem cells into chondrocytes. Methods Adipose-derived mesenchymal stem cells were transfected with lentiviral vector containing transforming growth factor-β3 gene, and transfection efficiency was detected by Flow cytometry and Confocal microscopy, CCK-8, RT-PCR and other related indicators of chondrogenic differentiation. Results There was no significant difference in the expression of EGFP green fluorescent protein between the multiplicity of infections of MOI=50 and MOI=100 at 96 h after transfection. The morphology of long spindle cells of transfected growth factor-β3 lentivirus-transfected adipose-derived mesenchymal stem cells was superior. Morphology of adipose-derived mesenchymal stem cells after transfection of lentiviral vector. Compared with the blank group, the number of cells after lentivirus transfection was not significantly reduced. Transfected adipose-derived mesenchymal stem cells containing transforming growth factor-β3 lentivirus can synthesize more cartilage-differentiated Aggrecan, SOX-9 and COL2 at the gene levels, which was significantly higher than those of the blank group(P<0.05), and more extracellular matrix was formed. Conclusion The TGF-β3 gene is introduced into cells by lentiviral vector, which is an ideal pathway for inducing chondrogenic differentiation of ADSCs, and can be used as a seed cell source for cartilage tissue engineering.
引文
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[4] NESTI L J,CATERSON E J,WANG M,et al.TGF-β1-stimulated osteoblasts require intracellular calcium signaling for enhanced α5integrin expression[J].Ann N Y Acad Sc,2002,961(1):178-182.
[5] 张冲,李莉.TGF-β1对大鼠肩袖损伤修复术后腱-骨愈合的影响[J].医用生物力学,2016,31(2):167-170.
[6] DAHLIN R L,NI M,MERETOJA V V,et al.TGF-β3-induced chondrogenesis in co-cultures of chondrocytes and mesenchymal stem cells on biodegradable scaffolds[J].Biomaterials,2014,35(1):123-132.
[7] WANKHADE U D,SHEN M,KOLHE R,et al.Advances in adipose-derived sem cells isolation,characterization,and application in regenerative tissue engineering[J].Stem Cells Int,2016,3206807.
[8] REZAI RAD M,BOHLOLI M,AKHAVAN RAHNAMA M,et al.Impact of tissue harvesting sites on the cellular behaviors of adipose-derived stem cells:implication for bone tissue engineering.[J].Stem Cells Int,2017:2156478.
[9] Stromps J P,Paul NE,Rath B,et al.Chondrogenic differentiation of human adipose-derived stem cells:a new path in articularcartilage defect management?[J].BioMed Res Int,2014,740926.
[10] PICANCO-CASTRO V,RUSSO-CARBOLANTE E M,COVAS D T.Advances in lentiviral vectors:a patent review[J].Recent Pat DNA Gene Seq,2012,6(2):82-90.
[11] LIU P,SUN L,CHEN H,et al.Lentiviral-mediated multiple gene transfer to chondrocytes promotes chondrocyte differentiation and bone formation in rabbit bone marrow-derived mesenchymal stem cells[J].Oncology Reports,2015,34(5):2618.
[12] SEGURA M M,MANGION M,GAILLET B.New developments in lentiviral vector design,production and purification[J].Expert Opin Biol Ther,2013,13(7):987-1011.
[13] GAO L,SHEU TJ,DONG Y,et al.TAK1 regulates Sox9 expression in chondrocytes and is essential forpostnatal development of the growth plate and articular cartilages[J].Cell Sci,2013,126:5704-5713.