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
2008年
47期
9389-9393
,共5页
刘雄英%蒋宁一%张绪超%周敦华%陈贵兵%胡莹莹%刘幸光%张弘%刘生%孟瑛%黄绍良
劉雄英%蔣寧一%張緒超%週敦華%陳貴兵%鬍瑩瑩%劉倖光%張弘%劉生%孟瑛%黃紹良
류웅영%장저일%장서초%주돈화%진귀병%호형형%류행광%장홍%류생%맹영%황소량
胚胎干细胞%分化%甲状腺细胞%促甲状腺素
胚胎榦細胞%分化%甲狀腺細胞%促甲狀腺素
배태간세포%분화%갑상선세포%촉갑상선소
背景:胚胎干细胞来源于发育早期囊胚的内细胞团,适当条件下能在体外维持未分化状态、正常二倍体核型及无限增殖能力,且具有多向分化潜能,能够发育分化成机体3个胚层的所有类型细胞.目的:观察体外定向诱导胚胎干细胞分化为甲状腺细胞的可行性及相关分子表达检测.设计、时间及地点:以细胞为对象的观察实验,于2004-01/2006-12在中山大学附属第二医院脐血库完成.材料:孕12.5～14.5 d的Balb/c孕鼠用于胚成纤维细胞饲养层的制备.E14小鼠胚胎干细胞细胞株由美国哈佛大学DLXu教授惠赠.成年昆明种小鼠用于甲状腺细胞的提取.方法:将制备的鼠胚成纤维饲养层接种于E14小鼠胚胎干细胞进行扩增培养.将脱离饲养层细胞后呈稳定克隆生长的胚胎干细胞诱导发育为胚胎体,再逐步添加促甲状腺素、胰岛素、碘化钾等共培养.以培养的成年昆明种小鼠甲状腺细胞为阳性对照.主要观察指标:①观察分化过程中细胞形态的变化.②免疫荧光法检测分化细胞标记物TSHR,PAX8,TTF-2,TTF-1的表达.③RT-PCR法检测分化细胞相关基因TSHR、PAX8,NIS,TPO,Tg的表达.结果:分化细胞边界清晰,呈圆形、类圆形、梭形或多角形贴壁生长.诱导培养第6天分化细胞中有甲状腺细胞特有基因PAX8,NIS,TPO,Tg,TSHR的表达,第8天检测到分化细胞中甲状腺细胞标记物TSHR,TIF-1,PAX8,TIF-2的表达,阳性细胞形态类似对照甲状腺细胞.结论:胚眙干细胞经胚胎体发育阶段,在特定条件下可定向分化为甲状腺细胞.
揹景:胚胎榦細胞來源于髮育早期囊胚的內細胞糰,適噹條件下能在體外維持未分化狀態、正常二倍體覈型及無限增殖能力,且具有多嚮分化潛能,能夠髮育分化成機體3箇胚層的所有類型細胞.目的:觀察體外定嚮誘導胚胎榦細胞分化為甲狀腺細胞的可行性及相關分子錶達檢測.設計、時間及地點:以細胞為對象的觀察實驗,于2004-01/2006-12在中山大學附屬第二醫院臍血庫完成.材料:孕12.5～14.5 d的Balb/c孕鼠用于胚成纖維細胞飼養層的製備.E14小鼠胚胎榦細胞細胞株由美國哈彿大學DLXu教授惠贈.成年昆明種小鼠用于甲狀腺細胞的提取.方法:將製備的鼠胚成纖維飼養層接種于E14小鼠胚胎榦細胞進行擴增培養.將脫離飼養層細胞後呈穩定剋隆生長的胚胎榦細胞誘導髮育為胚胎體,再逐步添加促甲狀腺素、胰島素、碘化鉀等共培養.以培養的成年昆明種小鼠甲狀腺細胞為暘性對照.主要觀察指標:①觀察分化過程中細胞形態的變化.②免疫熒光法檢測分化細胞標記物TSHR,PAX8,TTF-2,TTF-1的錶達.③RT-PCR法檢測分化細胞相關基因TSHR、PAX8,NIS,TPO,Tg的錶達.結果:分化細胞邊界清晰,呈圓形、類圓形、梭形或多角形貼壁生長.誘導培養第6天分化細胞中有甲狀腺細胞特有基因PAX8,NIS,TPO,Tg,TSHR的錶達,第8天檢測到分化細胞中甲狀腺細胞標記物TSHR,TIF-1,PAX8,TIF-2的錶達,暘性細胞形態類似對照甲狀腺細胞.結論:胚眙榦細胞經胚胎體髮育階段,在特定條件下可定嚮分化為甲狀腺細胞.
