中国组织工程研究与临床康复
中國組織工程研究與臨床康複
중국조직공정연구여림상강복
JOURNAL OF CLINICAL REHABILITATIVE TISSUE ENGINEERING RESEARCH
2009年
45期
8889-8894
,共6页
背景:研究表明FGF2,TGFβ/activin/nodal和IGF信号通路是人胚胎干细胞保持其多能性所必需的,然而直接添加外源性碱性成纤维细胞生长因子、转化生长因子β和胰岛素是否能够维持人胚胎干细胞的自我更新目前尚无报道.目的:拟建立人胚胎干细胞无饲养层无血清条件下的培养体系.设计,时间及地点:细胞学体外观察,于2007-09/2009-02在中国科学院昆明动物研究所完成.材料:12.5～13.5d龄清洁级ICR孕鼠2只,由昆明医学院动物中心提供.人胚胎干细胞株BG02购自美国Bresagen公司.方法:①消化离心BG02细胞,重悬于无饲养层过渡培养基后接种,常规培养5～7 d,挑去分化的人胚胎干细胞,加入分散酶消化,切割成小团块,离心重悬后按1:3比例重新接种预铺层粘连蛋白的4孔板,此时培养基换成无血清无饲养层培养基,由80%DMEM/F12、20%KSR、2 mmol/L glutamine、1%非必需氨基酸、0.1 mmol/L β-巯基乙醇、ITS(×1)、10~6 U/L青霉素、100 mg/L 链霉素、4 μg/L碱性成纤维细胞生长因子、0.12 μg/L转化生长因子β1组成.②无菌条件下取出ICR胎鼠,组织块胰酶消化法分离培养小鼠胚胎成纤维细胞,接种在0.1%明胶包被的6孔板中即为饲养层.将生长在小鼠胚胎成纤维细胞饲养层上的人胚胎干细胞株BG02预铺至有层粘连蛋白的培养板上,加入含碱性成纤维细胞生长因子、转化生长因子β1及ITS的无血清培养基连续培养.主要观察指标:观察BG02细胞在无血清无饲养层培养体系中的形态,采用细胞免疫组化法检测人胚胎干细胞特异性分子标志的表达,检测BG02细胞体外分化能力及其核型,比较BG02细胞在无血清无饲养层和小鼠胚胎成纤维细胞饲养层培养体系中的生长情况、细胞集落分化率.结果:在无血清无饲养层培养体系中,BG02细胞连续传代20代,细胞呈典型的人胚胎干细胞形态特征;BG02细胞表达SSEA-4,SSEA-3,TRA-1-60,TRA-1-81,Oct-4,但不表达SSEA-1;培养20 d后,BG02细胞进一步分化成3个胚层的细胞,分化细胞能够表达甲胎蛋白、巢蛋白、α-肌动蛋白;培养至20代细胞核型均正常(46XY).与在饲养层培养体系下比较,无血清无饲养层条件下BG02细胞BG02细胞生长速度减慢,细胞倍增时间明显延长(P＜0.05),集落分化率明显升高(P＜0.05).结论:实验初步建立了入胚胎干细胞无血清无饲养层的培养体系,添加碱性成纤维细胞生长因子、转化生长因子β1和ITS可以维持人胚胎干细胞的自我更新.
