兔髓核间充质干细胞与髓核细胞非接触式共培养的生物学效应
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摘要
[目的]通过非接触式共培养探索兔髓核间充质干细胞(NPMSCs)与兔髓核细胞(NPCs)的间接生物学效应。[方法]将第3代兔来源NPMSCs与NPCs进行非接触共培养,分设三组:NPCs/NPCs自身共培养对照组,NPMSCs/NPMSCs自身共培养对照组,NPMSCs/NPCs共培养组。分别在共培养3、5、7 d后,比较两种细胞增殖情况,ELISA检测上清液中转化生长因子β1(TGF-β1)、胰岛素样生长因子(IGF)的含量;采用RT-PCR检测共培养7 d后NPMSCs与NPCs的Col IIα1、AGG基因表达变化;免疫荧光染色检测NPMSCs中Col II的蛋白表达情况。[结果]在共培养第5 d和第7 d,共培养组NPCs的细胞数量均高于自身对照组(P<0.05),在第7d,共培养组NPMSCs的细胞数量均高于自身对照组;在共培养3、5、7 d后,共培养组上清液TGF-β1、IGF的含量均高于自身对照组(P<0.05);在培养7 d后,共培养组NPMSCs中Col II免疫荧光强度高于自身对照组,NPCs与NPMSCs的Col IIα1、AGG基因的表达较自身对照组显著升高(P<0.05)。[结论] NPMSCs与NPCs非接触共培养可以促进细胞因子分泌、细胞增殖、基质合成与分泌。
[Objective] To explore the indirect biological effects of non-contact coculture between rabbit-derived nucleus pulposus mesenchymal stem cells(NPMSCs) and rabbit nucleus pulposus cells(NPCs). [Method] The P3 generation rabbit-derived NPMSCs were co-cultured with NPCs by non-contacted ways, which were divided into three groups: NPCs/NPCs, NPMSCs/NPMSCs and NPMSCs/NPCs co-culture group. After 3, 5, and 7 days of co-culture, proliferation capacity of the two type of cells was tested. The content of transforming growth factor β1(TGF-β1) and insulin-like growth factor(IGF) in the supernatant was detected by ELISA. In addition, the gene expressions of Col IIα1 and AGG in NPMSCs and NPCs 7 days after co-culture were detected by using RT-PCR, while the protein expression of Col II in NPMSCs was assessed by immunofluorescence staining. [Results] At 5 and 7 days after coculture, the number of NPCs in the co-culture group was significantly higher than that in other control groups(P<0.05). Similarly, at 7 days of coculture, the number of NPMSCs in the coculture group was higher than that in other control groups. In addition, the content of TGF-β1 and IGF in the supernatant of the co-culture group was significantly higher than those in other two control groups at 3, 5 and 7 days(P<0.05). At 7 days, the intensity immunofluorescence of Col II was higher than that of control group, whereas the gene expression of Col IIα1 and AGG in NPCs and NPMSCs was significantly higher than that in control group(P<0.05). [Conclusion] Non-contact coculture of NPMSCs with NPCs do promote cytokine secretion, cell proliferation, matrix synthesis and secretion.
[Objective] To explore the indirect biological effects of non-contact coculture between rabbit-derived nucleus pulposus mesenchymal stem cells(NPMSCs) and rabbit nucleus pulposus cells(NPCs). [Method] The P3 generation rabbit-derived NPMSCs were co-cultured with NPCs by non-contacted ways, which were divided into three groups: NPCs/NPCs, NPMSCs/NPMSCs and NPMSCs/NPCs co-culture group. After 3, 5, and 7 days of co-culture, proliferation capacity of the two type of cells was tested. The content of transforming growth factor β1(TGF-β1) and insulin-like growth factor(IGF) in the supernatant was detected by ELISA. In addition, the gene expressions of Col IIα1 and AGG in NPMSCs and NPCs 7 days after co-culture were detected by using RT-PCR, while the protein expression of Col II in NPMSCs was assessed by immunofluorescence staining. [Results] At 5 and 7 days after coculture, the number of NPCs in the co-culture group was significantly higher than that in other control groups(P<0.05). Similarly, at 7 days of coculture, the number of NPMSCs in the coculture group was higher than that in other control groups. In addition, the content of TGF-β1 and IGF in the supernatant of the co-culture group was significantly higher than those in other two control groups at 3, 5 and 7 days(P<0.05). At 7 days, the intensity immunofluorescence of Col II was higher than that of control group, whereas the gene expression of Col IIα1 and AGG in NPCs and NPMSCs was significantly higher than that in control group(P<0.05). [Conclusion] Non-contact coculture of NPMSCs with NPCs do promote cytokine secretion, cell proliferation, matrix synthesis and secretion.
