犊牛脂肪干细胞的分离、培养与鉴定
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摘要
为给奶牛营养代谢病等相关研究提供种子细胞,本试验从犊牛肾周脂肪组织中分离出脂肪干细胞(adipose-derived stem cells,ADSCs),采用组织剪碎法结合0. 25%Ⅰ型胶原酶消化法消化、收集原代犊牛ADSCs,通过细胞形态学观察,使用免疫荧光染色法检测第5代ADSCs表面特异性蛋白,并使用CCK-8法测定第5、10代细胞生长曲线,观察第10代细胞的成脂、成骨分化能力。结果显示,分离出的犊牛ADSCs接种4 d后细胞呈成纤维细胞样生长;免疫荧光染色结果显示,分离的细胞Vimentin、CD34、CD44、CD90呈阳性,CD31、CD45呈阴性;该细胞在适当的诱导条件下可以定向分化为脂肪细胞和成骨细胞。本试验分离培养的细胞是犊牛脂肪干细胞,具有成脂、成骨分化能力。
In order to provide seed cells for related research on vegetative metabolic diseases of cows,adipose-derived stem cells( ADSCs) were eparated from perirenal adipose tissue of calves. ADSCs were collected by organization minced and 0. 25% collagenase Ⅰ digestion by different times. The separated cells were proved by the following aspects,observed the morphology,passage 5 of ADSCs were used to detect the surface-specific proteins,passage 5 and 10 were used to detect the growth curve by CCK-8,passage 10 were used to detect the ability of adipogenic and osteogenic differentiation. The results showed that the ADSCs from calves were grown as fibroblast-like after 4 days of inoculation. Immunofluorescence staining showed that the isolated cells expressed Vimentin,CD34,CD44,and CD90,but did not express CD31 and CD45. The cells can be differentiated into adipocytes and osteoblasts under appropriate induction conditions. The separated cells were ADSCs with the ability of adipogenic and osteogenic differentiation.
In order to provide seed cells for related research on vegetative metabolic diseases of cows,adipose-derived stem cells( ADSCs) were eparated from perirenal adipose tissue of calves. ADSCs were collected by organization minced and 0. 25% collagenase Ⅰ digestion by different times. The separated cells were proved by the following aspects,observed the morphology,passage 5 of ADSCs were used to detect the surface-specific proteins,passage 5 and 10 were used to detect the growth curve by CCK-8,passage 10 were used to detect the ability of adipogenic and osteogenic differentiation. The results showed that the ADSCs from calves were grown as fibroblast-like after 4 days of inoculation. Immunofluorescence staining showed that the isolated cells expressed Vimentin,CD34,CD44,and CD90,but did not express CD31 and CD45. The cells can be differentiated into adipocytes and osteoblasts under appropriate induction conditions. The separated cells were ADSCs with the ability of adipogenic and osteogenic differentiation.
引文
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[23]肖建红,张阳春,张常然,等.人皮下脂肪干细胞的成骨、成脂分化诱导及鉴定[J].中国组织工程研究,2015,19(32):5155-5161.XIAO J H,ZHANG Y C,ZHANG C R,et al.Human subcutaneous adipose-derived stem cells:osteoblastic/adipogenic differentiation and identification[J].Chinese Journal of Tissue Engineering Research,2015,19(32):5155-5161.(in Chinese with English abstract)
[24]张鉴清,季佳霖,崔新明,等.小鼠附睾脂肪干细胞的分离培养及鉴定[J].中国组织工程研究,2014,18(28):4535-4541.ZHAGN J Q,JI J L,CUI X M,et al.Isolation,culture and identification of adipose-derived stem cells from mouse epididymis[J].Chinese Journal of Tissue Engineering Research,2014,18(28):4535-4541.(in Chinese with English abstract)
[25]李玲慧,丁道芳,龚浩,等.大鼠腹腔脂肪干细胞的分离培养及诱导成骨[J].中国组织工程研究,2013,17(23):4232-4239.LI L H,DING D F,GONG H,et al.Isolation,culture and osteogenic differentiation of adipose-derived stem cells from the abdominal cavity of rats[J].Chinese Journal of Tissue Engineering Research,2013,17(23):4232-4239.(in Chinese with English abstract)
[2]王文杰,关伟军,马月辉,等.鸭胚脐带间充质干细胞的分离培养及其生物学特性[J].浙江农业学报,2015,27(11):1903-1909.WANG W J,GUAN W J,MA Y H,et al.Isolation,Culture and biological characteristics of umbilical cord mesenchymal stem cells from duck embryos[J].Acta Agriculturae Zhejiangensis,2015,27(11):1903-1909.(in Chinese with English abstract)
[3]LUE J,LIN G,NING H,et al.Transdifferentiation of adipose-derived stem cells into hepatocytes:a new approach[J].Liver International,2010,30(6):913-922.
