不同培养体系中施万细胞对BMSCs分化及分泌功能的影响
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摘要
目的观察不同培养体系中施万细胞(Schwann cells, SCs)对骨髓基质干细胞(bone marrow stromal cells, BMSCs)分化以及分泌功能的影响。方法从预变性SD大鼠坐骨神经中分离纯化SCs,采用全骨髓贴壁法分离培养SD大鼠BMSCs,均行免疫荧光鉴定。根据培养体系不同将实验分为3组:A组,SCs与PKH26标记的BMSCs在Transwell中接触共培养组;B组,SCs与PKH26标记的BMSCs在Transwell中非接触培养组;C组,PKH26标记的BMSCs单独培养组。采用Western blot检测各组S-100蛋白表达,应用ELISA法检测各组上清液中神经营养因子3(neurotrophins-3, NT-3)的含量。结果成功分离并培养SCs和BMSCs,免疫荧光鉴定SCs呈S-100阳性表达,BMSCs呈CD44和CD90阳性表达;Western blot检测显示,A组S-100蛋白表达水平明显高于B、C组(P<0.05),B组S-100蛋白表达水平高于C组(P<0.05);ELISA检测显示,A组上清液中NT-3含量显著高于B、C组,且呈现时间依赖性增高(P<0.05)。结论在接触共培养体系中,SCs能够有效诱导BMSCs向外周胶质细胞分化,并增强BMSCs的分泌功能。
Objective To explore the effects of Schwann cells(SCs) on differentiation and secretion of bone marrow stromal cells(BMSCs) in different culture systems. Methods SCs were extracted and puri?ed from the injured distal sciatic nerves of Sprague-Dawiey(SD) rats. BMSCs were isolated from bone marrow of SD rats and cultured in vitro. The cells were identi?ed by immuno?uorescent staining. According to the different culture systems, the experiments were divided into three groups: group A, PKH26-labeled BMSCs were co-cultured with SCs on the lower layer of transwell; group B, PKH26-labeled BMSCs and SCs were cultured on the upper and lower layers of transwell;group C, PKH26-labeled BMSCs were cultured on the lower layers of transwell. The protein expression of S-100 in each group was detected by Western blot. The content of neurotrophins-3(NT-3) in the supernatant of each group was detected by ELISA. Results SCs and BMSCs were isolated and cultured successfully. The identi?cation of SCs showed positive expression of S-100 and BMSCs showed positive expressions of CD44 and CD90. Western blot analysis showed that the expression level of S-100 in group A was significantly higher than that in the group B and C(P<0.05),and the level of S-100 in group B was higher than that in group C(P<0.05). ELISA showed that the content of NT-3 in supernatant of group A was significantly higher than that in the group B and C, and showed time-dependent increase(P<0.05). Conclusion In the contact co-culture system, SCs can effectively induce BMSCs differentiation into peripheral glial cells and enhance BMSCs secretory function.
Objective To explore the effects of Schwann cells(SCs) on differentiation and secretion of bone marrow stromal cells(BMSCs) in different culture systems. Methods SCs were extracted and puri?ed from the injured distal sciatic nerves of Sprague-Dawiey(SD) rats. BMSCs were isolated from bone marrow of SD rats and cultured in vitro. The cells were identi?ed by immuno?uorescent staining. According to the different culture systems, the experiments were divided into three groups: group A, PKH26-labeled BMSCs were co-cultured with SCs on the lower layer of transwell; group B, PKH26-labeled BMSCs and SCs were cultured on the upper and lower layers of transwell;group C, PKH26-labeled BMSCs were cultured on the lower layers of transwell. The protein expression of S-100 in each group was detected by Western blot. The content of neurotrophins-3(NT-3) in the supernatant of each group was detected by ELISA. Results SCs and BMSCs were isolated and cultured successfully. The identi?cation of SCs showed positive expression of S-100 and BMSCs showed positive expressions of CD44 and CD90. Western blot analysis showed that the expression level of S-100 in group A was significantly higher than that in the group B and C(P<0.05),and the level of S-100 in group B was higher than that in group C(P<0.05). ELISA showed that the content of NT-3 in supernatant of group A was significantly higher than that in the group B and C, and showed time-dependent increase(P<0.05). Conclusion In the contact co-culture system, SCs can effectively induce BMSCs differentiation into peripheral glial cells and enhance BMSCs secretory function.
