中华消化外科杂志
中華消化外科雜誌
중화소화외과잡지
CHINESE JOURNAL OF DIGESTIVE SURGERY
2014年
5期
358-363
,共6页
黄启科%党立力%阮柏%张卓超%刘杰%王兴%郑志刚%陶开山
黃啟科%黨立力%阮柏%張卓超%劉傑%王興%鄭誌剛%陶開山
황계과%당립력%원백%장탁초%류걸%왕흥%정지강%도개산
绿色荧光蛋白转基因小鼠%胎肝干细胞%分离%鉴定%示踪
綠色熒光蛋白轉基因小鼠%胎肝榦細胞%分離%鑒定%示蹤
록색형광단백전기인소서%태간간세포%분리%감정%시종
Green fluorescent protein transgenic mice%Fetal liver stem cell%Isolation%Identification%Tracing
目的 探讨绿色荧光蛋白转基因小鼠胎肝干细胞(FLSCs)在体内及体外定向分化的潜能.方法 取孕13d绿色荧光蛋白(GFP)转基因C57BL/6J小鼠胚胎肝脏,经机械消化和胶原酶消化获得FLSCs并扩大培养;成熟肝细胞来自于同品系小鼠成体肝脏,通过两步灌注胶原酶消化法提取,流式细胞仪鉴定其干细胞标志物的表达及自我增殖能力,RT-PCR及Western blot测定AFP表达;分别将FLSCs经肝细胞生长因子(HGF)及TGF-3诱导分化后,Real-time PCR检测Alb、角蛋白CK-7、8、19 mRNA以及Western blot检测Alb、角蛋白CK-7、8、19蛋白在诱导前后的表达变化,鉴定其向肝细胞和胆管细胞双向分化的能力;经尾静脉注射FLSCs到野生型C57BL/6J肝切除小鼠体内,观察其在肝内重构肝组织的能力.计量资料采用t检验,各时相点比较采用重复测量的方差分析,两两比较采用LSD-t检验.结果 成功克隆FLSCs细胞.流式细胞仪检测分离的FLSCs表达上皮细胞黏附分子(EpCAM)、CD133、CD49f干细胞标志物,其阳性表达率分别为78.6％±3.3％、31.7％±2.5％、47.6％±1.8％.AFP mRNA在FLSCs和成熟肝细胞中的相对表达量分别为0.640±0.115和0.053 ±0.012,蛋白相对表达量分别为1.383±0.265和0.243±0.227,两者比较,差异有统计学意义(t=8.772,5.354,P＜0.05).超低黏附培养皿培养FLPCs和成熟肝细胞,其形成克隆球数目分别为(234.0±57.0)个和(5.0±2.0)个,两者比较,差异有统计学意义(t=12.711,P＜0.05).体外HGF诱导FLSCs分化,Alb、CK-8 mRNA表达分别在8、10 d到达峰值,分别为0.851±0.030和0.771±0.031;两者蛋白水平于第10天达峰值,分别为4.573±0.015和1.562±0.029.体外TGF-β诱导FLSCs分化,CK-7、CK-19 mRNA在第12天达峰值,分别为15.454±2.152和10.675±1.822;两者蛋白水平于第14天达峰值,分别为3.423±0.105和1.892 ±0.081.在移植有FLSCs的肝切除小鼠体内可检测到表达增强型绿色荧光蛋白(EGFP)的成熟肝细胞及胆管细胞.结论 分离的绿色荧光蛋白转基因FLSCs能够稳定表达绿色荧光,具有显著的肝干细胞特征和向肝细胞和胆管细胞双向分化的潜能,且在体内研究中具有良好的示踪性.
