中华器官移植杂志
中華器官移植雜誌
중화기관이식잡지
CHINESE JOURNAL OF ORGAN TRANSPLANTATION
2008年
3期
159-163
,共5页
胎血%间质干细胞%细胞分化%肝细胞
胎血%間質榦細胞%細胞分化%肝細胞
태혈%간질간세포%세포분화%간세포
Fetal blood%Mesenchymal stem cells%Cell differentiation%Hepatocytes
目的 探讨脐血间充质干细胞(MSCs)在体外能否分化成肝细胞.方法 分离人脐血MSCs,培养传代,流式细胞仪进行细胞表面标志检测.取培养至第三代的细胞,接种于六孔板内,分两个阶段进行细胞分化的诱导,第一阶段采用含地塞米松(终浓度为0.5 μmol/L,下同)、肝细胞生长因子(HGF,10 ng/ml)、表皮生长因子(10 ng/ml)及1×ITS(胰岛素-转铁蛋白-硒)的F12培养基诱导2周,第二阶段用含地塞米松(0.5 μmol/L)、HGF(10 ng/ml)、抑瘤素M(10 ng/ml)及1×ITS的F12培养基继续诱导2周.诱导期间于倒置显微镜下观察细胞的形态变化;采用逆转录聚合酶链反应检测分化细胞的甲胎蛋白(AFP)、白蛋白、人细胞角蛋白18(CK-18)及酪氨酸氨基转移酶(TAT)基因的表达,以免疫荧光染色法检测分化细胞胞浆中AFP、白蛋白、CK-18的表达;用电子显微镜观察分化细胞的超微结构.结果 培养的脐血MSCs不表达CD14、CD34及CD45,也不表达CD49f、CD54及HLA-DR;部分表达CD106;强表达CD13、CD29及CD44.未分化的脐血MSCs不表达AFP、TAT及白蛋白基因,弱表达CK-18基因;诱导分化1周后可检测到AFP基因的表达,诱导分化4周后,不仅表达AFP、CK-18和白蛋白基因,还表达TAT基因.免疫荧光染色显示,未分化的MSCs胞浆中无AFP、白蛋白及CK-18的表达;分化细胞则可以检测到上述物质的表达.诱导至第4周的分化细胞,可见核仁大且明显,细胞核周围有板层状的内质网,胞浆中可见脂滴及簇状糖原,线粒体丰富,细胞表面有微绒毛.结论 脐血MSCs在合适的诱导条件下可以分化为肝样细胞.
目的 探討臍血間充質榦細胞(MSCs)在體外能否分化成肝細胞.方法 分離人臍血MSCs,培養傳代,流式細胞儀進行細胞錶麵標誌檢測.取培養至第三代的細胞,接種于六孔闆內,分兩箇階段進行細胞分化的誘導,第一階段採用含地塞米鬆(終濃度為0.5 μmol/L,下同)、肝細胞生長因子(HGF,10 ng/ml)、錶皮生長因子(10 ng/ml)及1×ITS(胰島素-轉鐵蛋白-硒)的F12培養基誘導2週,第二階段用含地塞米鬆(0.5 μmol/L)、HGF(10 ng/ml)、抑瘤素M(10 ng/ml)及1×ITS的F12培養基繼續誘導2週.誘導期間于倒置顯微鏡下觀察細胞的形態變化;採用逆轉錄聚閤酶鏈反應檢測分化細胞的甲胎蛋白(AFP)、白蛋白、人細胞角蛋白18(CK-18)及酪氨痠氨基轉移酶(TAT)基因的錶達,以免疫熒光染色法檢測分化細胞胞漿中AFP、白蛋白、CK-18的錶達;用電子顯微鏡觀察分化細胞的超微結構.結果 培養的臍血MSCs不錶達CD14、CD34及CD45,也不錶達CD49f、CD54及HLA-DR;部分錶達CD106;彊錶達CD13、CD29及CD44.未分化的臍血MSCs不錶達AFP、TAT及白蛋白基因,弱錶達CK-18基因;誘導分化1週後可檢測到AFP基因的錶達,誘導分化4週後,不僅錶達AFP、CK-18和白蛋白基因,還錶達TAT基因.免疫熒光染色顯示,未分化的MSCs胞漿中無AFP、白蛋白及CK-18的錶達;分化細胞則可以檢測到上述物質的錶達.誘導至第4週的分化細胞,可見覈仁大且明顯,細胞覈週圍有闆層狀的內質網,胞漿中可見脂滴及簇狀糖原,線粒體豐富,細胞錶麵有微絨毛.結論 臍血MSCs在閤適的誘導條件下可以分化為肝樣細胞.
