间介中胚层样细胞在肾脏再生中的应用
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摘要
目的体外诱导脂肪干细胞分化为间介中胚层样细胞,对肾脏脱细胞支架进行再细胞化,经共培养构建有部分肾脏结构的组织工程肾脏。方法手术获取Wistar大鼠腹股沟脂肪垫,从中分离、培养原代脂肪干细胞后对其进行干细胞特性鉴定。经过在不同阶段添加糖原合成酶激酶3抑制剂(CHIR99021)和成纤维生长因子9(FGF9),诱导脂肪干细胞分化为间介中胚层样细胞,并于诱导7 d后进行间介中胚层相关蛋白鉴定。将诱导成功的间介中胚层样细胞结合制备好的肾脏脱细胞支架进行共培养,10 d后获取再细胞化的组织工程肾脏,并对其进行肾脏分化的鉴定。结果获取的脂肪干细胞具有成骨成脂及成软骨分化的能力,能表达脂肪干细胞表面标志物。体外诱导7 d后得到的间介中胚层样细胞经过免疫荧光鉴定可以观察到锌指蛋白转录因子和配对盒基因-2的阳性表达,Western blot验证可得到同样的阳性结果。结合肾脏脱细胞支架后培养10 d,通过免疫荧光鉴定可以观察到Wilms肿瘤基因1、GATA连接蛋白-3和E-钙黏素的阳性表达。结论脂肪干细胞可被诱导分化为间介中胚层样细胞,诱导后的间介中胚层样细胞结合肾脏脱细胞支架可以观察到肾系分化的现象。
Objective To induce adipose-derived stem cells(ADSCs) to differentiate into intermediate mesoderm(IM)-like cells in vitro,with IM-like cells for recellularizing kidney scaffolds,and then to obtain a tissue-engineering kidney with renal structures and functions through co-culture. Methods After inguinal fat pads of Wistar rats were surgically harvested,the primary ADSCs were isolated,induced,and cultured for stem cell identification. ADSCs were inducted to differentiate into IM-like cells by adding glycogen synthase kinase-3 inhibitor(CHIR99021) and fibroblast growth factor 9(FGF9) at different stages. Seven days later,the IM-like cells were identified. The induced IM-like cells and well-prepared kidney decellularized scaffolds were co-cultured for 10 days to obtain recellularized tissue-engineered kidneys and their differentiation was identified. Results The ADSCs harvested had osteogenic and adipogenic abilities and could express the stem cell surface markers. After 7 days of in vitro induction,the positive expressions of odd-skipped related 1 and paired-box 2 were observed in IM-like cells by immunofluorescence technique. After 10 days of co-culture with kidney decellularized scaffolds,the positive expressions of Wilms' tumor 1,GATA-binding protein-3,and E-cadherin were observed by immunofluorescence technique. Conclusion ADSCs can be induced into IM-like cells,and renal cell differentiation can be observed through combining the induced IM-like cells with kidney decellularized scaffolds.
Objective To induce adipose-derived stem cells(ADSCs) to differentiate into intermediate mesoderm(IM)-like cells in vitro,with IM-like cells for recellularizing kidney scaffolds,and then to obtain a tissue-engineering kidney with renal structures and functions through co-culture. Methods After inguinal fat pads of Wistar rats were surgically harvested,the primary ADSCs were isolated,induced,and cultured for stem cell identification. ADSCs were inducted to differentiate into IM-like cells by adding glycogen synthase kinase-3 inhibitor(CHIR99021) and fibroblast growth factor 9(FGF9) at different stages. Seven days later,the IM-like cells were identified. The induced IM-like cells and well-prepared kidney decellularized scaffolds were co-cultured for 10 days to obtain recellularized tissue-engineered kidneys and their differentiation was identified. Results The ADSCs harvested had osteogenic and adipogenic abilities and could express the stem cell surface markers. After 7 days of in vitro induction,the positive expressions of odd-skipped related 1 and paired-box 2 were observed in IM-like cells by immunofluorescence technique. After 10 days of co-culture with kidney decellularized scaffolds,the positive expressions of Wilms' tumor 1,GATA-binding protein-3,and E-cadherin were observed by immunofluorescence technique. Conclusion ADSCs can be induced into IM-like cells,and renal cell differentiation can be observed through combining the induced IM-like cells with kidney decellularized scaffolds.
引文
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[2] Collins AJ,Foley RN,Herzog C,et al.Excerpts from the US Renal Data System 2009 Annual Data Report[J].Am J Kidney Dis,2010,55(1 Suppl 1):S1- S420,A6- A7.DOI:10.1053/j.ajkd.2009.10.009.
[3] Takasato M,Maier B,Little MH.Recreating kidney progenitors from pluripotent cells[J].Pediatr Nephrol,2014,29(4):543- 552.DOI:10.11604/pamj.2017.28.137.6287.
[4] Amine BHH,Haythem S,Kais H,et al.Pregnancy after renal transplantation:a retrospective study at the military hospital of Tunis from 1992 to 2011[J].Pan Afr Med J,2017,28:137.DOI:10.1007/s00467- 013- 2592- 7.
[5] Song JJ,Guyette JP,Gilpin SE,et al.Regeneration and experimental orthotopic transplantation of a bioengineered kidney[J].Nat Med,2013,19(5):646- 651.DOI:10.1038/nm.3154.