目的 探討綠色熒光蛋白轉基因小鼠胎肝榦細胞(FLSCs)在體內及體外定嚮分化的潛能.方法 取孕13d綠色熒光蛋白(GFP)轉基因C57BL/6J小鼠胚胎肝髒,經機械消化和膠原酶消化穫得FLSCs併擴大培養;成熟肝細胞來自于同品繫小鼠成體肝髒,通過兩步灌註膠原酶消化法提取,流式細胞儀鑒定其榦細胞標誌物的錶達及自我增殖能力,RT-PCR及Western blot測定AFP錶達;分彆將FLSCs經肝細胞生長因子(HGF)及TGF-3誘導分化後,Real-time PCR檢測Alb、角蛋白CK-7、8、19 mRNA以及Western blot檢測Alb、角蛋白CK-7、8、19蛋白在誘導前後的錶達變化,鑒定其嚮肝細胞和膽管細胞雙嚮分化的能力;經尾靜脈註射FLSCs到野生型C57BL/6J肝切除小鼠體內,觀察其在肝內重構肝組織的能力.計量資料採用t檢驗,各時相點比較採用重複測量的方差分析,兩兩比較採用LSD-t檢驗.結果 成功剋隆FLSCs細胞.流式細胞儀檢測分離的FLSCs錶達上皮細胞黏附分子(EpCAM)、CD133、CD49f榦細胞標誌物,其暘性錶達率分彆為78.6％±3.3％、31.7％±2.5％、47.6％±1.8％.AFP mRNA在FLSCs和成熟肝細胞中的相對錶達量分彆為0.640±0.115和0.053 ±0.012,蛋白相對錶達量分彆為1.383±0.265和0.243±0.227,兩者比較,差異有統計學意義(t=8.772,5.354,P＜0.05).超低黏附培養皿培養FLPCs和成熟肝細胞,其形成剋隆毬數目分彆為(234.0±57.0)箇和(5.0±2.0)箇,兩者比較,差異有統計學意義(t=12.711,P＜0.05).體外HGF誘導FLSCs分化,Alb、CK-8 mRNA錶達分彆在8、10 d到達峰值,分彆為0.851±0.030和0.771±0.031;兩者蛋白水平于第10天達峰值,分彆為4.573±0.015和1.562±0.029.體外TGF-β誘導FLSCs分化,CK-7、CK-19 mRNA在第12天達峰值,分彆為15.454±2.152和10.675±1.822;兩者蛋白水平于第14天達峰值,分彆為3.423±0.105和1.892 ±0.081.在移植有FLSCs的肝切除小鼠體內可檢測到錶達增彊型綠色熒光蛋白(EGFP)的成熟肝細胞及膽管細胞.結論 分離的綠色熒光蛋白轉基因FLSCs能夠穩定錶達綠色熒光,具有顯著的肝榦細胞特徵和嚮肝細胞和膽管細胞雙嚮分化的潛能,且在體內研究中具有良好的示蹤性.
목적 탐토록색형광단백전기인소서태간간세포(FLSCs)재체내급체외정향분화적잠능.방법 취잉13d록색형광단백(GFP)전기인C57BL/6J소서배태간장,경궤계소화화효원매소화획득FLSCs병확대배양;성숙간세포래자우동품계소서성체간장,통과량보관주효원매소화법제취,류식세포의감정기간세포표지물적표체급자아증식능력,RT-PCR급Western blot측정AFP표체;분별장FLSCs경간세포생장인자(HGF)급TGF-3유도분화후,Real-time PCR검측Alb、각단백CK-7、8、19 mRNA이급Western blot검측Alb、각단백CK-7、8、19단백재유도전후적표체변화,감정기향간세포화담관세포쌍향분화적능력;경미정맥주사FLSCs도야생형C57BL/6J간절제소서체내,관찰기재간내중구간조직적능력.계량자료채용t검험,각시상점비교채용중복측량적방차분석,량량비교채용LSD-t검험.결과 성공극륭FLSCs세포.류식세포의검측분리적FLSCs표체상피세포점부분자(EpCAM)、CD133、CD49f간세포표지물,기양성표체솔분별위78.6％±3.3％、31.7％±2.5％、47.6％±1.8％.AFP mRNA재FLSCs화성숙간세포중적상대표체량분별위0.640±0.115화0.053 ±0.012,단백상대표체량분별위1.383±0.265화0.243±0.227,량자비교,차이유통계학의의(t=8.772,5.354,P＜0.05).초저점부배양명배양FLPCs화성숙간세포,기형성극륭구수목분별위(234.0±57.0)개화(5.0±2.0)개,량자비교,차이유통계학의의(t=12.711,P＜0.05).체외HGF유도FLSCs분화,Alb、CK-8 mRNA표체분별재8、10 d도체봉치,분별위0.851±0.030화0.771±0.031;량자단백수평우제10천체봉치,분별위4.573±0.015화1.562±0.029.체외TGF-β유도FLSCs분화,CK-7、CK-19 mRNA재제12천체봉치,분별위15.454±2.152화10.675±1.822;량자단백수평우제14천체봉치,분별위3.423±0.105화1.892 ±0.081.재이식유FLSCs적간절제소서체내가검측도표체증강형록색형광단백(EGFP)적성숙간세포급담관세포.결론 분리적록색형광단백전기인FLSCs능구은정표체록색형광,구유현저적간간세포특정화향간세포화담관세포쌍향분화적잠능,차재체내연구중구유량호적시종성.
