体外诱导人羊水来源间充质干细胞向软骨细胞分化的实验研究
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摘要
研究目的
探讨分离、提纯和扩增孕中期羊水来源间充质干细胞(AF-MSCs)的方法,以及体外诱导AF-MSCs向软骨细胞分化的可能性。
研究方法
机械手段挑取孕中期人羊水细胞培养体系中的MSCs集落,培养扩增后,通过流式细胞术对表面抗原表达情况进行鉴定,并通过RT-PCR检测多能性基因Oct4及Nanog的表达情况。取第3代AF-MSCs,经TGF-β1、IGF-1、地塞米松等生长因子向软骨细胞方向诱导分化。倒置相差显微镜观察诱导后细胞形态的变化。诱导3周后,免疫荧光染色及RT-PCR分别检测软骨特异性基质CollengonⅡ、Aggrecan、Chondromudulin-1、S100的蛋白及其基因的表达情况。
研究结果
机械手段分离出的AF-MSCs为CD105、CD166阳性,CD29若阳性,CD34、CD45阴性,符合MSCs特点。AF-MSCs表达多能性基因Oct4,但不表达Nanog。诱导2周后,细胞开始呈多角形样改变;诱导后3周时,大部分细胞由长梭形转变为多角形。免疫荧光及RT-PCR检测显示诱导后的AF-MSCs软骨特异性基质CollengonⅡ、Aggrecan、Chondromudulin-1和S100的蛋白和基因呈阳性表达,而未经诱导培养的AF-MSCs呈阴性表达。
研究结论
机械分离法可有效获得孕中期羊水来源间充质干细胞,经TGF-β1,IGF-1,地塞米松联合诱导3周后,AF-MSCs可以分化为软骨细胞样细胞。
Objective
To investigate the method of isolation,purification and amplification of messenchymal stem cells(MSCs) derived from second-trimester human amniotic fluid and the possibility of AF-MSCs induced into chondrocyte-like cells in vitro.
Methods
Fibroblast-like MSCs were separated mechanically from cultured amnioticytes, expanded and analyzed its surface antigens by flow cytometry.The multipotiential marker Oct4 and Nanog was tested by RT-PCR.After 3 passages AF-MSCs were shifted to chondrocyte medium(IMDM supplemented with 10%FBS,1%Amino-max-100,TGF-β1,IGF-1,Dex etc)for 3 weeks.The morphology of induced cells were evaluated by contrast microscopy.The positive cells expressing chondrocyte special extracellular matrix such as CollagenⅡ,Aggrecan,Chondromodulin-1,S100 were confirmed by immunofluorescence. RT-PCR was used to study the mRNA expression of CollagenⅡ,Aggrecan, Chondromodullin-1,S100.
Results
AF-MSCs could be successfully isolated,purified and enriched from the culture of amniocytes using mechanical methods.The expanded MSCs were positive for CD105, CD166,low positive for CD29,but negative for CD34,CD45,that was consistent with the regular characters of MSC.RT-PCR analysis showed the expression of multipotiential marker Oct4,but not for Nanog.After induction by chondrocyte medium for 3 weeks, most AF-MSCs presented typical morphological features of chondrocyte.The immunofluorescence and RT-PCR analysis showed the expression of CollagenⅡ, Aggrecan,Chondromodulin-1,S100 in the induced group.
Conclusions
Mechanical method is an effective approach to isolate MSCs from second-trimester human amniotic fluid.AF-MSCs can be induced into chondrocyte-like cells after 3 weeks' chondrogenic induction.