引文
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[16]Chen X,Zhu L,Wu G,et al.A comparison between nucleus pulposus-derived stem cell transplantation and nucleus pulposus cell transplantation for the treatment of intervertebral disc degeneration in a rabbit model[J].Int j Surg(London,England),2016,28:77-82.
[17]Shen Q,Zhang L,Chai B,et al.Isolation and characterization of mesenchymal stem-like cells from human nucleus pulposus tissue[J].Sci China Life Sci,2015,58(5):509-511.
[18]Tao Y,Zhou X,Liang C,et al.TGF-beta3 and IGF-1 synergy ameliorates nucleus pulposus mesenchymal stem cell differentiation towards the nucleus pulposus cell type through MAPK/ERK signaling[J].Growth Factors,2015:1-11.
[19]Liu S,Liang H,Lee S,et al.Isolation and identification of stem cells from degenerated human intervertebral discs and their migration characteristics[J].Acta Bioch Biophysica Sinica,2016,49(2):101-109.
[20]Tao YQ,Liang CZ,Li H,et al.Potential of co-culture of nucleus pulposus mesenchymal stem cells and nucleus pulposus cells in hyperosmotic microenvironment for intervertebral disc regeneration[J].Cell Biol Int,2013,37(8):826-834.
[21]Abilez OJ,Wu JC.Stem cell isolation:Differential stickiness[J].Nat Mater,2013,12(6):474-476.
[22]Sakai D,Nakamura Y,Nakai T,et al.Exhaustion of nucleus pulposus progenitor cells with ageing and degeneration of the intervertebral disc[J].Nature Communications,2012,3:1264.
[23]Jia Z,Yang P,Wu Y,et al.Comparison of biological characteristics of nucleus pulposus mesenchymal stem cells derived from nondegenerative and degenerative human nucleus pulposus[J].Exp Ther Med,2017,13(6):3574-3580.
[24]Johnstone B,Bayliss MT.The large proteoglycans of the human intervertebral disc.Changes in their biosynthesis and structure with age,topography,and pathology[J].Spine.1995,20(6):674-684.2018-09-21
[2]Grad S,Peroglio M,Li Z,et al.Endogenous cell homing for intervertebral disk regeneration[J].J Am Acad Orthop Surg,2015,23(4):264-266.
[3]Le Maitre CL,Baird P,Freemont AJ,et al.An in vitro study investigating the survival and phenotype of mesenchymal stem cells following injection into nucleus pulposus tissue[J].Arthritis Res Ther,2009,11(1):R20.
[4]Feng G,Zhao X,Liu H,et al.Transplantation of mesenchymal stem cells and nucleus pulposus cells in a degenerative disc model in rabbits:a comparison of 2 cell types as potential candidates for disc regeneration[J].J Neurosurg Spine,2011,14(3):322-329.
[5]Wei A,Tao H,Chung SA,et al.The fate of transplanted xenogeneic bone marrow-derived stem cells in rat intervertebral discs[J].JOrthop Res,2009,27(3):374-379.
[6]Chen X,Zhu L,Wu G,et al.A comparison between nucleus pulposus-derived stem cell transplantation and nucleus pulposus cell transplantation for the treatment of intervertebral disc degeneration in a rabbit model[J].Int J Surg,2016,28:77-82.
[7]Wei A,Chung SA,Tao H,et al.Differentiation of rodent bone marrow mesenchymal stem cells into intervertebral disc-like cells following coculture with rat disc tissue[J].Tissue Engine Part A,2009,15(9):2581-2595.
