CAJ | 학술논문

背景:肝细胞自身增殖能力有限,近几年关于各类干细胞向肝细胞分化的成功报道很多,包括胚胎干细胞、骨髓细胞、胰腺干细胞、神经干细胞及各种来源的间充质干细胞等.目的:探讨应用多种生长分化因子体外联合诱导人脐血间充质干细胞向肝样细胞分化的可行性.设计、时间及地点:细胞学体外观察,于2005-10/2006-04在暨南大学医学院血液病研究所完成.材料:胎儿脐带血来源于顺产及剖腹产的健康孕妇,由暨南大学第一附属医院产科提供,产妇均签署知情同意书.方法:体外分离培养人脐血间充质干细胞,胰酶-EDTA消化传代.取传至第3代细胞,按5×10~4/cm~2接种,48 h后去除原培养基,PBS洗涤后,第1阶段用含地塞米松、肝细胞生长因子、表皮生长因子、ITS的F12培养基诱导2周,第2阶段用含地塞米松、肝细胞生长因子、致瘤素M、ITS的F12培养基继续诱导2周.主要观察指标:流式细胞仪检测脐血间充质干细胞表面标志的表达,RT-PCR检测肝细胞相关基因的表达,免疫荧光染色检测肝细胞相关蛋白的表达,透射电镜观察细胞超微结构.结果:培养的细胞不表达造血细胞系标志CD34,CD45,CDl4;亦不表达CD54,CD49f,HLA-DR;部分表达内皮细胞标志CD106;强表达CD29,CD44及CDl3.诱导4周后,甲胎蛋白、白蛋白、ck-18及tat基因均呈阳性表达;免疫荧光染色显示细胞浆中甲胎蛋白、白蛋白、ck-18均呈阳性;细胞胞浆中出现脂滴及糖原的沉积,细胞表面有微绒毛,出现双核细胞,初步具备了肝细胞的超微结构特征.结论:联合应用地塞米松、肝细胞生长因子、表皮生长因子、致瘤素M及ITS等多种生长分化因子,可在体外成功诱导人脐血间充质干细胞向肝样细胞分化.
배경:간세포자신증식능력유한,근궤년관우각류간세포향간세포분화적성공보도흔다,포괄배태간세포、골수세포、이선간세포、신경간세포급각충래원적간충질간세포등.목적:탐토응용다충생장분화인자체외연합유도인제혈간충질간세포향간양세포분화적가행성.설계、시간급지점:세포학체외관찰,우2005-10/2006-04재기남대학의학원혈액병연구소완성.재료:태인제대혈래원우순산급부복산적건강잉부,유기남대학제일부속의원산과제공,산부균첨서지정동의서.방법:체외분리배양인제혈간충질간세포,이매-EDTA소화전대.취전지제3대세포,안5×10~4/cm~2접충,48 h후거제원배양기,PBS세조후,제1계단용함지새미송、간세포생장인자、표피생장인자、ITS적F12배양기유도2주,제2계단용함지새미송、간세포생장인자、치류소M、ITS적F12배양기계속유도2주.주요관찰지표:류식세포의검측제혈간충질간세포표면표지적표체,RT-PCR검측간세포상관기인적표체,면역형광염색검측간세포상관단백적표체,투사전경관찰세포초미결구.결과:배양적세포불표체조혈세포계표지CD34,CD45,CDl4;역불표체CD54,CD49f,HLA-DR;부분표체내피세포표지CD106;강표체CD29,CD44급CDl3.유도4주후,갑태단백、백단백、ck-18급tat기인균정양성표체;면역형광염색현시세포장중갑태단백、백단백、ck-18균정양성;세포포장중출현지적급당원적침적,세포표면유미융모,출현쌍핵세포,초보구비료간세포적초미결구특정.결론:연합응용지새미송、간세포생장인자、표피생장인자、치류소M급ITS등다충생장분화인자,가재체외성공유도인제혈간충질간세포향간양세포분화.
BACKGROUND: Reproductive activity of hepatocytes is limited. There are numerous studies concerning stem cells differentiation into hepatocytes, including embryonic stem cells, bone marrow cells, pancreas stem cells, neural stem cells, various sources of mesenchymal stem cells.OBJECTIVE: To explore the possibility of induction of mesenchymal stem cells (MSCs) from umbilical cord blood (UCB) into hepatocyte-like cells in vitro culture.DESIGN, TIME AND SETTING: A cytological in vitro study was performed at the Institute of Hematology, Medical College of Jinan University from October 2005 to April 2006.MATERIALS: Fetus cord blood was obtained from spontaneous delivery and caesarean delivered healthy pregnant women at the Department of Obstetrics and Gynecology, First Affiliated Hospital of Jinan University. The parturients signed the informed consents.METHODS: UCB-MSCs were incubated in vitro, and digested in trypsin-EDTA. The third passage of cells at 5 × 10~4 cells/cm~2 wereinoculated. Original medium was removed 48 hours later. Cells were washed in phosphate-buffered saline. In the first phase, cells were incubated in F12 medium supplemented with dexamethasone, hepatocyte growth factor, epidermal growth factor and 1 ×its for 2 weeks. In the second phase, cells were incubated in F12 medium containing dexamethasone, hepatocyte growth factor,oncostatin M and 1 × ITS for two weeks.MAIN OUTCOME MEASURES: The following parameters were measured: expression of surface marker of UCB-MSCs using flow cytometry, expression of related gene and protein of hepatocytes following induction respectively using RT-PCR and immunofluorescence staining.RESULTS: No CD34/CD45/CD14 of hematopoietic markers were detected, either no the CD54, CD49f, HLA-DR were found. The low expression of CD106 and high expression of CD29, CD44, CD13 were found. The gene expression of a-fetoprotein, albumin,CK-18 and TAT were discovered, and three kind of protein a-fetoprotein, albumin, CK-18 were positively observed in cytoplasm after 4 weeks of induction using immunofluorescence staining.CONCLUSION: UCB-MSCs are able to differentiate into hepatocyte-like cells in vitro culture following combination with many growth differentiation factors, such as dexamethasone, hepatocyte growth factor, epidermal growth factor, tumorigenesis M and ITS.