Objective To investigate the potential of directed differentiation of fetal liver stem cells (FLSCs) in mice with sponataneous green fluorescence in vitro and in vivo.Methods FLSCs were taken from green fluorescent protein transgenic mice at embryonic day 13.After mechanical dissection and enzyme digestion with collagenase,FLSCs were further cultured.The expression of stem cell related markers and the self-renewal ability were identified by flow cytometry.The expression of alpha fetoprotein (AFP) was detected by RT-PCR and Western-blot.FLSCs were induced to differentiation by hepatic growth factor (HGF) and transforming growth factor β (TGF-β),respectively,and the ability of dual-direction (hepatocyte and cholangiocyte) differentiation was measured by exploring the expression alteration of Albumin (Alb),cytokeratin (CK)-7,8,19 by using RT-PCR and the expression alteration of CK-7,8,19 and Alb by using Western blot.Suspended FLSCs were injected to wild type C57BL/6J mouce hepatectomy model via caudal vein,and the ability of hepatic tissue reconstruction was observed.All data were analyzed using the analysis of variance or LSD-t test.Results FLSCs cells were successfully cloned.Stem cell related markers including epithelial cell adhesion molecule (EpCAM),CD133 and CD49f were detected by flow cytometry.The positive expressions of EpCAM,CD133 and CD49f were 78.6％ ± 3.3％,31.7％ ± 2.5％ and 47.6％ ± 1.8％,respectively.The relative mRNA and protein expression levels of AFP in FLSCs and mature hepatocytes were 0.640 ± 0.115,0.053 ± 0.012 and 1.383 ± 0.265,0.243 ± 0.227,there were significant differences in AFP expression between FLSCs and mature hepatocytes (t =8.772,5.354,P ＜ 0.05).FLSCs and mature hepatocytes were both cultured in ultra-low attachment dishes for 2 weeks.There was a significant difference in the clone formation rate beteween the FLSCs and mature hepatocytes,which were 234.0 ± 57.0 and 5.0 ± 2.0,respectively (t =12.711,P ＜ 0.05).After induced differentiation by HGF in vitro,the expression level of Alb and CK-8 in fetal liver stem cells peaked at the 8th day and the 10th day,which were 0.851 ± 0.030 and 0.771 ± 0.031,respectively.The protein expression levels of Alb and CK-8 in FLSCs peaked at the 10th day,which were 4.573 ±0.015 and 1.562 ±0.029,respectively.After the induction of differentiation by TGF-3 in vitro,the mRNA expression levels of CK-7 and CK-19 in FLSCs peaked at the 12th day,which were 15.454 ±2.152 and 10.675 ± 1.822,respectively.The mRNA expression levels of CK-7 and CK-19 in FLSCs peaked at the 14th day,which were 3.423 ± 0.105 and 1.892 ± 0.081,respectively.EGFP could be detected in both hepatoeyte and cholangiocyte after transplanting FLSCs to mouse hepatectomy model.Conclusions The FLSCs isolated from C57BL/6JEGFP mice can stably express the green fluorescence protein.These cells possess the traits of hepatic stem cells and have the potential ability of dual-direction differention into both hepatocytes and cholangiocytes in vitro and in vivo.Furthermore,FLSCs derived from green fluorecent protein transgenic mice have a good tracing effect in vivo.