揹景:研究錶明FGF2,TGFβ/activin/nodal和IGF信號通路是人胚胎榦細胞保持其多能性所必需的,然而直接添加外源性堿性成纖維細胞生長因子、轉化生長因子β和胰島素是否能夠維持人胚胎榦細胞的自我更新目前尚無報道.目的:擬建立人胚胎榦細胞無飼養層無血清條件下的培養體繫.設計,時間及地點:細胞學體外觀察,于2007-09/2009-02在中國科學院昆明動物研究所完成.材料:12.5～13.5d齡清潔級ICR孕鼠2隻,由昆明醫學院動物中心提供.人胚胎榦細胞株BG02購自美國Bresagen公司.方法:①消化離心BG02細胞,重懸于無飼養層過渡培養基後接種,常規培養5～7 d,挑去分化的人胚胎榦細胞,加入分散酶消化,切割成小糰塊,離心重懸後按1:3比例重新接種預鋪層粘連蛋白的4孔闆,此時培養基換成無血清無飼養層培養基,由80%DMEM/F12、20%KSR、2 mmol/L glutamine、1%非必需氨基痠、0.1 mmol/L β-巰基乙醇、ITS(×1)、10~6 U/L青黴素、100 mg/L 鏈黴素、4 μg/L堿性成纖維細胞生長因子、0.12 μg/L轉化生長因子β1組成.②無菌條件下取齣ICR胎鼠,組織塊胰酶消化法分離培養小鼠胚胎成纖維細胞,接種在0.1%明膠包被的6孔闆中即為飼養層.將生長在小鼠胚胎成纖維細胞飼養層上的人胚胎榦細胞株BG02預鋪至有層粘連蛋白的培養闆上,加入含堿性成纖維細胞生長因子、轉化生長因子β1及ITS的無血清培養基連續培養.主要觀察指標:觀察BG02細胞在無血清無飼養層培養體繫中的形態,採用細胞免疫組化法檢測人胚胎榦細胞特異性分子標誌的錶達,檢測BG02細胞體外分化能力及其覈型,比較BG02細胞在無血清無飼養層和小鼠胚胎成纖維細胞飼養層培養體繫中的生長情況、細胞集落分化率.結果:在無血清無飼養層培養體繫中,BG02細胞連續傳代20代,細胞呈典型的人胚胎榦細胞形態特徵;BG02細胞錶達SSEA-4,SSEA-3,TRA-1-60,TRA-1-81,Oct-4,但不錶達SSEA-1;培養20 d後,BG02細胞進一步分化成3箇胚層的細胞,分化細胞能夠錶達甲胎蛋白、巢蛋白、α-肌動蛋白;培養至20代細胞覈型均正常(46XY).與在飼養層培養體繫下比較,無血清無飼養層條件下BG02細胞BG02細胞生長速度減慢,細胞倍增時間明顯延長(P＜0.05),集落分化率明顯升高(P＜0.05).結論:實驗初步建立瞭入胚胎榦細胞無血清無飼養層的培養體繫,添加堿性成纖維細胞生長因子、轉化生長因子β1和ITS可以維持人胚胎榦細胞的自我更新.
배경:연구표명FGF2,TGFβ/activin/nodal화IGF신호통로시인배태간세포보지기다능성소필수적,연이직접첨가외원성감성성섬유세포생장인자、전화생장인자β화이도소시부능구유지인배태간세포적자아경신목전상무보도.목적:의건립인배태간세포무사양층무혈청조건하적배양체계.설계,시간급지점:세포학체외관찰,우2007-09/2009-02재중국과학원곤명동물연구소완성.재료:12.5～13.5d령청길급ICR잉서2지,유곤명의학원동물중심제공.인배태간세포주BG02구자미국Bresagen공사.방법:①소화리심BG02세포,중현우무사양층과도배양기후접충,상규배양5～7 d,도거분화적인배태간세포,가입분산매소화,절할성소단괴,리심중현후안1:3비례중신접충예포층점련단백적4공판,차시배양기환성무혈청무사양층배양기,유80%DMEM/F12、20%KSR、2 mmol/L glutamine、1%비필수안기산、0.1 mmol/L β-구기을순、ITS(×1)、10~6 U/L청매소、100 mg/L 련매소、4 μg/L감성성섬유세포생장인자、0.12 μg/L전화생장인자β1조성.②무균조건하취출ICR태서,조직괴이매소화법분리배양소서배태성섬유세포,접충재0.1%명효포피적6공판중즉위사양층.장생장재소서배태성섬유세포사양층상적인배태간세포주BG02예포지유층점련단백적배양판상,가입함감성성섬유세포생장인자、전화생장인자β1급ITS적무혈청배양기련속배양.주요관찰지표:관찰BG02세포재무혈청무사양층배양체계중적형태,채용세포면역조화법검측인배태간세포특이성분자표지적표체,검측BG02세포체외분화능력급기핵형,비교BG02세포재무혈청무사양층화소서배태성섬유세포사양층배양체계중적생장정황、세포집락분화솔.결과:재무혈청무사양층배양체계중,BG02세포련속전대20대,세포정전형적인배태간세포형태특정;BG02세포표체SSEA-4,SSEA-3,TRA-1-60,TRA-1-81,Oct-4,단불표체SSEA-1;배양20 d후,BG02세포진일보분화성3개배층적세포,분화세포능구표체갑태단백、소단백、α-기동단백;배양지20대세포핵형균정상(46XY).여재사양층배양체계하비교,무혈청무사양층조건하BG02세포BG02세포생장속도감만,세포배증시간명현연장(P＜0.05),집락분화솔명현승고(P＜0.05).결론:실험초보건립료입배태간세포무혈청무사양층적배양체계,첨가감성성섬유세포생장인자、전화생장인자β1화ITS가이유지인배태간세포적자아경신.