목적 탐토제혈간충질간세포(MSCs)재체외능부분화성간세포.방법 분리인제혈MSCs,배양전대,류식세포의진행세포표면표지검측.취배양지제삼대적세포,접충우륙공판내,분량개계단진행세포분화적유도,제일계단채용함지새미송(종농도위0.5 μmol/L,하동)、간세포생장인자(HGF,10 ng/ml)、표피생장인자(10 ng/ml)급1×ITS(이도소-전철단백-서)적F12배양기유도2주,제이계단용함지새미송(0.5 μmol/L)、HGF(10 ng/ml)、억류소M(10 ng/ml)급1×ITS적F12배양기계속유도2주.유도기간우도치현미경하관찰세포적형태변화;채용역전록취합매련반응검측분화세포적갑태단백(AFP)、백단백、인세포각단백18(CK-18)급락안산안기전이매(TAT)기인적표체,이면역형광염색법검측분화세포포장중AFP、백단백、CK-18적표체;용전자현미경관찰분화세포적초미결구.결과 배양적제혈MSCs불표체CD14、CD34급CD45,야불표체CD49f、CD54급HLA-DR;부분표체CD106;강표체CD13、CD29급CD44.미분화적제혈MSCs불표체AFP、TAT급백단백기인,약표체CK-18기인;유도분화1주후가검측도AFP기인적표체,유도분화4주후,불부표체AFP、CK-18화백단백기인,환표체TAT기인.면역형광염색현시,미분화적MSCs포장중무AFP、백단백급CK-18적표체;분화세포칙가이검측도상술물질적표체.유도지제4주적분화세포,가견핵인대차명현,세포핵주위유판층상적내질망,포장중가견지적급족상당원,선립체봉부,세포표면유미융모.결론 제혈MSCs재합괄적유도조건하가이분화위간양세포.
Objective To discuss the possibility of differentiation of mesenchymal stem cells(MSCs)from umbilical cord blood(UCB)into hepatocyte-like cells in the in vitro culture.Methods MSCs were isolated from UCB,cultured and passaged.The surface markers were examined by flow cytometry.When cells were cultured to the third passage,they were inoculated into a 6-well plate.A two-stage induction method was used:MSCs in the first phase were cultured in DMEM/F12 medium supplemented with dexamethasone(final concentration of 0.5 μmol/L,the same below),hepatocyte growth factor(HGF,10 ng/ml),epidermal growth factor(10 ng/m1)and 1×insulin-transferrin-Se(ITS)for two weeks,then in DMEM/F12 supplemented with 0.5 μmol/L dexamethasone,10 ng/ml HGF,1×ITS,10 ng/ml Oncostatin M for another two weeks.Morphological changes were observed under a microscope.The gene expression correlated with hepatocytes was detected by using RT-PCR.Immunofluorescence staining was used to identify the expression of specific protein related to hepatocytes(AFP,Albumin,CK-18).Ultrastructure was detected under an electron microscope.Results In the cultured MSCs from UCB,CD34/CD45/CD14,CD54,CD49f and HLA-DR were not detected,there was low expression of CD106 and strong expression of CD29,CD44 and CD13.The gene expression of AFP,albumin,CK-18 and TAT was discovered and three kinds of protein AFP,albumin and CK-18 were positively showed in cytoplasm after 4 weeks'induction.The hepatin granules and fatty drops in cytoplasm of cells induced for 4 weeks were found under an electron microscope.Conclusion The MSCs fromUCB can differentiate into hepatocyte-like ceils in the in vitro culture under some conditions.