探讨分离、提纯和扩增孕中期羊水来源间充质干细胞(AF-MSCs)的方法,以及体外诱导AF-MSCs向软骨细胞分化的可能性。
研究方法
机械手段挑取孕中期人羊水细胞培养体系中的MSCs集落,培养扩增后,通过流式细胞术对表面抗原表达情况进行鉴定,并通过RT-PCR检测多能性基因Oct4及Nanog的表达情况。取第3代AF-MSCs,经TGF-β1、IGF-1、地塞米松等生长因子向软骨细胞方向诱导分化。倒置相差显微镜观察诱导后细胞形态的变化。诱导3周后,免疫荧光染色及RT-PCR分别检测软骨特异性基质CollengonⅡ、Aggrecan、Chondromudulin-1、S100的蛋白及其基因的表达情况。
研究结果
机械手段分离出的AF-MSCs为CD105、CD166阳性,CD29若阳性,CD34、CD45阴性,符合MSCs特点。AF-MSCs表达多能性基因Oct4,但不表达Nanog。诱导2周后,细胞开始呈多角形样改变;诱导后3周时,大部分细胞由长梭形转变为多角形。免疫荧光及RT-PCR检测显示诱导后的AF-MSCs软骨特异性基质CollengonⅡ、Aggrecan、Chondromudulin-1和S100的蛋白和基因呈阳性表达,而未经诱导培养的AF-MSCs呈阴性表达。
研究结论
机械分离法可有效获得孕中期羊水来源间充质干细胞,经TGF-β1,IGF-1,地塞米松联合诱导3周后,AF-MSCs可以分化为软骨细胞样细胞。
Objective
To investigate the method of isolation,purification and amplification of messenchymal stem cells(MSCs) derived from second-trimester human amniotic fluid and the possibility of AF-MSCs induced into chondrocyte-like cells in vitro.
Methods
Fibroblast-like MSCs were separated mechanically from cultured amnioticytes, expanded and analyzed its surface antigens by flow cytometry.The multipotiential marker Oct4 and Nanog was tested by RT-PCR.After 3 passages AF-MSCs were shifted to chondrocyte medium(IMDM supplemented with 10%FBS,1%Amino-max-100,TGF-β1,IGF-1,Dex etc)for 3 weeks.The morphology of induced cells were evaluated by contrast microscopy.The positive cells expressing chondrocyte special extracellular matrix such as CollagenⅡ,Aggrecan,Chondromodulin-1,S100 were confirmed by immunofluorescence. RT-PCR was used to study the mRNA expression of CollagenⅡ,Aggrecan, Chondromodullin-1,S100.
Results
AF-MSCs could be successfully isolated,purified and enriched from the culture of amniocytes using mechanical methods.The expanded MSCs were positive for CD105, CD166,low positive for CD29,but negative for CD34,CD45,that was consistent with the regular characters of MSC.RT-PCR analysis showed the expression of multipotiential marker Oct4,but not for Nanog.After induction by chondrocyte medium for 3 weeks, most AF-MSCs presented typical morphological features of chondrocyte.The immunofluorescence and RT-PCR analysis showed the expression of CollagenⅡ, Aggrecan,Chondromodulin-1,S100 in the induced group.
Conclusions
Mechanical method is an effective approach to isolate MSCs from second-trimester human amniotic fluid.AF-MSCs can be induced into chondrocyte-like cells after 3 weeks' chondrogenic induction.
引文
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2 Kaviani A,Perry T,Dzakovic A,et al.The amniotic fluid as a source of cells for fetal tissue engineering[J].Ped Surgery,2008,36(ll):1662-1665.
3 Chacko DM,Das AV,Zhao X,et al.Transplantation of ocular stem cells:the role of injury in incorporation and differentiation of grafted cells in the retina[J].Vision Res,2003,43(12):937-946.
4 Fuchs JR,Kaviani A,Oh JT,et al.Diaphragmatic reconstruction with autologous tendon engineered from mesenchymal amniocytes[J].Pediatric Surg,2004,39(6):834-838.
5 Kunisaki SM,Fuchs JR,Kaviani A,et al.Diaphragmatic repair through fetal tissue engineering:a comparison between mesenchymal amniocyte and myoblast-based constructs[J].Pediatric Surg,2006:41(1):34-39.