引文
[1]Scherer SS:The biology and pathobiology of schwann cells.Current opinion in neurology 1997,10:386-397.
[2]Kitada M,Murakami T,Wakao S,et al.Direct conversion of adult human skin fibroblasts into functional schwann cells that achieve robust recovery of the severed peripheral nerve in rats[J].Glia,2019.
[3]Zhang J,Zhao F,Wu G,et al.Functional and histologica improvement of the injured spinal cord following transplantation of schwann cells transfected with nrg1gene[J].Anatomical record,2010,293:1933-1946.
[4]Zhang JF,Zhao FS,Wu G,et al.Therapeutic effect of cotransplantation of neuregulin-1-transfected schwann cells and bone marrow stromal cells on spinal cord hemisection syndrome[J].Neuroscience letters,2011,497:128-133.
[5]Zolocinska A.The expression of marker genes during the differentiation of mesenchymal stromal cells[J].Advances in clinical and experimental medicine:official organ Wroclaw Medical University,2018,27:717-723.
[6]Polymeri A,Giannobile WV,Kaigler D.Bone marrow stromal stem cells in tissue engineering and regenerative medicine[J].Hormone and metabolic research=Hormon-und Stoffwechselforschung=Hormones et metabolisme,2016,48:700-713.
[7]赵富生,武庚,武杨,等.雪旺细胞对不同年龄大鼠BMSCs分化的影响[J].中国修复重建外科杂志,2011,25(2):160-165.
[8]Robinson LR.Predicting recovery from peripheral nerve trauma[J].Physical medicine and rehabilitation clinics of North America,2018,29:721-733.
[9]Ji WC,Zhang XW,Qiu YS.Selected suitable seed cell,scaffold and growth factor could maximize the repair effect using tissue engineering method in spinal cord injury[J].World journal of experimental medicine,2016,6:58-62.
[10]Faroni A,Mobasseri SA,Kingham PJ,et al.Periphera nerve regeneration:Experimental strategies and future perspectives[J].Advanced drug delivery reviews,2015,82-83:160-167.
[11]Li H,Ghazanfari R,Zacharaki D,et al.Isolation and characterization of primary bone marrow mesenchyma stromal cells[J].Annals of the New York Academy of Sciences,2016,1370:109-118.
[12]Berebichez-Fridman R,Montero-Olvera PR.Sources and clinical applications of mesenchymal stem cells:State-ofthe-art review[J].Sultan Qaboos University medical journal,2018,18:e264-e277.
[13]Shimizu S,Kitada M,Ishikawa H,et al.Peripheral nerve regeneration by the in vitro differentiated-human bone marrow stromal cells with schwann cell property[J].Biochemical and biophysical research communicatio ns,2007,359:915-920.
[14]Bianco P,Riminucci M,Gronthos S,et al.Bone marrow stromal stem cells:Nature,biology,and potentia applications[J].Stem cells,2001,19:180-192.
[15]陈欢意,牛平,赵帅,等.碱性成纤维细胞生长因子预诱导对骨髓基质干细胞向多巴胺能神经元分化的影响[J].临床神经病学杂志,2017,20(4):274-277.
[16]杨琴,杨军,谢鹏,等.刺五加体外诱导骨髓基质细胞分化为神经元样细胞的实验研究[J].南方医科大学学报,2009,29(3):487-493.
[17]黄家贵,沈长波,刘舒,等.白藜芦醇诱导大鼠骨髓基质细胞的神经元样细胞分化伴Shh信号激[J].第三军医大学学报,2013,35(4):280-283.
[18]贾晓旭,朱金墙,王硕,等.神经营养因子在神经损伤中的作用及其中药调节[J].天津中医药大学学报,2016,35(5):356-360.
[19]Keefe KM,Sheikh IS,Smith GM.Targeting neurotrophins to specific populations of neurons:Ngf,bdnf,and nt-3and their relevance for treatment of spinal cord injury[J].International journal of molecular sciences,2017,18.
[20]Luo XY,Meng XJ,Cao DC,et al.Transplantation of bone marrow mesenchymal stromal cells attenuates liver fibrosis in mice by regulating macrophage subtypes[J].Stem cel research&therapy,2019,10:16.
[21]Mung KL,Tsui YP,Tai EW,et al.Rapid and efficient generation of neural progenitors from adult bone marrow stromal cells by hypoxic preconditioning[J].Stem cel research&therapy 2016,7:146.
