近交系五指山小型猪脂肪间充质干细胞分离培养及生物学特性
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摘要
目的:建立近交系五指山小型猪(inbreed line miniature pig of Wuzhishan,ILMW)脂肪间充质干细胞(adiposederived mesenchymal stem cells,ASCs)体外分离培养和鉴定方法。方法:选取6月龄健康ILMW 3头,从颈部皮下无菌获取脂肪组织约100 g,采用0.1%I型胶原酶消化组织,获得ILMW-ASCs,并进行体外培养和增殖;取第3,5,8,13代细胞进行如下实验:测量细胞大小,观察细胞形态,检测细胞表型、细胞周期、细胞凋亡,并进行细胞诱导分化和绘制细胞增殖曲线。结果:体外分离培养的ILMW-ASCs在体外贴壁,呈成纤维样或旋涡状生长;原代细胞培养36 h出现贴壁,第5天进入对数增殖期,约7 d后80%的细胞汇合。ILMW-ASCs细胞直径及体积分别为(17.00±0.54)μm和(2.58±0.24)×10–9 L。细胞表面抗原CD29,CD44和CD90呈高表达,不同代数之间的差异不明显(均P>0.05);CD45,CD8a和HLA-DR呈低表达,与第3代相比,随着代数的增加细胞表面抗原的阳性率逐渐增高(均P<0.05)。ILMW-ASCs成脂诱导结果为油红O染色呈阳性;ILMW-ASCs成骨诱导结果为茜素红染色呈阳性。ILMW-ASCs的第13代细胞出现凋亡和衰老,与较低培养代数相比细胞周期S期的比例下降(均P<0.05)。结论:ILMW-ASCs取材方便,贴壁时间早,细胞增殖活跃,传代周期短,传代后能保持稳定的生物学特性,是猪来源ASCs的最佳选择之一,可为大动物疾病模型和组织器官修复的细胞治疗提供候选干细胞来源。
Objective: To establish a method for isolation, culture and identification of adipose-derived mesenchymal stem cells(ASCs) from the inbreed line miniature pig of Wuzhishan(ILMW).Methods: A total of 100 g adipose tissues were obtained from subcutaneous tissues of neck in sixmonth old healthy ILMW(3 samples, male). ASCs from ILMW(ILMW-ASCs) were isolated from adipose tissues through 0.1% collagenase digestion. The cells at the 3 rd, 5 th, 8 th, 13 th passages were collected. Cell morphology, size, phenotype, cell cycle, and apoptosis were monitored. Cell differentiation was induced and cell proliferation curve was drawn.Results: The ILMW-ASCs, fibroblast-like or whirlpool-like, began the adherence at 36 h and entered a logarithmic phase in the 5 th day. Eighty percent of them were fused in the 7 th day. The average diameter and volume of ILMW-ASCs were(17.00±0.54) μm and(2.58±0.24)×10–9 L,respectively. The expressions of CD29, CD44 and CD90 were positive, and there was no significant difference between the different passages(all P>0.05). The expressions of CD45, CD8 a and HLADR were increased with the increase in passages after the 3 th passage(all P<0.05). The adipogenic induction of ILMW-ASCs was observed by positive oil red O staining, and the osteogenic induction of ILMW-ASCs was determined by positive alizarin red staining. Apoptosis and senescence occurred in the 13 passage of ILMW-ASCs, and the proportion of S phase of cell cycle was lower than that in lower passages(all P<0.05).Conclusion: ILMW-ASCs are one of the best choice for porcine ASCs, which might provide a source of candidate stem cells for therapy of large animal disease models and tissue or organ repairment.