6 鄂征．组织培养和分子细胞生物学技术[M]．北京：北京出版社,2001．168-169．
7 Prusa AR,Hengstschlager M.Amniotic fluid cells and human stem cell researchra new connection[J].Med Sci Monit,2002,8(11):253-257.
8 Tsai MS,Lee JL,Chang YJ,et al.Isolation of human multipotent mesenchymal stem cells from second trimester amniotic fluid using a novel two-stage culture protocol[J].Hum Reprod,2004,9(6):1450-1457.
9 Tsai MS,Hwang SM,Tsai YL,et al.Clonal Amniotic Fluid-Derived Stem Cells Express Characteristics of Both Mesenchymal and Neural Stem Cells[J].Biology of Reproduction,2006,74(24),545-551.
10 In't Anker PS,Scherjon SA,Kleijburg-van der Keur C,et al.Amniotic fluid as a novel source of mesenchymal stem cells for therapeutic transplantation[J].Blood 2003,102(2):1548-1549.
11 Sartore S,Maddalena L,Annalisa A,et al.Amniotic mesenchymal cells autotransplanted in a porcine model of cardiac ischemia do not differentiate to cardiogenic phenotypes[J].Europ J Card Surg,2005,24(28):677-684.
12 Bossolasco P,Montemurro T,Cova L.et al.Molecular and phenotypic characterization of human amniotic fluid cells and their differentiation potential[J].Cell Res 2006,16(4):329-336.
13 Pesce M,Anastassiadis K,Scholer HR.Oct-4:lessons of totipotency from embryonic stem cells[J].Cells Tissues O狓?s,2005,165(3-4):144-152.
14 Prusa AR,Marton E,Rosner M,et al.Oct-4 expressing cells in human amniotic fluid:a new source for stem cell research?[J].Hum Reprod,2003,18(7):1489-1493.
15 Tsai MS,Hwang SM,Tsai YL,et al.Clonal amniotic fluid-derived stem cells express characteristics of both mesenchymal and neural stem cells[J].Biol.Reprod,2006,74(3):545-551.
16 Kim J,Lee Y,Kim H,et al.Human amniotic fluid-derived stem cells have characteristics of multipotent stem cells[J].Cell Prolif,2007,40(l):75-90.
17 Tsai MS,Hwang SM,Tsai YL,et al.Clonal amniotic fluid-derived stem cells express characteristics of both mesenchymal and neural stem cells[J].Bio Reprod,2006,74(3):545-551.
18 De Gemmis P,Lapucci C,Bertelli M,et al.A real-time PCR approach to evaluate adipogenic potential of amniotic fluid-derived human mesenchymal stem cells[J].Stem Cells Dev,2006,15(5):719-728.
19 Prusa AR,Marton E,Rosner M,et al.Neurogenic cells in human amniotic fluid[J].Am J Obstet Gynecol,2004,191(1):309-314.
20 Tsai MS,Lee JL,Chang YJ,et al.Isolation of human multipotent mesenchymal stem cells from second-trimester amniotic fluid using a novel two-stage culture protocol[J].Hum report,2004,19(6):1450-1456.
21 Shakibaei M.Inhibition of chondrogenesis by integrin antibody in vitro[J].Exp Cell Res,2002,240(13):95-106.
22 Qureshi HY,Ricci G,Zafarullah M.Smad signaling pathway is a pivotal component of tissue inhibitor of metalloproteinases-3 regulation by transforming growth factor beta in human chondrocytes[J].Biochim Biophys Acta,2008,1783(9):1605-1612..
23 Kolambkar YM,Peister A,Soker S,Atala A,Guldberg RE.Chondrogenic differentiation of amniotic fluid-derived stem cells[J].Mol Histol,2007,38(5):405-413.