[4]ZUK P A,ZHU M,MIZUNO H,et al.Multilineage cells from human adipose tissue:implications for cell-based therapies[J].Tissue Engineering Part A,2001,7(2):211-228.
[5]韩正滨.脂肪源性干细胞的多向分化潜力及应用前景[J].生物工程学报,2007,23(2):195-200.HAN Z B,CHEN H X,DENG J X.Multi-differentiation potential and application prospect of adipose-derived stem cells[J].Chinese Journal of Biotechnology,2007,23(2):195-200.(in Chinese with English abstract)
[6]赵娜.脂肪干细胞诱导分化的现状及前景[J].中国组织工程研究,2015,19(6):969-974.ZHAO N.Induced differentiation of adipose-derived stem cells[J].Chinese Journal of Tissue Engineering Research,2015,19(6):969-974.(in Chinese with English abstract)
[7]GHADERI G M,SAHEBGHADAM L A,KORDI T D,et al.The enhancement of differentiating adipose derived mesenchymal stem cells toward hepatocyte like cells using gelatin cryogel scaffold[J].Biochemical and Biophysical Research Communications,2017,491(4):1000-1006.
[8]QU C Q,ZHANG G H,ZHANG L J,et al.Osteogenic and adipogenic potential of porcine adipose mesenchymal stem cells[J].In Vitro Cellular&Developmental Biology-Animal,2007,43(2):95-100.
[9]GUAN X,WANG N,CUI F,et al.Caveolin 1 is essential in the differentiation of human adipose derived stem cells into hepatocyte like cells via an MAPK pathway dependent mechanism[J].Molecular Medicine Reports,2016,13(2):1487-1494.
[10]殷立恒.PRL、GC对奶牛脂肪细胞和肝细胞脂代谢的影响[D].长春:吉林大学,2015.YIN L H.Effects of PRL and GC on lipid metabolism in fat cells and hepatocytes of dairy cows[D].Changchun:Jilin U-niversity,2015.(in Chinese with English abstract)
[11]张世奇.PLIN1对奶牛脂肪细胞脂代谢及炎性细胞因子合成的影响[D].长春:吉林大学,2017.ZHANG S Q.Effects of PLIN1 on fat metabolism and inflammatory cytokines synthesis in dairy fat cells[D].Changchun:Jilin University,2017.(in Chinese with English abstract)
[12]李春明,刘毅.人脂肪间充质干细胞冻存方法的改进[J].中国美容医学杂志,2007,16(8):1026-1028.LI C M,LIU Y.Modification of in situ cryopreservation of human adipose mesenchymalstem cells[J].Chinese Journal of Aesthetic Medicine,2007,16(8):1026-1028.(in Chinese with English abstract)
[13]AUST L,DEVLIN B,FOSTER S J,et al.Yield of human adipose-derived adult stem cells from liposuction aspirates[J].Cytotherapy,2004,6(1):7-14.
[14]BAKSH D,SONG L,TUAN R S.Adult mesenchymal stem cells:characterization,differentiation,and application in cell and gene therapy[J].Journal of Cellular and Molecular Medicine,2004,8(3):301-316.