배경:배태간세포래원우발육조기낭배적내세포단,괄당조건하능재체외유지미분화상태、정상이배체핵형급무한증식능력,차구유다향분화잠능,능구발육분화성궤체3개배층적소유류형세포.목적:관찰체외정향유도배태간세포분화위갑상선세포적가행성급상관분자표체검측.설계、시간급지점:이세포위대상적관찰실험,우2004-01/2006-12재중산대학부속제이의원제혈고완성.재료:잉12.5～14.5 d적Balb/c잉서용우배성섬유세포사양층적제비.E14소서배태간세포세포주유미국합불대학DLXu교수혜증.성년곤명충소서용우갑상선세포적제취.방법:장제비적서배성섬유사양층접충우E14소서배태간세포진행확증배양.장탈리사양층세포후정은정극륭생장적배태간세포유도발육위배태체,재축보첨가촉갑상선소、이도소、전화갑등공배양.이배양적성년곤명충소서갑상선세포위양성대조.주요관찰지표:①관찰분화과정중세포형태적변화.②면역형광법검측분화세포표기물TSHR,PAX8,TTF-2,TTF-1적표체.③RT-PCR법검측분화세포상관기인TSHR、PAX8,NIS,TPO,Tg적표체.결과:분화세포변계청석,정원형、류원형、사형혹다각형첩벽생장.유도배양제6천분화세포중유갑상선세포특유기인PAX8,NIS,TPO,Tg,TSHR적표체,제8천검측도분화세포중갑상선세포표기물TSHR,TIF-1,PAX8,TIF-2적표체,양성세포형태유사대조갑상선세포.결론:배이간세포경배태체발육계단,재특정조건하가정향분화위갑상선세포.
BACKGROUND: Embryonic stem cells fESCs) develop from early blastular inner cell mass.Under proper condition,ESCs can maintain undifferentiated state in vitro,normal diploid nuclear type,and proliferative potential.In addition,ESCs possess multi-directional differentiation capacity and can differentiate into all sorts of cells of three germ layers. OBJECTIVE: To investigate the feasibility of directed differentiation of ESCs towards thyroid cells in vitro as well as related molecular expression. DESIGN,TIME AND SETTING: A cell-based observational experiment was performed at the Bank of Cord Blood,Second Hospital Affiliated to Sun Yat-sen University between January 2004 and December 2006.MATERIALS: Balb/c pregnant mice at pregnancy 12.5-14.5 days were used for preparation of embryonic fibroblast feeder layer.E14 mouse embryonic stem cell (ESC) strains were gifted by professor Xu from Harvard University.METHODS: Murine El4 ESCs were cultured in methylcellulose semisolid medium to form embryoid bodies (Ebs).These Ebs were transferred for further inductive culture with the stepwise addition of growth factors- thyrotropin (TSH),insulin and kalium iodidum (KI).The cultured thyroid cells of adult Kunming mice were taken as positive control.MAIN OUTCOME MEASURES: ①Cellular morphological change in the process of differentiation.② Detection of expression levels of TSHR,PAX8,TTF-2,and TIF-1 by immunofluorescence assay.③ Detection of expression levels of TSHR,PAX8,NIS,TPO,and Tg by reverse transcription-polymerase chain reaction (RT-PCR).RESULTS: The differentiated cells had clear boundary,exhibiting round,oval,shuttle-shaped,or polygon adhesive growth.On day 6 of inductive ifferentiation,the differentiated cells showed the expression of PAX8,NIS,TPO,Tg,and TSHR,the specific gene of thyroid ceils.On day 8 of inductive differentiation,the expression of TSHR,TIF-1,PAX8,and TIF-2 was detectable in the differentiated cells with morphous similar to thyroid cells.CONCLUSION: ESCs can differentiate towards thyroid cells under given inductive conditions.