24 Elisseeff J,Mcintosh W,Fu K,et al.Controlled release of IGF-1 and TGF-betal in a photopolymerizing hydrogel for cartilage tissue engineering[J].Orthop Res,2004,19(6):1098-1104.
25 Matsuda C,Takagi M,Hattori T,Wakitani S,Yoshida T.Differentiation of Human Bone Marrow Mesenchymal Stem Cells to Chondrocytes for Construction of Three-dimensional Cartilage Tissue[J].Cytotechnology,2005,47(3):7-11.
26 Na K,Kim S,Sun BK,Woo DG,Yang HN,Chung HM,Park KH.Bioimaging of dexamethasone and TGF beta-1 and its biological activities of chondrogenic differentiation in hydrogel constructs[J].Biomed Mater Res A,2008,87(2):283-289.
1 Arshak R,Alexanian.Epigenetic Modifiers Promote Efficient Generation of Neural-Like Cells From Bone Marrow-Derived Mesenchymal Cells Grown in Neural Environment[J].Cell Biochem,2007,100(2):362-371.
2 Moo-I1 Kang,Hye-Soo Kim,Yu-Chae Jung,et al.Transitional CpG Methylation Between Promoters and Retroelements of Tissue-Specific Genes During Human Mesenchymal Cell Differentiation[J].Cell Biochem,2007,102(1):224-239.
3 Azuara V,Perry P,Sauer S,et al.Chromatin signatures of pluripotent cell lines[J].Nat Cell Biol,2006;8(5):532-538.
4 Bernstein BE,Mikkelsen TS,Xie X,et al.A bivalent chromatin structure marks key developmental genes in embryonic stem cells[J].Cell,2006,125(2):315-326.
5 Spivakov M,Fisher AG.Epigenetic signatures of stem-cell identity[J].Nat Rev Genet,2007,8(4):263-271.
6 IValk-Lingbeek ME,Bruggeman SW,van Lohuizen M.Stem cells and cancer:the polycomb connection[J].Cell,2004,118(4):409-418.
7 Vire,E.Brenner C,Deplus R,et al.The Polycomb group protein EZH2 directly controls DNA methylation[J].Natur,2006,439(7078):871-874.
8 Lee TI,Jenner RG,Boyer LA,et al.Control of developmental regulators by Polycomb in human embryonic stem cells[J].Cell,2006,125(2):301-313.
9 Boyer LA,Plath K,Zeitlinger J,et al.Polycomb complexes repress developmental regulators in murine embryonic stem cells[J].Nature,2006,441(7091):349-353.
10 Jorgensen HF,Giadrossi S,Casanova M.et al.Stem cells primed for action:polycomb repressive complexes restrain the expression of lineage-specific regulators in embryonic stem cells[J].Cell Cycle,2006,5(13):1411-1414.
11 Bracken AP,Dietrich N,Pasini D,et al.Genome-wide mapping of Polycomb target genes unravels their roles in cell fate transitions[J].Genes 2006,20(9):1123-1136.
12 Min J,Zhang Y,Xu RM.Structural basis for specific binding of Polycomb chromodomain to histone H3 methylated at Lys 27[J].Genes,2003,17(15):1823-1828.
13 Mohd-Sarip A,van der Knaap JA,Wyman C,et al.Architecture of a Polycomb nucleoprotein complex[J].Mol Cell,2006,24(l):91-100.
14 Zhang H,Christoforou A,Aravind L,et al.The C elegans Polycomb gene SOP-2 encodes an RNA binding protein[J].Mol Cell,2004,14(6):841-847.
15 Lee TI,Jenner RG,Boyer LA,et al.Control of developmental regulators by Polycomb in human embryonic stem cells[J].Cell,2006,125(2):301-313.
16 Chen X,Hiller M,Sancak Y,et al.Tissue-specific TAFs counteract Polycomb to turn on terminal differentiation[J].Science,2005,310(5749):869-872.
