兔膀胱脱细胞基质对人骨髓间充质干细胞增殖、分化的影响
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摘要
目的研究兔膀胱脱细胞基质(BAMG)对人骨髓间充质干细胞(hBMSCs)增殖及分化能力的影响,并探讨其构建膀胱修复材料的可行性。方法采用正常人骨髓体外分离培养原代hBMSCs。用兔膀胱构建BAMG,制备BAMG浸泡液培养基,并以BAMG浸泡液培养基为基础配制成骨、成脂、成平滑肌分化的诱导剂。将hBMSCs分别用正常培养液、正常诱导液、BAMG浸泡液及BAMG诱导液培养。采用苏木素-伊红(HE)与Masson三色染色法进行组织形态鉴定。采用流式细胞术及活细胞荧光染料羧基荧光素二醋酸盐琥珀酰亚胺酯(CFSE)标记法检测BAMG浸泡液培养基对hBMSCs的生长毒性;分别观察以BAMG浸泡液为基础的诱导剂对hBMSCs成骨、成脂、成平滑肌的分化能力的影响。结果成功构建BAMG标本,与未处理的膀胱组织相比,已去除绝大多数细胞核,组织结构更加疏松、多孔。BAMG浸泡液与正常培养基对hBMSCs增殖无差异。诱导后的hBMSCs仍能高效分化为成骨、成脂、成平滑肌细胞。结论 BAMG不影响hBMSCs的增殖和分化能力,可作为支架材料,应用于膀胱组织工程中。
Objective To investigate the influence of rabbit bladder acellular matrix graft( BAMG) on proliferation and differentiation of human bone marrow mesenchymal stem cells( hBMSCs) and explore the feasibility of building bladder repair materials by it. Methods Primary hBMSCs were isolated and cultured with normal human bone marrow in vitro.BAMG was built with rabbit bladder. BAMG soaking solution culture medium was established,and the inducers of differentiation into osteoblasts,adipoblasts and smooth muscle were prepared based on the BAMG soaking solution. The hBMSCs were respectively cultured in normal culture medium,normal inducers culture medium,BAMG soaking solution culture medium and BAMG inducers solution. Hematoxylin-eosin( HE) and Masson tricolor staining were used to identify the tissue morphology. The Flow cytometry and living cell fluorescent dye-carboxyl fluorescein diacetate( CFSE)-labeling method were used to detect the growth toxicity of BAMG soaking solution culture medium on hBMSCs. The effects of inducers based on the BAMG soaking solution culture medium on the abilities of differentiation into osteoblasts,adipoblasts and smooth muscle were respectively observed. Results BAMG specimen was constructed successfully. Compared with notreatment bladder tissues,most of the BAMG cell nuclei were removed,and its tissue structure was more loose and porous.There was no significant difference in proliferation of hBMSCs for BAMG soaking solution culture medium and normal culture medium. The hBMSCs after induction can still be efficiently differentiated into osteoblasts,adipoblasts and smooth muscle. Conclusion BAMG does not affect the abilities of proliferation and differentiation of hBMSCs,so it can be used as a scaffold material in the application of bladder tissue engineering.
Objective To investigate the influence of rabbit bladder acellular matrix graft( BAMG) on proliferation and differentiation of human bone marrow mesenchymal stem cells( hBMSCs) and explore the feasibility of building bladder repair materials by it. Methods Primary hBMSCs were isolated and cultured with normal human bone marrow in vitro.BAMG was built with rabbit bladder. BAMG soaking solution culture medium was established,and the inducers of differentiation into osteoblasts,adipoblasts and smooth muscle were prepared based on the BAMG soaking solution. The hBMSCs were respectively cultured in normal culture medium,normal inducers culture medium,BAMG soaking solution culture medium and BAMG inducers solution. Hematoxylin-eosin( HE) and Masson tricolor staining were used to identify the tissue morphology. The Flow cytometry and living cell fluorescent dye-carboxyl fluorescein diacetate( CFSE)-labeling method were used to detect the growth toxicity of BAMG soaking solution culture medium on hBMSCs. The effects of inducers based on the BAMG soaking solution culture medium on the abilities of differentiation into osteoblasts,adipoblasts and smooth muscle were respectively observed. Results BAMG specimen was constructed successfully. Compared with notreatment bladder tissues,most of the BAMG cell nuclei were removed,and its tissue structure was more loose and porous.There was no significant difference in proliferation of hBMSCs for BAMG soaking solution culture medium and normal culture medium. The hBMSCs after induction can still be efficiently differentiated into osteoblasts,adipoblasts and smooth muscle. Conclusion BAMG does not affect the abilities of proliferation and differentiation of hBMSCs,so it can be used as a scaffold material in the application of bladder tissue engineering.
引文
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[12]Gigante A,Torcianti M,Boldrini E,et al.Vitamin K and D association stimulates in vitro osteoblast differentiation of fracture site derived human mesenchymal stem cells[J].J Biol Regul Homeost Agents,2008,22(1):35-44.
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[2]Simaioforidis V,de Jonge P,Sloff M,et al.Ureteral tissue engineering:where are we and how to proceed[J].Tissue Eng Part B Rev,2013,19(5):413-419.
[3]Zhu WD,Xu YM,Feng C,et al.Different bladder defects reconstructed with bladder acellular matrix grafts in a rabbit model[J].Urologe A,2011,50(11):1420-1425.
[4]谭云鹤,种铁,杨平,等.人骨髓间充质干细胞生物学特性及腺病毒介导的GFP标记[J].西安交通大学学报(医学版),2012,33(5):587-592.
[5]Bharadwaj S,Liu G,Shi Y,et al.Multipotential differentiation of human urine-derived stem cells:potential for therapeutic applications in urology[J].Stem Cells,2013,31(9):1840-1856.
[6]Hosseini SM,Talaei-Khozani T,Sani M,et al.Differentiation of human breast-milk stem cells to neural stem cells and neurons[J].Neurol Res Int,2014,2014:807896.
[7]Anumanthan G,Makari JH,Honea L,et al.Directed differentiation of bone marrow derived mesenchymal stem cells into bladder urothelium[J].J Urol,2008,180 Suppl 4:1778-1783.
[8]Tian H,Bharadwaj S,Liu Y,et al.Differentiation of human bone marrow mesenchymal stem cells into bladder cells:potential for urological tissue engineering[J].Tissue Eng Part A,2010,16(5):1769-1779.
[9]Ziaran S,Galambosová M,Danisovic L.Tissue engineering of urethra:Systematic review of recent literature[J].Exp Biol Med(Maywood),2017,242(18):1772-1785.
[10]赵阳,吴稼晟,周哲,等.人脂肪来源干细胞与膀胱脱细胞基质-丝素蛋白双层支架的生物相容性研究[J].组织工程与重建外科,2014,10(6):309-313.
[11]范雪梅,徐惠成,王朝丽.脱细胞膀胱基质补片的生物相容性研究[J].重庆医学,2014,43(3):298-300,303.
[12]Gigante A,Torcianti M,Boldrini E,et al.Vitamin K and D association stimulates in vitro osteoblast differentiation of fracture site derived human mesenchymal stem cells[J].J Biol Regul Homeost Agents,2008,22(1):35-44.
[13]Yang YJ,Li XL,Xue Y,et al.Bone marrow cells differentiation into organ cells using stem cell therapy[J].Eur Rev Med Pharmacol Sci,2016,20(13):2899-2907.
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