[1]罗茜,王斌,雷增杰,等.肝星状细胞具有向肝细胞样细胞分化的干细胞潜能[J].第三军医大学学报,2013,35(07):604-608.
 Luo Xi,Wang Bin,Lei Zengjie,et al.Hepatic stellate cells might possess potential capabilities of stem cells to differentiate into hepatocyte-like cells in vitro[J].J Third Mil Med Univ,2013,35(07):604-608.
点击复制

肝星状细胞具有向肝细胞样细胞分化的干细胞潜能(/HTML )
分享到:

《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
35卷
期数:
2013年第07期
页码:
604-608
栏目:
论著
出版日期:
2013-04-15

文章信息/Info

Title:
Hepatic stellate cells might possess potential capabilities of stem cells to differentiate into hepatocyte-like cells in vitro
作者:
罗茜王斌雷增杰李青李砚熊吉陈潇迪张霹雲魏艳玲王军陈东风
第三军医大学大坪医院野战外科研究所消化内科
Author(s):
Luo Xi Wang Bin Lei Zengjie Li Qing Li Yan Xiong Ji Chen Xiaodi Zhang Piyun Wei Yanling Wang Jun Chen Dongfeng
Department of Gastroenterology, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, 400042, China
关键词:
肝星状细胞干细胞细胞分离诱导分化
Keywords:
hepatic stellate cells stem cells isolation induction differentiation
分类号:
R322.47; R329-33; R392.12
文献标志码:
A
摘要:
目的      探讨肝星状细胞是否能体外诱导分化为肝细胞样细胞以及在这过程中某些干细胞标记物的表达和变化。      方法       采用标准胶原酶灌注联合percoll密度梯度离心法提取并纯化原代大鼠肝星状细胞,免疫荧光鉴定细胞表型;将原代肝星状细胞分为对照组和实验组进行体外培养,对照组肝星状细胞不加任何细胞因子培养24 h,实验组肝星状细胞加入bFGF 20 μg/L、FGF4 20 μg/L、HGF 20 μg/L、IL-6 1 μg/L细胞因子诱导7 d;倒置相差显微镜下观察诱导细胞形态的变化,Real-time PCR和细胞免疫荧光技术检测肝星状细胞诱导前后肝细胞ALB、AFP mRNA和蛋白的表达情况,以及诱导前后干细胞标记物mRNA和蛋白水平的表达及变化。      结果       ①通过胶原酶灌注联合percoll密度梯度离心法获得的大鼠肝星状细胞,其特异性蛋白Desmin和GFAP表达阳性率≥95%。②实验组大鼠肝星状细胞经过bFGF、FGF4、HGF、IL-6等细胞因子诱导7 d后,与对照组细胞相比细胞形态由星形变为类圆形或多角形,转录水平出现肝细胞特异性标记物ALB的表达(P<0.01)和AFP的表达(P<0.05),同时实验组ALB和AFP蛋白表达呈阳性,而对照组则呈阴性。③实验组大鼠肝星状细胞经过bFGF、FGF4、HGF、IL-6等细胞因子诱导7 d后,与对照组相比干细胞标记物P75NTR转录水平表达下降(P<0.05),CD90、c-kit、sox-2、oct-4等转录水平表达显著下降(P<0.01),并伴有干细胞标记物蛋白水平的表达下调或无表达。      结论       初步证明肝星状细胞可能具有潜在的干细胞特性,在体外可诱导分化形成肝细胞样细胞,在诱导过程中肝星状细胞的干细胞标记物明显下调。
Abstract:
Objective       To investigate whether hepatic stellate cells (HSCs) can differentiate to hepatocyte-like cells in vitro, and the expression and changes of some stem cell markers during the induction.       Methods       Primary rat HSCs were isolated by the standard collagenase perfusion and purified by density gradient centrifugation, and then identified with fluorescent immunocytochemistry for Desmin and GFAP. The HSCs were treated with the cytokines as bFGF (20 μg/L), FGF4 (20 μg/L), HGF (20 μg/L) and IL-6 (1 μg/L) for 7 d served as experimental group, while those cultured without any cytokine for 24 h served as control group. The morphological changes of HSCs were examined by inverted phase contrast microscopy, and the hepatocyte-like phenotypes were determined by detecting the mRNA and protein expression of AFP, ALB, and other stem cell markers expression by real time-PCR and immunocytochemical assay.       Results       Our primary culture method obtained a large number of primary rat HSCs, with a positive rate to Desmin and GFAP of over 95%. After a combined induction of bFGF, FGF4, HGF and IL-6 for 7 d, HSCs showed a dramatically increased expression of AFP and ALB (P<0.05) at mRNA and protein levels, with the shape changing from star-like to rotund or polygonal. But no such change was found in control cells. The combined induction also resulted in a significant decrease in mRNA expression of p75NTR (P<0.05) and CD90, c-kit, sox-2 and oct-4 (P<0.01). The expression of these markers at protein level was also significantly decreased or even undetectable.       Conclusion       HSCs might be of the potential as stem cells to differentiate into hepatocyte-like cells in vitro. During the induction, the expression of some stem cell markers is significantly downregulated.