17 Chen CZ,Li L,Lodish HF.MicroRNAs modulate hematopoietic lineage differentiation[J].Science,2004,303(5654):83-86.
18 Martinez J,Patkaniowska A,Urlaub H,et al.Single-stranded antisense siRNAs guide target RNA cleavage in RNAi[J].Cell,2002,10(5):563-574.
19 Kanellopoulou C,Muljo S,Kung A,et al.Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing[J].Genes,2006,19(4):489-501.
20 Lin Song and Rocky S.Tuan MicroRNAs and Cell Differentiation in Mammalian Development[J].Birth Defects Research,2006,78(2):140-149.
21 Esau C,Kang X,Peralta E,et al.MicroRNA-143 regulates adipocyte differentiation[J].Biol Chem,2004,279(50):52361-52365.
22 Kawasaki H,Taira K.Transcriptional gene silencing by short interfering RNAs[J].Curr Opin Mol Ther,2005,7(2):125-131.
23 Zaehres H,Lensh MW,Daheron L,et al.High-efficiency RNA interference in human embryonic stem cells[J].Stem Cells,2005,23(3):299～305.
24 Xu Y,Su H,Sani Mirmalek,Yang X,et al.The use of small interfering RNAs to inhibit adipocyte differentiation in human preadipocytes and fetal-femur-derived mesenchymal cells[J].Exp Cell Res,2006,312(10):1856-1864.
25 Boyer LA,Plath K,Zeitlinger J,et al.Polycomb complexes repress developmental regulators in murine embryonic stem cells[J].Nature,2006,441(7091):349-353.
26 Meshorer E,Yellajoshula D,George Eet al.Hyperdynamic plasticity of chromatin proteins in pluripotent embryonic stem cells[J].Dev Cell,2006,10(1):105-116.
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2 Kaviani A,Perry T,Dzakovic A,et al.The amniotic fluid as a source of cells for fetal tissue engineering[J].Ped Surgery,2008,36(ll):1662-1665.
3 Chacko DM,Das AV,Zhao X,et al.Transplantation of ocular stem cells:the role of injury in incorporation and differentiation of grafted cells in the retina[J].Vision Res,2003,43(12):937-946.
4 Fuchs JR,Kaviani A,Oh JT,et al.Diaphragmatic reconstruction with autologous tendon engineered from mesenchymal amniocytes[J].Pediatric Surg,2004,39(6):834-838.
5 Kunisaki SM,Fuchs JR,Kaviani A,et al.Diaphragmatic repair through fetal tissue engineering:a comparison between mesenchymal amniocyte and myoblast-based constructs[J].Pediatric Surg,2006:41(1):34-39.
6 鄂征．组织培养和分子细胞生物学技术[M]．北京：北京出版社,2001．168-169．
7 Prusa AR,Hengstschlager M.Amniotic fluid cells and human stem cell researchra new connection[J].Med Sci Monit,2002,8(11):253-257.
8 Tsai MS,Lee JL,Chang YJ,et al.Isolation of human multipotent mesenchymal stem cells from second trimester amniotic fluid using a novel two-stage culture protocol[J].Hum Reprod,2004,9(6):1450-1457.
9 Tsai MS,Hwang SM,Tsai YL,et al.Clonal Amniotic Fluid-Derived Stem Cells Express Characteristics of Both Mesenchymal and Neural Stem Cells[J].Biology of Reproduction,2006,74(24),545-551.
10 In't Anker PS,Scherjon SA,Kleijburg-van der Keur C,et al.Amniotic fluid as a novel source of mesenchymal stem cells for therapeutic transplantation[J].Blood 2003,102(2):1548-1549.
11 Sartore S,Maddalena L,Annalisa A,et al.Amniotic mesenchymal cells autotransplanted in a porcine model of cardiac ischemia do not differentiate to cardiogenic phenotypes[J].Europ J Card Surg,2005,24(28):677-684.