[8]Lehmann TP,Filipiak K,Juzwa W,et al.Coculture of human nucleus pulposus cells with multipotent mesenchymal stromal cells from human bone marrow reveals formation of tunnelling nanotubes[J].Molecular Med Rep,2014,9(2):574-582.
[9]Pierantozzi E,Badin M,Vezzani B,et al.Human pericytes isolated from adipose tissue have better differentiation abilities than their mesenchymal stem cell counterparts[J].Cell Tissue Res,2015,361(3):769-778.
[10]Yi Z,Guanjun T,Lin C,et al.Effects of transplantation of htimp1-expressing bone marrow mesenchymal stem cells on the extracellular matrix of degenerative intervertebral discs in an in vivo rabbit model[J].Spine,2014,Epub ahead of print.
[11]Pettine K,Suzuki R,Sand T,et al.Treatment of discogenic back pain with autologous bone marrow concentrate injection with minimum two year follow-up[J].Int Orthop,2015,40(1):135-140.
[12]Serigano K,Sakai D,Hiyama A,et al.Effect of cell number on mesenchymal stem cell transplantation in a canine disc degeneration model[J].J Orthop Res,2010,28(10):1267-1275.
[13]Liu K,Chen Z,Luo XW,et al.Determination of the potential of induced pluripotent stem cells to differentiate into mouse nucleus pulposus cells in vitro[J].Genetics Molecular Res,2015,14(4):12394-12405.
[14]Zhang Y,Chee A,Shi P,et al.Allogeneic articular chondrocyte transplantation downregulates interleukin 8 gene expression in the degenerating rabbit intervertebral disk in vivo[J].Am J Physical Med Rehabili,2015,94(7):530-538.
[15]Han B,Wang HC,Li H,et al.Nucleus pulposus mesenchymal stem cells in acidic conditions mimicking degenerative intervertebral discs give better performance than adipose tissue-derived mesenchymal stem cells[J].Cells Tissues Organs,2014,199(5-6):342-352.
[16]Chen X,Zhu L,Wu G,et al.A comparison between nucleus pulposus-derived stem cell transplantation and nucleus pulposus cell transplantation for the treatment of intervertebral disc degeneration in a rabbit model[J].Int j Surg(London,England),2016,28:77-82.
[17]Shen Q,Zhang L,Chai B,et al.Isolation and characterization of mesenchymal stem-like cells from human nucleus pulposus tissue[J].Sci China Life Sci,2015,58(5):509-511.
[18]Tao Y,Zhou X,Liang C,et al.TGF-beta3 and IGF-1 synergy ameliorates nucleus pulposus mesenchymal stem cell differentiation towards the nucleus pulposus cell type through MAPK/ERK signaling[J].Growth Factors,2015:1-11.
[19]Liu S,Liang H,Lee S,et al.Isolation and identification of stem cells from degenerated human intervertebral discs and their migration characteristics[J].Acta Bioch Biophysica Sinica,2016,49(2):101-109.
[20]Tao YQ,Liang CZ,Li H,et al.Potential of co-culture of nucleus pulposus mesenchymal stem cells and nucleus pulposus cells in hyperosmotic microenvironment for intervertebral disc regeneration[J].Cell Biol Int,2013,37(8):826-834.
[21]Abilez OJ,Wu JC.Stem cell isolation:Differential stickiness[J].Nat Mater,2013,12(6):474-476.
[22]Sakai D,Nakamura Y,Nakai T,et al.Exhaustion of nucleus pulposus progenitor cells with ageing and degeneration of the intervertebral disc[J].Nature Communications,2012,3:1264.
[23]Jia Z,Yang P,Wu Y,et al.Comparison of biological characteristics of nucleus pulposus mesenchymal stem cells derived from nondegenerative and degenerative human nucleus pulposus[J].Exp Ther Med,2017,13(6):3574-3580.
[24]Johnstone B,Bayliss MT.The large proteoglycans of the human intervertebral disc.Changes in their biosynthesis and structure with age,topography,and pathology[J].Spine.1995,20(6):674-684.2018-09-21