参考文献/References:

罗茜, 王斌, 雷增杰, 等. 肝星状细胞具有向肝细胞样细胞分化的干细胞潜能[J].第三军医大学学报,2013,35(7):604-608.

相似文献/References:

[1]蒋娟,易亭伍,张瑜,等.非小细胞肺癌的肿瘤干细胞与非肿瘤干细胞中表皮生长因子受体基因异质性的研究[J].第三军医大学学报,2012,34(20):2039.
 Jiang Juan,Yi Tingwu,Zhang Yu,et al.Genetic heterogeneity of EGFR in cancer and non-cancer stem cells from non-small cell lung cancer[J].J Third Mil Med Univ,2012,34(07):2039.
[2]陈太邦,赵建华,王永飞,等.体外树突状细胞通过NT-3上调P-ERK1/2的表达促进神经干/祖细胞分化[J].第三军医大学学报,2012,34(23):2368.
 Chen Taibang,Zhao Jianhua,Wang Yongfei,et al.Dendritic cells promote neural stem/progenitor cells differentiation through NT-3 up-regulating p-ERK1/2 in vitro[J].J Third Mil Med Univ,2012,34(07):2368.
[3]钱莘,安江洪,敖绪军,等.小鼠支气管肺泡干细胞的培养鉴定和分选[J].第三军医大学学报,2008,30(09):775.
 QIAN Shen,AN Jiang-hong,AO Xu-jun,et al.Cultivation, identification and sorting of bronchoalveolar stem cells derived from mouse lung [J].J Third Mil Med Univ,2008,30(07):775.
[4]卞修武.对肿瘤研究前沿之肿瘤干细胞的几点认识[J].第三军医大学学报,2008,30(11):995.
[5]黄宏,朱方强,孙宏振,等.脂肪来源干细胞体外成骨诱导和成脂诱导分化[J].第三军医大学学报,2008,30(13):1219.
 HUANG Hong,ZHU Fang-qiang,SUN Hong-zhen,et al.Differentiation of swine adipose tissue-derived stromal cells into adipocytes and osteoblasts in vitro[J].J Third Mil Med Univ,2008,30(07):1219.
[6]江德鹏,向静,文亮,等.创伤修复中白细胞蛋白酶抑制因子和中性粒细胞趋化因子差异表达的研究[J].第三军医大学学报,2006,28(17):1742.
[7]徐远旭,杨恬,黄恩毅,等.环氧合酶-2在毛囊皮脂腺单位的表达及其意义[J].第三军医大学学报,2006,28(11):1170.
[8]呙琳琳,戴立里,唐静.丹参素对PDGF-BB刺激下肝星状细胞的抑制作用[J].第三军医大学学报,2011,33(15):1610.
 Guo Linlin,Dai Lili,Tang Jing.Salvianic acid A (Danshensu) inhibits proliferation and activation in rat hepatic stellate cells induced by platelet derived growth factor-BB[J].J Third Mil Med Univ,2011,33(07):1610.
[9]王军民,姚希贤,杨书良.丹参对AngⅡ刺激鼠HSCs活化Ca2+效应抑制作用的随机对照试验[J].第三军医大学学报,2005,27(08):756.
[10]阮艳,蹇锐,程小星,等.Bmi-1调节干性相关分子转录的初步分析[J].第三军医大学学报,2009,31(09):768.
 RUAN Yan,JIAN Rui,CHENG Xiao-xing,et al.Transcriptional regulatory effects of Bmi-1 on stemness-associated genes in embryonic stem cells[J].J Third Mil Med Univ,2009,31(07):768.

更新日期/Last Update: 2013-04-07