12 Bossolasco P,Montemurro T,Cova L.et al.Molecular and phenotypic characterization of human amniotic fluid cells and their differentiation potential[J].Cell Res 2006,16(4):329-336.
13 Pesce M,Anastassiadis K,Scholer HR.Oct-4:lessons of totipotency from embryonic stem cells[J].Cells Tissues O狓?s,2005,165(3-4):144-152.
14 Prusa AR,Marton E,Rosner M,et al.Oct-4 expressing cells in human amniotic fluid:a new source for stem cell research?[J].Hum Reprod,2003,18(7):1489-1493.
15 Tsai MS,Hwang SM,Tsai YL,et al.Clonal amniotic fluid-derived stem cells express characteristics of both mesenchymal and neural stem cells[J].Biol.Reprod,2006,74(3):545-551.
16 Kim J,Lee Y,Kim H,et al.Human amniotic fluid-derived stem cells have characteristics of multipotent stem cells[J].Cell Prolif,2007,40(l):75-90.
17 Tsai MS,Hwang SM,Tsai YL,et al.Clonal amniotic fluid-derived stem cells express characteristics of both mesenchymal and neural stem cells[J].Bio Reprod,2006,74(3):545-551.
18 De Gemmis P,Lapucci C,Bertelli M,et al.A real-time PCR approach to evaluate adipogenic potential of amniotic fluid-derived human mesenchymal stem cells[J].Stem Cells Dev,2006,15(5):719-728.
19 Prusa AR,Marton E,Rosner M,et al.Neurogenic cells in human amniotic fluid[J].Am J Obstet Gynecol,2004,191(1):309-314.
20 Tsai MS,Lee JL,Chang YJ,et al.Isolation of human multipotent mesenchymal stem cells from second-trimester amniotic fluid using a novel two-stage culture protocol[J].Hum report,2004,19(6):1450-1456.
21 Shakibaei M.Inhibition of chondrogenesis by integrin antibody in vitro[J].Exp Cell Res,2002,240(13):95-106.
22 Qureshi HY,Ricci G,Zafarullah M.Smad signaling pathway is a pivotal component of tissue inhibitor of metalloproteinases-3 regulation by transforming growth factor beta in human chondrocytes[J].Biochim Biophys Acta,2008,1783(9):1605-1612..
23 Kolambkar YM,Peister A,Soker S,Atala A,Guldberg RE.Chondrogenic differentiation of amniotic fluid-derived stem cells[J].Mol Histol,2007,38(5):405-413.
24 Elisseeff J,Mcintosh W,Fu K,et al.Controlled release of IGF-1 and TGF-betal in a photopolymerizing hydrogel for cartilage tissue engineering[J].Orthop Res,2004,19(6):1098-1104.
25 Matsuda C,Takagi M,Hattori T,Wakitani S,Yoshida T.Differentiation of Human Bone Marrow Mesenchymal Stem Cells to Chondrocytes for Construction of Three-dimensional Cartilage Tissue[J].Cytotechnology,2005,47(3):7-11.
26 Na K,Kim S,Sun BK,Woo DG,Yang HN,Chung HM,Park KH.Bioimaging of dexamethasone and TGF beta-1 and its biological activities of chondrogenic differentiation in hydrogel constructs[J].Biomed Mater Res A,2008,87(2):283-289.
1 Arshak R,Alexanian.Epigenetic Modifiers Promote Efficient Generation of Neural-Like Cells From Bone Marrow-Derived Mesenchymal Cells Grown in Neural Environment[J].Cell Biochem,2007,100(2):362-371.
2 Moo-I1 Kang,Hye-Soo Kim,Yu-Chae Jung,et al.Transitional CpG Methylation Between Promoters and Retroelements of Tissue-Specific Genes During Human Mesenchymal Cell Differentiation[J].Cell Biochem,2007,102(1):224-239.