Objective: To establish a method for isolation, culture and identification of adipose-derived mesenchymal stem cells(ASCs) from the inbreed line miniature pig of Wuzhishan(ILMW).Methods: A total of 100 g adipose tissues were obtained from subcutaneous tissues of neck in sixmonth old healthy ILMW(3 samples, male). ASCs from ILMW(ILMW-ASCs) were isolated from adipose tissues through 0.1% collagenase digestion. The cells at the 3 rd, 5 th, 8 th, 13 th passages were collected. Cell morphology, size, phenotype, cell cycle, and apoptosis were monitored. Cell differentiation was induced and cell proliferation curve was drawn.Results: The ILMW-ASCs, fibroblast-like or whirlpool-like, began the adherence at 36 h and entered a logarithmic phase in the 5 th day. Eighty percent of them were fused in the 7 th day. The average diameter and volume of ILMW-ASCs were(17.00±0.54) μm and(2.58±0.24)×10–9 L,respectively. The expressions of CD29, CD44 and CD90 were positive, and there was no significant difference between the different passages(all P>0.05). The expressions of CD45, CD8 a and HLADR were increased with the increase in passages after the 3 th passage(all P<0.05). The adipogenic induction of ILMW-ASCs was observed by positive oil red O staining, and the osteogenic induction of ILMW-ASCs was determined by positive alizarin red staining. Apoptosis and senescence occurred in the 13 passage of ILMW-ASCs, and the proportion of S phase of cell cycle was lower than that in lower passages(all P<0.05).Conclusion: ILMW-ASCs are one of the best choice for porcine ASCs, which might provide a source of candidate stem cells for therapy of large animal disease models and tissue or organ repairment.
引文
[1]Walters EM,Wolf E,Whyte JJ,et al.Completion of the swine genome will simplify the production of swine as a large animal biomedical model[J].BMC Med Genomics,2012,5:55-59.
[2]Monaco E,Bionaz M,Rodriguez-Zas S,et al.Transcriptomics compar ison between porcine adipose and bone marrow mesenchymal stem cells during in vitro osteogenic and adipogenic differentiation[J].PLoS One,2012,7(3):e32481.
[3]王昕,曹红鹤,耿社民,等.利用微卫星标记对中国4种小型猪的遗传多样性研究[J].畜牧兽医学报,2002,33(6):530-532.WANG Xin,CAO Honghe,GENG Shemin,et al.Chinese miniature pigs’genetic diversity by using micro-satellite marker[J].Acta Veterinariaet Zootechnica Sinica,2002,33(6):530-532.
[4]Zuk PA,Zhu M,Ashjian P,et al.Human adipose tissue is a source of multipotent stem cells[J].Mol Biol Cell,2002,13(12):4279-4295.
[5]Dai R,Wang Z,Samanipour R,et al.Adipose-derived stem cells for tissue engineering and regenerative medicine applications[J].Stem Cells Int,2016,2016:6737345.
[6]Tsuji W,Rubin JP,Marra KG.Adipose-derived stem cells:Implications in tissue regeneration[J].World J Stem Cells,2014,6(3):312-321.
[7]Bajek A,Gurtowska N,Olkowska J,et al.Adipose-derived stem cells as a tool in cell-based therapies[J].Arch Immunol Ther Exp,2016,64(6):443-454.
[8]Hirsch T,Rothoeft T,Teig N,et al.Regeneration of the entire human epidermis using transgenic stem cells[J].Nature,2017,551(7680):327-332.
[9]Hodges WM,O'Brien F,Fulzele S,et al.Function of microRNAs in the osteogenic differentiation and therapeutic application of adiposederived stem cells(ASCs)[J].Int J Mol Sci,2017,18(12):2597-2617.
[10]Ashraf S,Han IB,Park H,et al.Role of RHEB in regulating differentiation fate of mesenchymal stem cells for cartilage and bone regeneration[J].Int J Mol Sci,2017,18(4):880-893.
[11]Bertozzi N,Simonacci F,Grieco MP,et al.The biological and clinical basis for the use of adipose-derived stem cells in the field of wound healing[J].Ann Med Surg(Lond),2017,20:41-48.
[12]Lee SC,Jeong HJ,Lee SK,et al.Hypoxic conditioned medium from human adipose-derived stem cells promotes mouse liver regeneration through JAK/STAT3 signaling[J].Stem Cells Transl Med,2016,5(6):816-825.
[13]Hsueh YY,Chang YJ,Huang CW,et al.Synergy of endothelial and neural progenitor cells from adipose-derived stem cells to preserve neurovascular structures in rat hypoxic-ischemic brain injury[J].Sci Rep,2015,5:14985.
