CAJ | 학술논문

背景:目前研究认为在胚胎发育后的多种组织中存在一类干细胞群体,可分化为不同胚层的组织细胞;但又不同于胚胎干细胞,随着妊娠期间胚胎的发育胚胎干细胞会逐渐失去部分分化潜能,会出现一些特殊表型或分子标记,如CD105,称其为胚胎后亚全能干细胞.目的:根据胚胎后亚全能干细胞表达CD105的特性分离胎儿骨髓源性胚胎后亚全能干细胞,经体内外诱导使其分化为肝细胞样细胞并检测其功能.设计、时间及地点:动物随机分组,细胞分子生物学实验,于2003-03/2005-03在天津市第三中心医院卫生部人工细胞工程技术研究中心完成.材料:取22周龄左右胎儿股骨和胫骨骨髓分离出胚胎后亚全能干细胞.以雌性成年SCID鼠作为干细胞移植的受体.CD105免疫磁珠为德国Miltenyi Biotec产品.鼠抗人白蛋白抗体为美国Sigma产品.碱性成纤维细胞生长因子、肝细胞生长因子为英国PEPROTECH产品.方法:利用密度梯度离心结合免疫磁珠方法分离胎儿骨髓CD105(+)胚胎后亚全能干细胞,体外培养,用含30 μg/L肝细胞生长因20 μg/L碱性成纤维细胞生长因子的诱导培养基将其向肝细胞样细胞诱导分化.将24只SCID鼠随机分为干细胞治疗组和对照组,每组12只,均按800 mg/kg的剂量向腹腔内注射D-氨基半乳糖制备肝损伤模型.造模次日,干细胞移植组鼠在肝原位输注106左右CD105(+)细胞,对照组分别输注106左右的CD105(-)细胞或同等体积的培养液.主要观察指标:于干细胞移植后2,7d,1,3个月对肝脏组织行免疫组化检测人源白蛋白表达.结果:免疫磁珠筛选后的细胞免疫细胞化学检测CD105呈弱阳性表达:细胞在对数生长期的倍增时间为30 h左右:约传10代后进入衰退期.SCID鼠移植胚胎后亚全能干细胞3个月后在小鼠肝脏中可见有点状或小灶状的人白蛋白表达,对照组未见表达.结论:来源于胎儿骨髓的胚胎后亚全能干细胞可以在肝脏微环境下转化为肝细胞样细胞. 揹景:目前研究認為在胚胎髮育後的多種組織中存在一類榦細胞群體,可分化為不同胚層的組織細胞;但又不同于胚胎榦細胞,隨著妊娠期間胚胎的髮育胚胎榦細胞會逐漸失去部分分化潛能,會齣現一些特殊錶型或分子標記,如CD105,稱其為胚胎後亞全能榦細胞.目的:根據胚胎後亞全能榦細胞錶達CD105的特性分離胎兒骨髓源性胚胎後亞全能榦細胞,經體內外誘導使其分化為肝細胞樣細胞併檢測其功能.設計、時間及地點:動物隨機分組,細胞分子生物學實驗,于2003-03/2005-03在天津市第三中心醫院衛生部人工細胞工程技術研究中心完成.材料:取22週齡左右胎兒股骨和脛骨骨髓分離齣胚胎後亞全能榦細胞.以雌性成年SCID鼠作為榦細胞移植的受體.CD105免疫磁珠為德國Miltenyi Biotec產品.鼠抗人白蛋白抗體為美國Sigma產品.堿性成纖維細胞生長因子、肝細胞生長因子為英國PEPROTECH產品.方法:利用密度梯度離心結閤免疫磁珠方法分離胎兒骨髓CD105(+)胚胎後亞全能榦細胞,體外培養,用含30 μg/L肝細胞生長因20 μg/L堿性成纖維細胞生長因子的誘導培養基將其嚮肝細胞樣細胞誘導分化.將24隻SCID鼠隨機分為榦細胞治療組和對照組,每組12隻,均按800 mg/kg的劑量嚮腹腔內註射D-氨基半乳糖製備肝損傷模型.造模次日,榦細胞移植組鼠在肝原位輸註106左右CD105(+)細胞,對照組分彆輸註106左右的CD105(-)細胞或同等體積的培養液.主要觀察指標:于榦細胞移植後2,7d,1,3箇月對肝髒組織行免疫組化檢測人源白蛋白錶達.結果:免疫磁珠篩選後的細胞免疫細胞化學檢測CD105呈弱暘性錶達:細胞在對數生長期的倍增時間為30 h左右:約傳10代後進入衰退期.SCID鼠移植胚胎後亞全能榦細胞3箇月後在小鼠肝髒中可見有點狀或小竈狀的人白蛋白錶達,對照組未見錶達.結論:來源于胎兒骨髓的胚胎後亞全能榦細胞可以在肝髒微環境下轉化為肝細胞樣細胞.
