文献类型：期刊文献

中文题名：脐血CD34^+细胞体外诱导肝样细胞的研究

英文题名：RESEARCH ON IN VITRO DIFFERENTIATION OF HUMAN UMBILICAL CORD BLOOD-DERIVED CD34^+ CELLS INTO HEPATOCYTE-LIKE CELLS

作者：陈祎祺[1];蔡海波[1];谭文松[1]

机构：[1]华东理工大学生物反应器工程国家重点实验室,上海200237

年份：2008

卷号：22

期号：6

起止页码：747

中文期刊名：中国修复重建外科杂志

外文期刊名：Chinese Journal of Reparative and Reconstructive Surgery

收录：MEDLINE(收录号：18630578);CSTPCD;;Scopus;北大核心:【北大核心2004】；CSCD:【CSCD2011_2012】；PubMed;

基金：上海市生物医药重大科技攻关资助项目(05DZ19328)~~

语种：中文

中文关键词：脐血CD34^+细胞;体外培养;肝样细胞;转分化;细胞因子

外文关键词：Human umbilical cord blood-derived CD34^＋ cells In vitro culture Hepatocyte-like cells Transdifferentiate Cytokine

摘要：目的探讨通过细胞因子及其组合体外诱导脐血CD34+细胞转分化成肝样细胞的效果。方法采用密度梯度离心法分离脐血单个核细胞(mononuclear cell,MNC),从MNC中获得富集CD34+细胞。选用人白血病抑制因子、制瘤素M、bFGF、aFGF、肝细胞生长因子、EGF及干细胞生长因子7种细胞因子,浓度分别为10、10、10、10、20、20和50 ng/mL,设计了49种细胞因子组合,分别采用这些细胞因子组合体外培养脐血CD34+细胞,培养周期为28 d。以新鲜脐血CD34+细胞作为对照。每7天检测诱导后细胞中细胞角蛋白19(cytokeratin19,CK-19)、CK-18、谷氨酰胺合成酶(glutamine synthetase,GS)、人血清白蛋白(albumin,ALB)以及甲胎蛋白(α-fetoprotein,AFP)的mRNA转录情况,并应用免疫荧光法、过碘酸-雪夫(periodic acid-schiff,PAS)染色与吲哚菁绿(indocyanine green,ICG)染色法检测细胞分泌ALB、合成糖原以及解毒能力,以此判断是否存在脐血CD34+细胞体外向肝样细胞的转分化。结果在经细胞因子诱导后的CD34+细胞中均可检测到人肝细胞所能表达的CK-19、CK-18和GS的mRNA条带,未能检测出肝细胞特征性的ALB与AFP的mRNA条带;而新鲜脐血CD34+细胞中以上5种蛋白的mRNA均未能检出。免疫荧光染色观察示诱导前后的细胞中均未能检测到ALB的表达,且均不能有效吞噬ICG染料。PAS反应检测结果显示,经细胞因子诱导后的细胞紫红色糖原沉积区域较新鲜脐血CD34+细胞增大,出现紫红色区域的细胞增多。结论脐血CD34+细胞体外经细胞因子组合诱导后,虽有肝细胞相关蛋白mRNA表达,但未能转分化为肝样细胞。
Objective To observe the effect of cytokines and combinations in the inducement of human umbilical cord blood-derived CD34^＋ cells into hepatocyte-like cells. Methods The mononuclear cells （MNCs） were derived by density gradient centrifugation and the CD34^＋ cells were sperated from MNCs. The human umbilical cord blood-derived CD34^＋ cells were cultivated through 49 different combinations of cytokines including leukemia inhibitor factor （LIF）, oncostatin M, bFGF, aFGF, hepatocyte growth factor, EGF and stem cell factor for 28 days, and the concentrations of the cytokines were 10, 10, 10, 10, 20, 20 and 50 ng/mL, respectively. The mRNAs of cytokeratin 19 （CK-19）, CK-18, glutamine synthetase （GS）, human albumin （ALB） and a-fetoprotein （AFP） were detected every seven days. The ALB secretion ability, detoxification ability and hepatin synthesis ability of the induced cells were detected by immunofluorescence assay, indocyanine green .（ICG） and periodic acid-schiff assay, respectively. The fresh umbilical cord blood-derived CD34^＋ cells were detected at the same time as a control. Results The mRNAs of CK-19, CK-18 and GS could be transcribed in all the induced cells, but the transcription of the mRNAs of ALB and AFP which was the special mark of mature hepatocyte and 1 iver stem cell, respectively, was not found. All the mRNAs could not be found in freshly isolated umbilical cord blood-derived CD34^＋ cells. All the cells induced in vitro could not release ALB, and not help the detoxification of ICG which was the fundamental function of mature 1 iver cells. These results were the same in the control group. The hepatin synthesis ability of all the induced cells increased by comparison to the fresh ones. Conclusion Though some mRNAs of proteins which are transcribed in hepatocytes can be found in the induced cells, umbilical cord blood-derived CD34^＋ cells could not be transdifferentiated into hepatocyte-like cells through cytokines in vitro.