3 Azuara V,Perry P,Sauer S,et al.Chromatin signatures of pluripotent cell lines[J].Nat Cell Biol,2006;8(5):532-538.
4 Bernstein BE,Mikkelsen TS,Xie X,et al.A bivalent chromatin structure marks key developmental genes in embryonic stem cells[J].Cell,2006,125(2):315-326.
5 Spivakov M,Fisher AG.Epigenetic signatures of stem-cell identity[J].Nat Rev Genet,2007,8(4):263-271.
6 IValk-Lingbeek ME,Bruggeman SW,van Lohuizen M.Stem cells and cancer:the polycomb connection[J].Cell,2004,118(4):409-418.
7 Vire,E.Brenner C,Deplus R,et al.The Polycomb group protein EZH2 directly controls DNA methylation[J].Natur,2006,439(7078):871-874.
8 Lee TI,Jenner RG,Boyer LA,et al.Control of developmental regulators by Polycomb in human embryonic stem cells[J].Cell,2006,125(2):301-313.
9 Boyer LA,Plath K,Zeitlinger J,et al.Polycomb complexes repress developmental regulators in murine embryonic stem cells[J].Nature,2006,441(7091):349-353.
10 Jorgensen HF,Giadrossi S,Casanova M.et al.Stem cells primed for action:polycomb repressive complexes restrain the expression of lineage-specific regulators in embryonic stem cells[J].Cell Cycle,2006,5(13):1411-1414.
11 Bracken AP,Dietrich N,Pasini D,et al.Genome-wide mapping of Polycomb target genes unravels their roles in cell fate transitions[J].Genes 2006,20(9):1123-1136.
12 Min J,Zhang Y,Xu RM.Structural basis for specific binding of Polycomb chromodomain to histone H3 methylated at Lys 27[J].Genes,2003,17(15):1823-1828.
13 Mohd-Sarip A,van der Knaap JA,Wyman C,et al.Architecture of a Polycomb nucleoprotein complex[J].Mol Cell,2006,24(l):91-100.
14 Zhang H,Christoforou A,Aravind L,et al.The C elegans Polycomb gene SOP-2 encodes an RNA binding protein[J].Mol Cell,2004,14(6):841-847.
15 Lee TI,Jenner RG,Boyer LA,et al.Control of developmental regulators by Polycomb in human embryonic stem cells[J].Cell,2006,125(2):301-313.
16 Chen X,Hiller M,Sancak Y,et al.Tissue-specific TAFs counteract Polycomb to turn on terminal differentiation[J].Science,2005,310(5749):869-872.
17 Chen CZ,Li L,Lodish HF.MicroRNAs modulate hematopoietic lineage differentiation[J].Science,2004,303(5654):83-86.
18 Martinez J,Patkaniowska A,Urlaub H,et al.Single-stranded antisense siRNAs guide target RNA cleavage in RNAi[J].Cell,2002,10(5):563-574.
19 Kanellopoulou C,Muljo S,Kung A,et al.Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing[J].Genes,2006,19(4):489-501.
20 Lin Song and Rocky S.Tuan MicroRNAs and Cell Differentiation in Mammalian Development[J].Birth Defects Research,2006,78(2):140-149.
21 Esau C,Kang X,Peralta E,et al.MicroRNA-143 regulates adipocyte differentiation[J].Biol Chem,2004,279(50):52361-52365.
22 Kawasaki H,Taira K.Transcriptional gene silencing by short interfering RNAs[J].Curr Opin Mol Ther,2005,7(2):125-131.
23 Zaehres H,Lensh MW,Daheron L,et al.High-efficiency RNA interference in human embryonic stem cells[J].Stem Cells,2005,23(3):299～305.
24 Xu Y,Su H,Sani Mirmalek,Yang X,et al.The use of small interfering RNAs to inhibit adipocyte differentiation in human preadipocytes and fetal-femur-derived mesenchymal cells[J].Exp Cell Res,2006,312(10):1856-1864.
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