배경:목전연구인위재배태발육후적다충조직중존재일류간세포군체,가분화위불동배층적조직세포;단우불동우배태간세포,수착임신기간배태적발육배태간세포회축점실거부분분화잠능,회출현일사특수표형혹분자표기,여CD105,칭기위배태후아전능간세포.목적:근거배태후아전능간세포표체CD105적특성분리태인골수원성배태후아전능간세포,경체내외유도사기분화위간세포양세포병검측기공능.설계、시간급지점:동물수궤분조,세포분자생물학실험,우2003-03/2005-03재천진시제삼중심의원위생부인공세포공정기술연구중심완성.재료:취22주령좌우태인고골화경골골수분리출배태후아전능간세포.이자성성년SCID서작위간세포이식적수체.CD105면역자주위덕국Miltenyi Biotec산품.서항인백단백항체위미국Sigma산품.감성성섬유세포생장인자、간세포생장인자위영국PEPROTECH산품.방법:이용밀도제도리심결합면역자주방법분리태인골수CD105(+)배태후아전능간세포,체외배양,용함30 μg/L간세포생장인20 μg/L감성성섬유세포생장인자적유도배양기장기향간세포양세포유도분화.장24지SCID서수궤분위간세포치료조화대조조,매조12지,균안800 mg/kg적제량향복강내주사D-안기반유당제비간손상모형.조모차일,간세포이식조서재간원위수주106좌우CD105(+)세포,대조조분별수주106좌우적CD105(-)세포혹동등체적적배양액.주요관찰지표:우간세포이식후2,7d,1,3개월대간장조직행면역조화검측인원백단백표체.결과:면역자주사선후적세포면역세포화학검측CD105정약양성표체:세포재대수생장기적배증시간위30 h좌우:약전10대후진입쇠퇴기.SCID서이식배태후아전능간세포3개월후재소서간장중가견유점상혹소조상적인백단백표체,대조조미견표체.결론:래원우태인골수적배태후아전능간세포가이재간장미배경하전화위간세포양세포.
BACKGROUND: At present, studies show that a kind of stem cell community which in many kinds of organizations can differentiate into tissue cells of different embryonic layers; but those are different from embryonic stem cells, embryonic stem cell will lose the part differentiation potential gradually during the development of pregnancy, and will present some special phenotypes or the molecular markers, as CD105 and so on, will cell it postembryonic pluripotent stem cells.OBJECTIVE: To study the isolation of postembryonic pluripotent stem cells from fetal bone marrow, proliferative culture in vitro, induction and differentiation; transplantation to the liver of SCID mice with hepatic failure, and detect therapy effects.DESIGN, TIME AND SETTING: Cell observation and animal randomization experiment which was completed in the Ministry of Health of Cell Engineering Technology Research Center, Tianjin Third Central Hospital from March 2003 to March 2005.MATERIALS: The postembryonic pluripotent stem cells were extracted from thighbone and shinbone of 22-week old fetuses under sterile circumstance. Adult female SCID mice ware regarded as the recipients. CD105 immunornagnetic beads were provided by Miltenyi Biotec, Germany; mouse-anti-human albumin by Sigma, USA; basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF) by PEPROTECH, UK.METHODS: Postembryonic pluripotant stem cells obtained from fetal bone marrow were isolated using density gradient centrifugation and micromagnetic beads technique. The hepatocyte-like cells were induced and differentiated with culture media containing HGF (30 ng/mL) and bFGF (20 ng/mL). Twenty-four SCID mice were randomly divided into experimental group and control group with 12 mice in each group. Hepatic injury models were established with intraperitoneal injection of D-galactosamine On the next day, about 106 CD105(+) cells were perfused into liver in situ in the experimental group, and about 106 CD105(-) cells or isovolumic culture medium were perfused in the control group.MAIN OUTCOME MEASURES: Two, seven days, one and three months after the transplantation of cells, human albumin expression in the liver tissue was detected by immunohistochemistry.RESULTS: The immunocytochemical assay of the cells after micromagnetic beads selection showed that the CD105 expression was slightly positive; the doubling time of the cells in the logarithmic growth period was around 30 hours; after being expanded for 10 population doublings, the cells entered decline period. The cells were transplanted into SCID mice's liver; 3 months later, the human serum albumin in the mouse liver was assessed by using monoclonal antibody of mouse-anti-human serum albumin, dotted or small focal expression of the protein could be detected. However, any expression was not observed in the control group.CONCLUSION: The bone marrow-derived pluripotent stem cells are able to transform to hepatocytas in the hepatic microenvironment.