[15]SAKAGUCHI Y,SEKIYA I,YAGISHITA K,et a.Comparison of human stem cells derived from various mesenchymal tissues:superiority of synovium as a cell source[J].Arthritis Rheum,2010,52(8):2521-2529.
[16]QIN Y H,CHEN G H,BIAN S Y,et al.In vitro culture and identification of adipose tissue-derived mesenchymal stem cells in rats[J].Journal of Clinical Rehabilitative Tissue Engineering Research,2007,11(33):6701-6705.
[17]王清富,陈庄洪,蔡贤华,等.兔脂肪干细胞的多向诱导分化[J].中国组织工程研究,2012,16(49):9162-9167.WANG Q F,CHEN Z H,CAI X H,et al.Multidirectional induced differentiation of adipose-derived stem cells in rabbits[J].Chinese Journal of Tissue Engineering Research,2012,16(49):9162-9167.(in Chinese with English abstract)
[18]任宇,吴海青,马玉珍,等.牛脂肪间充质干细胞的分离、培养与鉴定[J].生物工程学报,2010,26(12):1645-1651.REN Y,WU H Q,MA Y Z,et al.Isolation,cultivation and identification of adipose-derived stem cell in bovines[J].Chinese Journal of Biotechnology,2010,26(12):1645-1651.(in Chinese with English abstract)
[19]刘真真,李海洋,邓彦飞,等.家畜脂肪干细胞研究进展[J].基因组学与应用生物学,2014,33(1):205-214.LIU Z Z,LI H Y,DENG Y F,et al.The advance in livestock adipose-derived stem cells[J].Genomics and Applied Biology,2014,33(1):205-214.(in Chinese with English abstract)
[20]DOMINICI M,LE BLANC K,MUELLER I,et al.Minimal criteria for defining multipotent mesenchymal stromal cells.The International society for cellular therapy position statement[J].Cytotherapy,2006,8(4):315-317.
[21]陈犹白,陈聪慧,韩岩,等.脂肪干细胞表型和标记物的研究进展[J].中国美容医学,2016,25(3):91-100.CHEN Y B,CHEN C H,HAN Y,et al.A review on phenotype and markers of adipose-derived stem cells[J].Chinese Journal of Aesthetic Medicine,2016,25(3):91-100.(in Chinese with English abstract)
[22]GEORGIEV-HRISTOV T,GARCA-ARRANZ M,GARCA-OLMO D.Adipose tissue-derived products for complex fistula treatment[J].Techniques in Coloproctology,2013,17(6):675-676.
[23]肖建红,张阳春,张常然,等.人皮下脂肪干细胞的成骨、成脂分化诱导及鉴定[J].中国组织工程研究,2015,19(32):5155-5161.XIAO J H,ZHANG Y C,ZHANG C R,et al.Human subcutaneous adipose-derived stem cells:osteoblastic/adipogenic differentiation and identification[J].Chinese Journal of Tissue Engineering Research,2015,19(32):5155-5161.(in Chinese with English abstract)
[24]张鉴清,季佳霖,崔新明,等.小鼠附睾脂肪干细胞的分离培养及鉴定[J].中国组织工程研究,2014,18(28):4535-4541.ZHAGN J Q,JI J L,CUI X M,et al.Isolation,culture and identification of adipose-derived stem cells from mouse epididymis[J].Chinese Journal of Tissue Engineering Research,2014,18(28):4535-4541.(in Chinese with English abstract)
[25]李玲慧,丁道芳,龚浩,等.大鼠腹腔脂肪干细胞的分离培养及诱导成骨[J].中国组织工程研究,2013,17(23):4232-4239.LI L H,DING D F,GONG H,et al.Isolation,culture and osteogenic differentiation of adipose-derived stem cells from the abdominal cavity of rats[J].Chinese Journal of Tissue Engineering Research,2013,17(23):4232-4239.(in Chinese with English abstract)