[2]Kitada M,Murakami T,Wakao S,et al.Direct conversion of adult human skin fibroblasts into functional schwann cells that achieve robust recovery of the severed peripheral nerve in rats[J].Glia,2019.
[3]Zhang J,Zhao F,Wu G,et al.Functional and histologica improvement of the injured spinal cord following transplantation of schwann cells transfected with nrg1gene[J].Anatomical record,2010,293:1933-1946.
[4]Zhang JF,Zhao FS,Wu G,et al.Therapeutic effect of cotransplantation of neuregulin-1-transfected schwann cells and bone marrow stromal cells on spinal cord hemisection syndrome[J].Neuroscience letters,2011,497:128-133.
[5]Zolocinska A.The expression of marker genes during the differentiation of mesenchymal stromal cells[J].Advances in clinical and experimental medicine:official organ Wroclaw Medical University,2018,27:717-723.
[6]Polymeri A,Giannobile WV,Kaigler D.Bone marrow stromal stem cells in tissue engineering and regenerative medicine[J].Hormone and metabolic research=Hormon-und Stoffwechselforschung=Hormones et metabolisme,2016,48:700-713.
[7]赵富生,武庚,武杨,等.雪旺细胞对不同年龄大鼠BMSCs分化的影响[J].中国修复重建外科杂志,2011,25(2):160-165.
[8]Robinson LR.Predicting recovery from peripheral nerve trauma[J].Physical medicine and rehabilitation clinics of North America,2018,29:721-733.
[9]Ji WC,Zhang XW,Qiu YS.Selected suitable seed cell,scaffold and growth factor could maximize the repair effect using tissue engineering method in spinal cord injury[J].World journal of experimental medicine,2016,6:58-62.
[10]Faroni A,Mobasseri SA,Kingham PJ,et al.Periphera nerve regeneration:Experimental strategies and future perspectives[J].Advanced drug delivery reviews,2015,82-83:160-167.
[11]Li H,Ghazanfari R,Zacharaki D,et al.Isolation and characterization of primary bone marrow mesenchyma stromal cells[J].Annals of the New York Academy of Sciences,2016,1370:109-118.
[12]Berebichez-Fridman R,Montero-Olvera PR.Sources and clinical applications of mesenchymal stem cells:State-ofthe-art review[J].Sultan Qaboos University medical journal,2018,18:e264-e277.
[13]Shimizu S,Kitada M,Ishikawa H,et al.Peripheral nerve regeneration by the in vitro differentiated-human bone marrow stromal cells with schwann cell property[J].Biochemical and biophysical research communicatio ns,2007,359:915-920.
[14]Bianco P,Riminucci M,Gronthos S,et al.Bone marrow stromal stem cells:Nature,biology,and potentia applications[J].Stem cells,2001,19:180-192.
[15]陈欢意,牛平,赵帅,等.碱性成纤维细胞生长因子预诱导对骨髓基质干细胞向多巴胺能神经元分化的影响[J].临床神经病学杂志,2017,20(4):274-277.
[16]杨琴,杨军,谢鹏,等.刺五加体外诱导骨髓基质细胞分化为神经元样细胞的实验研究[J].南方医科大学学报,2009,29(3):487-493.
[17]黄家贵,沈长波,刘舒,等.白藜芦醇诱导大鼠骨髓基质细胞的神经元样细胞分化伴Shh信号激[J].第三军医大学学报,2013,35(4):280-283.
[18]贾晓旭,朱金墙,王硕,等.神经营养因子在神经损伤中的作用及其中药调节[J].天津中医药大学学报,2016,35(5):356-360.
[19]Keefe KM,Sheikh IS,Smith GM.Targeting neurotrophins to specific populations of neurons:Ngf,bdnf,and nt-3and their relevance for treatment of spinal cord injury[J].International journal of molecular sciences,2017,18.
[20]Luo XY,Meng XJ,Cao DC,et al.Transplantation of bone marrow mesenchymal stromal cells attenuates liver fibrosis in mice by regulating macrophage subtypes[J].Stem cel research&therapy,2019,10:16.
[21]Mung KL,Tsui YP,Tai EW,et al.Rapid and efficient generation of neural progenitors from adult bone marrow stromal cells by hypoxic preconditioning[J].Stem cel research&therapy 2016,7:146.