[14]Davy PM,Lye KD,Mathews J,et al.Human adipose stem cell and ASC-derived cardiac progenitor cellular therapy improves outcomes in a murine model of myocardial infarction[J].Stem Cells Cloning,2015,8:135-148.
[15]Forcales SV.Potential of adipose-derived stem cells in muscular regenerative therapies[J].Front Aging Neurosci,2015,7:123.
[16]Banyard DA,Salibian AA,Widgerow AD.Implications for human adipose-derived stem cells in plastic surgery[J].J Cell Mol Med,2015,19(1):21-30.
[17]张青峰,冯书堂.小型猪品系五指山猪(WZSP)的研究进展[J].安徽农学通报,2007,13(14):161-162.ZHANG Qingfeng,FENG Shutang.Research progress of miniature pig strain Wuzhishan pig(WZSP)[J].Anhui Agricultural Science Bulletin,2007,13(14):161-162.
[18]向杨,邢健生,白志明,等.近交系五指山小型猪骨髓间充质干细胞分离培养及生物特性[J].中南大学学报(医学版),2015,40(3):261-268.XIANG Yang,XING Jiansheng,BAI Zhiming,et al.Separation culture and biological characteristics analysis for bone marrowderived mesenchymal stem cells from inbreed line miniature pig of Wuzhishan[J].Journal of Central South University.Medical Science,2015,40(3):261-268.
[19]成钢,冯书堂.五指山猪近交系精原干细胞体外培养研究[J].生物工程学报,2006,22(4):689-693.CHENG Gang,FENG Shutang.Studies on spermatogonial stem cells cultured in vitro of Wuzhishan mini porcine[J].Chinese Journal of Biotechnology,2006,22(4):689-693.
[20]向杨,龙家乐,邢健生,等.近交系五指山小型猪骨髓间充质干细胞体外分泌肝细胞生长因子延缓慢性肾脏病纤维化进展[J].中南大学学报(医学版),2016,41(12):1260-1269.XIANG Yang,LONG Jiale,XING Jiansheng,et al.BM-MSCs from Wuzhishan mini-pigs delay the progress of renal fibrosis induced by chronic kidney disease through autocrine hepatocyte growth factor in vitro[J].Journal of Central South University.Medical Science,2016,41(12):1260-1269.
[21]Xiang Y,Bai ZM,Zhang SF,et al.Lead adsorption,anticoagulation and in vivo toxicity studies on the new magnetic nanomaterial Fe3O4@SiO2@DMSA as a hemoperfusion adsorbent[J].Nanomedicine,2017,13(4):1341-1351.
[22]田玉龙,钟红珊.五指山小型猪在心血管系统疾病建模中的应用[J].介入放射学杂志,2016,25(4):363-366.TIAN Yulong,ZHONG Hongshan.Application of Wuzhishan miniature pigs in establishing experimental models of cardiovascular system[J].Journal of Intervent Radiology,2016,25(4):363-366.
[23]Dariolli R,Bassaneze V,Nakamuta JS,et al.Porcine adipose tissue-derived mesenchymal stem cells retain their proliferative characteristics,senescence,karyotype and plasticity after long-term cryopreservation[J].PLoS One,2013,8(7):e67939.
[24]尹巧香,王恒,裴志勇,等.猪的脂肪和骨髓来源间充质干细胞培养特性比较[J].生物医学工程与临床,2013,17(6):523-527.YIN Qiaoxiang,WANG Heng,PEI Zhiyong,et al.Comparison of culture characteristics between swine bone marrow and adipose tissue-derived mesenchymal stem cells[J].Biomedical Engineering and Clinical,2013,17(6):523-527.
[25]Williams KJ,Picou AA,Kish SL,et al.Isolation and characterization of porcine adipose tissue-derived adult stem cells[J].Cells Tissues Organs,2008,188(3):251-258.
[26]Bourin P,Bunnell BA,Casteilla L,et al.Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells:a joint statement of the International Federation for Adipose Therapeutics and Science(IFATS)and the International Society for Cellular Therapy(ISCT)[J].Cytotherapy,2013,15(6):641-648.