[6] Huang SJ,Fu RH,Shyu WC,et al.Adipose-derived stem cells:isolation,characterization,and differentiation potential[J].Cell Transplant,2013,22(4):701- 709.DOI:10.3727/096368912X655127.
[7] Lee J,Abdeen AA,Tang X,et al.Matrix directed adipogenesis and neurogenesis of mesenchymal stem cells derived from adipose tissue and bone marrow[J].Acta Biomater,2016,42:46- 55.DOI:10.1016/j.actbio.2016.06.037.
[8] Takasato M,Er PX,Chiu HS,et al.Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis[J].Nature,2015,526(7574):564- 568.DOI:10.1038/nature 15695.
[9] 肖枫林,李清刚,傅博,等.骨髓间充质干细胞构建类肾器官实验[J].解放军医学院学报,2017,(10):964- 967.DOI:10.3969/j.issn.2095- 5227.2017.10.015.
[10] Bansal P,Ajay D.Laboratory dialysis-past,present and future[J].Recent Pat Biotechnol,2012,6(1):32- 44.DOI:10.2174/187220812799789217.
[11] Jha V,Garcia-Garcia G,Iseki K,et al.Chronic kidney disease:global dimension and perspectives[J].Lancet,2013,382(9888):260- 272.DOI:10.1016/S0140- 6736(13)60687-X.
[12] Al-Awqati Q,Oliver JA.Stem cells in the kidney[J].Kidney Int,2002,61(2):387- 395.DOI:10.1046/j.1523- 1755.2002.00164.x.
[13] Sollazzo V,Lucchese A,Palmieri A,et al.Polylactide-polyglycolide resorbable plates stimulates adipose tissue-derived stem cells towards osteoblasts differentiation[J].Int J Immunopathol Pharmacol,2011,24(2 Suppl):59- 64.DOI:10.1177/03946320110240S211.
[14] Lee HJ,Jung J,Cho KJ,et al.Comparison of in vitro hepatogenic differentiation potential between various placenta-derived stem cells and other adult stem cells as an alternative source of functional hepatocytes[J].Differentiation,2012,84(3):223- 231.DOI:10.1016/j.diff.2012.05.007.
[15] Sun Y,Zhang M,Ji S,et al.Induction differentiation of rabbit adiposederived stromal cells into insulinproducing cells in vitro[J].Mol Med Rep,2015,12(5):6835- 6840.DOI:10.3892/mmr.2015.4305.
[16] Zhang HT,Liu ZL,Yao XQ,et al.Neural differentiation ability of mesenchymal stromal cells from bone marrow and adipose tissue:a comparative study[J].Cytotherapy,2012,14(10):1203- 1214.DOI:10.3109/14653249.2012.711470.
[17] Choi YS,Dusting GJ,Stubbs S,et al.Differentiation of human adipose-derived stem cells into beating cardiomyocytes[J].J Cell Mol Med,2010,14(4):878- 889.DOI:10.1111/j.1582- 4934.2010.01009.x.
[18] Lu W,Yu J,Zhang Y,et al.Mixture of fibroblasts and adipose tissue-derived stem cells can improve epidermal morphogenesis of tissue-engineered skin[J].Cells Tissues Organs,2012,195(3):197- 206.DOI:10.1159/000324921.
[19] Bonandrini B,Figliuzzi M,Papadimou E,et al.Recellularization of well-preserved acellular kidney scaffold using embryonic stem cells[J].Tissue Eng Part A,2014,20(9- 10):1486- 1498.DOI:10.1089/ten.TEA.2013.0269.
[20] Gomes ME,Rodrigues MT,Domingues RMA,et al.Tissue engineering and regenerative medicine:new trends and directions-a year in review[J].Tissue Eng Part B Rev,2017,23(3):211- 224.DOI:10.1089/ten.TEB.2017.0081.
[21] Usui J,Kobayashi T,Yamaguchi T,et al.Generation of kidney from pluripotent stem cells via blastocyst complementation[J].Am J Pathol,2012,180(6):2417- 2426.DOI:10.1016/j.ajpath.2012.03.007.
[22] Hinata N,Suzuki R,Ishizawa A,et al.Fetal development of the mesonephric artery in humans with reference to replacement by the adrenal and renal arteries[J].Ann Anat,2015,202:8- 17.DOI:10.1016/j.aanat.2015.07.005.
[23] Humes HD,MacKay SM,Funke AJ,et al.Tissue engineering of a bioartificial renal tubule assist device:in vitro transport and metabolic characteristics[J].Kidney Int,1999,55(6):2502- 2514.DOI:10.1046/j.1523- 1755.1999.00486.x.
[24] Guan Y,Liu S,Sun C,et al.The effective bioengineering method of implantation decellularized renal extracellular matrix scaffolds[J].Oncotarget,2015,6(34):36126- 36138.DOI:10.18632/oncotarget.5304.
[25] Sambi M,Chow T,Whiteley J,et al.Acellular mouse kidney ECM can be used as a three-dimensional substrate to test the differentiation potential of embryonic stem cell derived renal progenitors[J].Stem Cell Rev,2017,13(4):513- 531.DOI:10.1007/s12015- 016- 9712- 2.