BACKGROUND:FGF2,TGFβ/activin/nodal and IGF signal pathways are necessary for keeping functions of human embryonic stem calls,but there was no report concerning whether addition of exogenous basic fibroblast growth factor,transforming growth factor β and insulin can maintain self-renewal of human embryonic stern cells.OBJECTIVE:To establish a feeder layer-and serum-free culture system of human embryonic stem cells.DESIGN,TIME AND SETTING:The cytological in vitro study was performed at the Kunming Animal Institute of Chinese Academy of Sciences from September 2007 to February 2009.MATERIALS:Pregnancy 12.5 or 13.5-day ICR strain mica (clean grade) were provided by the Animal Center of Kunming Medical College.Human embryonic stem cell line BG02 was purchased from Bresagen,USA.METHODS:BG02 cells were digested,centrifuged,resuspended in feeder layer-free medium,and then incubated for 5-7 days.Differentiated human embryonic stem cells were removed.Dispase was added for digestion.Samples were cut into blocks,centrifuged,resuspended,and then incubated at 1:3 in a 4-well plate coated with laminin in feeder layer-free and serum-free medium,supplemented with 80% DMEM/F12,20% KSR,2 mmol/L glutamine,1% non-essential amino acid,0.1 mmol/L β-mercaptoethanol,insulin-transferrin-selenium (ITS) (×1),10~6 U/L penicillin,100 mg/L streptomycin,4 μg/L basic fibroblast growth factor (bFGF) and 0.12 μg/L transforming growth factor-β1 (TGF-β1).ICR fetus mica were sterilely obtained to culture mouse embryonic flbroblasts by tissue pancreatin digestion method.These cells were incubated in a 6-well plate coated with 0.1% gelatin.Human embryonic stem cell line BG02 cultured on mouse embryonic fibroblasts feeder layers was transferred to laminin-coated plates in serum-free medium containing bFGF,TGFβ1 and ITS.MAIN OUTCOME MEASURES:The morphology of BG02 cells in feeder layer-and serum-free condition was observed.The specific molecular markers of human embryonic stem cells were determined by immunohistochemical staining.The karyotype and differentiation ability in vitro of BG02 cells were analyzed.The differences in the proliferation and the differentiated rate of BG02 clumps in feeder layer-and serum-free condition or mouse embryonic fibroblast feeder layer condition were compared.RESULTS:BG02 calls had been continuously cultured for at least 20 passages in feeder layer-and serum-free culture condition.BG02 clones in this culture system had typical human embryonic stem cell morphology.BG02 cells all expressed SSEA-4,SSEA-3,TRA-1-60,TRA-1-81,Oct-4,but did not express SSEA-1.After 20 days of culture,BG02 cells had the capacity to differentiate into derivatives of embryonic endoderm,mesoderm,and ectoderm.Differentiated cells could express alpha-fetoprotein,nestin,α-actin.At passage 20,call karyotype was normal (46XY).The growth of BG02 cells cultured in feeder layer-and serum-free condition was more slowly compared with mouse embryonic fibroblast feeder,and doubling time was significantly prolonged (P ＜ 0.05).Differentiation rate of the colonies was significantly increased (P ＜ 0.05).CONCLUSION:A feeder layer-and serum-free condition for culture of BG02 cells has been established.The addition of bFGF,TGFβ1 and ITS can maintain the self-renewal of BG02 cells.