[27]Kim D,Monaco E,Maki A,et al.Morphological and transcriptomic comparison of adipose and bone marrow derived porcine stem cells[J].Cell Tissue Res,2010,341(3):359.
[28]Niada S,Ferreira LM,Arrigoni E,et al.Porcine adipose-derived stem cells from buccal fat pad and subcutaneous adipose tissue for future preclinical studies in oral surgery[J].Stem Cell Res Ther,2013,4(6):148.
[29]Wang KH,Kao AP,Hsuan WC,et al.Optimizing proliferation and characterization of multipotent stem cells from porcine adipose tissues[J].Biotechnol Appl Biochem,2008,51(pt4):159-166.
[2]Monaco E,Bionaz M,Rodriguez-Zas S,et al.Transcriptomics compar ison between porcine adipose and bone marrow mesenchymal stem cells during in vitro osteogenic and adipogenic differentiation[J].PLoS One,2012,7(3):e32481.
[3]王昕,曹红鹤,耿社民,等.利用微卫星标记对中国4种小型猪的遗传多样性研究[J].畜牧兽医学报,2002,33(6):530-532.WANG Xin,CAO Honghe,GENG Shemin,et al.Chinese miniature pigs’genetic diversity by using micro-satellite marker[J].Acta Veterinariaet Zootechnica Sinica,2002,33(6):530-532.
[4]Zuk PA,Zhu M,Ashjian P,et al.Human adipose tissue is a source of multipotent stem cells[J].Mol Biol Cell,2002,13(12):4279-4295.
[5]Dai R,Wang Z,Samanipour R,et al.Adipose-derived stem cells for tissue engineering and regenerative medicine applications[J].Stem Cells Int,2016,2016:6737345.
[6]Tsuji W,Rubin JP,Marra KG.Adipose-derived stem cells:Implications in tissue regeneration[J].World J Stem Cells,2014,6(3):312-321.
[7]Bajek A,Gurtowska N,Olkowska J,et al.Adipose-derived stem cells as a tool in cell-based therapies[J].Arch Immunol Ther Exp,2016,64(6):443-454.
[8]Hirsch T,Rothoeft T,Teig N,et al.Regeneration of the entire human epidermis using transgenic stem cells[J].Nature,2017,551(7680):327-332.
[9]Hodges WM,O'Brien F,Fulzele S,et al.Function of microRNAs in the osteogenic differentiation and therapeutic application of adiposederived stem cells(ASCs)[J].Int J Mol Sci,2017,18(12):2597-2617.
[10]Ashraf S,Han IB,Park H,et al.Role of RHEB in regulating differentiation fate of mesenchymal stem cells for cartilage and bone regeneration[J].Int J Mol Sci,2017,18(4):880-893.
[11]Bertozzi N,Simonacci F,Grieco MP,et al.The biological and clinical basis for the use of adipose-derived stem cells in the field of wound healing[J].Ann Med Surg(Lond),2017,20:41-48.
[12]Lee SC,Jeong HJ,Lee SK,et al.Hypoxic conditioned medium from human adipose-derived stem cells promotes mouse liver regeneration through JAK/STAT3 signaling[J].Stem Cells Transl Med,2016,5(6):816-825.
[13]Hsueh YY,Chang YJ,Huang CW,et al.Synergy of endothelial and neural progenitor cells from adipose-derived stem cells to preserve neurovascular structures in rat hypoxic-ischemic brain injury[J].Sci Rep,2015,5:14985.
[14]Davy PM,Lye KD,Mathews J,et al.Human adipose stem cell and ASC-derived cardiac progenitor cellular therapy improves outcomes in a murine model of myocardial infarction[J].Stem Cells Cloning,2015,8:135-148.
[15]Forcales SV.Potential of adipose-derived stem cells in muscular regenerative therapies[J].Front Aging Neurosci,2015,7:123.
[16]Banyard DA,Salibian AA,Widgerow AD.Implications for human adipose-derived stem cells in plastic surgery[J].J Cell Mol Med,2015,19(1):21-30.
