课题背景：课题已获四川省教育厅2003年科研资助，正在积极申请国家自然科学基金的资助。前期已经成功的完成了骨髓间充质干细胞的体外培养、鉴定、诱导分化及诱导分化后的细胞特异性标志物的检测，现阶段处于动物实验过程，在肝纤维化大鼠模型的建立及骨髓间充质干细胞与诱导后细胞移植的基础上，正在进行移植后各项指标的检测及病理学标本检测，用以验证诱导后细胞对肝纤维化的治疗作用。

应用要点：目前分离骨髓间充质干细胞的方法主要有免疫磁珠法、流式细胞仪分离法、贴壁培养筛选法、密度梯度离心法，本实验采用裂解红细胞与贴壁培养法相结合，可避免由于骨髓间充质干细胞的丢失造成的细胞难以增殖，提高骨髓间充质干细胞的培养成功率，此外还可缩短骨髓间充质干细胞原代培养的时间。

重要的概念：甲胎蛋白是由肝前体细胞分泌的一种胞浆蛋白，随着细胞逐渐成熟而消失，成熟肝细胞不表达。白蛋白是主要由成熟肝细胞分泌的一种胞浆蛋白，是反映肝细胞代谢活性的一个特异指标。在诱导过程中，甲胎蛋白表达逐渐降低与白蛋白表达逐渐增高能反映肝细胞由幼稚到成熟的过程。

摘要
背景：肝细胞移植是治疗终末期肝功能衰竭的有效方法之一，却面临细胞来源缺乏、体外难以大量增殖、传代后无法保持其原有特性、免疫排斥等因素的限制，寻找新的肝细胞来源尤为迫切。
目的：探讨肝细胞生长因子、酸性成纤维细胞生长因子及抑瘤素M诱导骨髓间充质干细胞分化为肝细胞的能力。
设计、时间及地点：细胞对照观察，于2006-06/2007-03在泸州医学院附属医院实验室完成。
材料：清洁级雄性Wistar大鼠24只用于制备骨髓间充质干细胞。红细胞裂解液由泸州医学院附属医院传染免疫实验室配制。重组鼠肝细胞生长因子、抑瘤素M为美国R&D产品，酸性成纤维细胞生长因子由美国Peprotech生产。
方法：采用红细胞裂解+贴壁法分离培养大鼠骨髓间充质干细胞，胰蛋白酶消化后传代。传至第4代，以5×107 L-1接种，设立6组向肝细胞诱导分化：第1组作为对照，不加任何细胞因子；第2组加入肝细胞生长因子、酸性成纤维细胞生长因子；第3组加入肝细胞生长因子、抑瘤素M；第4组加入酸性成纤维细胞生长因子、抑瘤素M；第5组加入肝细胞生长因子、酸性成纤维细胞生长因子、抑瘤素M；第6组于1~9 d加入肝细胞生长因子，9~18 d加入酸性成纤维细胞生长因子，9~21 d加入抑瘤素M。每组所加入的各种细胞因子终末浓度均为20 μg/L。
主要观察指标：采用溴甲酚绿法、化学发光法、RT-PCR法检测肝细胞特异性标志物白蛋白、甲胎蛋白的表达。采用谷氨酸脱氢酶法检测尿素含量。
结果：诱导0，7，14，21 d，未加入诱导因子的对照组及酸性成纤维细胞生长因子+抑瘤素M组无白蛋白、尿素表达，肝细胞生长因子+酸性成纤维细胞生长因子+抑瘤素M组培养基上清液中甲胎蛋白、白蛋白、尿素含量均显著高于其余因子组(P < 0.05)。
结论：酸性成纤维细胞生长因子与抑瘤素M共同作用无法诱导骨髓间充质干细胞向肝细胞分化，二者联合肝细胞生长因子可成功诱导骨髓间充质干细胞分化为肝细胞。分时段加入此三种细胞因子，可能影响了细胞因子间的相互作用而导致分化率降低。
关键词：骨髓间充质干细胞；细胞因子；肝细胞

王忠琼，李昌平，杜光红，钟晓琳，陈霞.多种细胞因子诱导骨髓间充质干细胞向肝细胞的分化[J].中国组织工程研究与临床康复，2008，12(21):4035-4038 [www.zglckf.com/zglckf/ejournal/upfiles/08-21/21k-4035(ps).pdf]

Several kinds of cytokines induce differentiation of bone marrow mesenchymal stem cells into hepatocytes

Abstract

BACKGROUND: Hepatocellular transplantation is an effective method to treat terminal stage liver function failure. However, hepatocellular transplantation is limited by deficiency of cell source, in vitro proliferation, difficult to keep original feature after passage and immunologic rejection. It is urgent to search for a new hepatocyte source.
OBJECTIVE: To investigate the differentiation ability of bone marrow mesenchymal stem cells (BMSCs) into hepatocytes induced by hepatocyte growth factor, acid fibroblast growth factor and oncostatin M.
DESIGN, TIME AND SETTING: The cell control experiment was performed at the Laboratory of Hospital Affiliated to Luzhou Medical College from June 2006 to March 2007.
MATERIALS: Twenty-four male Wistar rats of clean grade were selected to collect BMSCS. Red cell lysate was prepared by Laboratory of Infection Immunity of Hospital Affiliated to Luzhou Medical College. Hepatocyte growth factor and oncostatin M were purchased from R&D, USA. Acid fibroblast growth factor was bought from Peprotech, USA.
METHODS: Rat BMSCs were separated by splitting red cells with adherent culture, digested by trypsin and then passaged. At the fourth passage, cells at the density of 5×107 L-1 were incubated and divided into 6 groups. BMSCs in the group 1 were considered to controls, without any cytokines. BMSCs in the group 2 were incubated with hepatocyte growth factor and acid fibroblast growth factor. BMSCs in the group 3 were incubated with hepatocyte growth factor and oncostatin M. BMSCs in the group 4 were incubated with acid fibroblast growth factor and oncostatin M. BMSCs in the group 5 were incubated with hepatocyte growth factor, acid fibroblast growth factor and oncostatin M. BMSCs in the group 6 were incubated with hepatocyte growth factor from days 1 to 9, with acid fibroblast growth factor from days 9 to 18 and with oncostatin M from days 9 to 21. End level of various cytokines was 20 μg/L in each group.
MAIN OUTCOME MEASURES: Albumin and alpha fetoprotein were measured by bromocresol green method, chemi-luminescent technique and reverse transcription-polymerase chain reaction method. Urea levels were examined by glutamic acid dehydrogenase method.
RESULTS: At days 0, 7, 14 and 21, albumin and urea were negative expression in the control group and group 4. Alpha fetoprotein, albumin and urea were significantly higher in the group 5 than in other groups (P < 0.05).
CONCLUSION: Combination of acid fibroblast growth factor and oncostatin M cannot induce the differentiation of BMSCs into hepatocytes. Their combination with hepatocyte growth factor can successfully induce the differentiation of BMSCs into hepatocytes. Addition of three cytokines in different time can decrease differentiation ratio.

Wang ZQ, Li CP, Du GH, Zhong XL, Chen X.Several kinds of cytokines induce differentiation of bone marrow mesenchymal stem cells into hepatocytes.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(21):4035-4038(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-21/21k-4035(ps).pdf]