[17]张青峰,冯书堂.小型猪品系五指山猪(WZSP)的研究进展[J].安徽农学通报,2007,13(14):161-162.ZHANG Qingfeng,FENG Shutang.Research progress of miniature pig strain Wuzhishan pig(WZSP)[J].Anhui Agricultural Science Bulletin,2007,13(14):161-162.
[18]向杨,邢健生,白志明,等.近交系五指山小型猪骨髓间充质干细胞分离培养及生物特性[J].中南大学学报(医学版),2015,40(3):261-268.XIANG Yang,XING Jiansheng,BAI Zhiming,et al.Separation culture and biological characteristics analysis for bone marrowderived mesenchymal stem cells from inbreed line miniature pig of Wuzhishan[J].Journal of Central South University.Medical Science,2015,40(3):261-268.
[19]成钢,冯书堂.五指山猪近交系精原干细胞体外培养研究[J].生物工程学报,2006,22(4):689-693.CHENG Gang,FENG Shutang.Studies on spermatogonial stem cells cultured in vitro of Wuzhishan mini porcine[J].Chinese Journal of Biotechnology,2006,22(4):689-693.
[20]向杨,龙家乐,邢健生,等.近交系五指山小型猪骨髓间充质干细胞体外分泌肝细胞生长因子延缓慢性肾脏病纤维化进展[J].中南大学学报(医学版),2016,41(12):1260-1269.XIANG Yang,LONG Jiale,XING Jiansheng,et al.BM-MSCs from Wuzhishan mini-pigs delay the progress of renal fibrosis induced by chronic kidney disease through autocrine hepatocyte growth factor in vitro[J].Journal of Central South University.Medical Science,2016,41(12):1260-1269.
[21]Xiang Y,Bai ZM,Zhang SF,et al.Lead adsorption,anticoagulation and in vivo toxicity studies on the new magnetic nanomaterial Fe3O4@SiO2@DMSA as a hemoperfusion adsorbent[J].Nanomedicine,2017,13(4):1341-1351.
[22]田玉龙,钟红珊.五指山小型猪在心血管系统疾病建模中的应用[J].介入放射学杂志,2016,25(4):363-366.TIAN Yulong,ZHONG Hongshan.Application of Wuzhishan miniature pigs in establishing experimental models of cardiovascular system[J].Journal of Intervent Radiology,2016,25(4):363-366.
[23]Dariolli R,Bassaneze V,Nakamuta JS,et al.Porcine adipose tissue-derived mesenchymal stem cells retain their proliferative characteristics,senescence,karyotype and plasticity after long-term cryopreservation[J].PLoS One,2013,8(7):e67939.
[24]尹巧香,王恒,裴志勇,等.猪的脂肪和骨髓来源间充质干细胞培养特性比较[J].生物医学工程与临床,2013,17(6):523-527.YIN Qiaoxiang,WANG Heng,PEI Zhiyong,et al.Comparison of culture characteristics between swine bone marrow and adipose tissue-derived mesenchymal stem cells[J].Biomedical Engineering and Clinical,2013,17(6):523-527.
[25]Williams KJ,Picou AA,Kish SL,et al.Isolation and characterization of porcine adipose tissue-derived adult stem cells[J].Cells Tissues Organs,2008,188(3):251-258.
[26]Bourin P,Bunnell BA,Casteilla L,et al.Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells:a joint statement of the International Federation for Adipose Therapeutics and Science(IFATS)and the International Society for Cellular Therapy(ISCT)[J].Cytotherapy,2013,15(6):641-648.
[27]Kim D,Monaco E,Maki A,et al.Morphological and transcriptomic comparison of adipose and bone marrow derived porcine stem cells[J].Cell Tissue Res,2010,341(3):359.
[28]Niada S,Ferreira LM,Arrigoni E,et al.Porcine adipose-derived stem cells from buccal fat pad and subcutaneous adipose tissue for future preclinical studies in oral surgery[J].Stem Cell Res Ther,2013,4(6):148.
[29]Wang KH,Kao AP,Hsuan WC,et al.Optimizing proliferation and characterization of multipotent stem cells from porcine adipose tissues[J].Biotechnol Appl Biochem,2008,51(pt4):159-166.
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