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
目的：鉴于目前对体外培养诱导骨髓间充质干细胞向肝样细胞分化的条件掌握尚不成熟，尤其是无血清培养。实验拟将临床常用微创治疗同细胞移植结合，观察荷瘤兔骨髓间充质干细胞在射频消融兔肝肿瘤自体血清诱导下向肝样细胞的分化,寻求一种自体肝组织的修复方法。
方法:实验于2006-09/2007-09 在西安交通大学医学院中心实验室完成。实验室为教育部基因与环境研究重点实验室。①实验材料：健康新西兰大白兔,二三月龄，雌雄不限，体质量1.5~2.0 kg，由西安交通大学医学院动物试验中心提供，实验过程中对动物处置符合动物伦理学标准。荷瘤种兔包块由西安交通大学第一医院介入中心刘亚民教授馈赠。②实验方法：取荷瘤种兔瘤组织块，在兔在肝脏右叶做一直径为2~3 mm的隧道，将肿瘤组织块植入隧道底部制备肝肿瘤动物模型。射频消融治疗灭活荷瘤兔肝脏肿瘤及边缘1 cm正常肝脏组织,于72 h后取全血及骨髓。应用体外细胞培养技术自骨髓血中分离、纯化兔骨髓间充质干细胞后,取P2细胞在4种不同培养基培育：对照组：含体积分数为0.1胎牛血清的低糖DMEM培养液；射频兔肝肿瘤治疗血清组：无血清低糖DMEM培养液加入射频兔肝肿瘤治疗血清；射频肝肿瘤治疗血清热灭活组：无血清低糖DMEM培养液加入射频肝肿瘤治疗灭活血清；生长因子组：含0.1胎牛血清的低糖DMEM培养液加入肝细胞生长因子及表皮细胞生长因子。③实验评估：倒置显微镜下观查细胞形态变化，流式细胞仪测定细胞周期并观察其增殖及生长特征,免疫荧光法检测肝细胞标志物白蛋白及CK18表达，鉴定细胞性质及功能。
结果:①原代、传代培养及流式细胞仪结果显示, 射频肝肿瘤治疗兔血清及肝细胞生长因子、表皮生长因子对兔骨髓间充质干细胞的诱导能明显促进细胞的增殖，S+G2+M期细胞百分数明显增高，2周后免疫荧光染色可见分化细胞表达肝细胞标志物CK18和白蛋白。②射频肝肿瘤治疗兔灭活血清及胎牛血清能使兔骨髓间充质干细胞生长，但不如射频肝肿瘤治疗兔血清及肝细胞生长因子、皮细胞生长因子促进细胞增殖明显（P < 0.01），传代培养2周，细胞形态未见明显变化，未见肝细胞标志物CK18和白蛋白表达。
结论: 肿瘤射频消融兔血清可激活、诱导自体骨髓间充质干细胞增殖并向肝样细胞分化,使射频消融肿瘤治疗的同时行肝样细胞移植修复肝损伤成为可能。
关键词：射频消融；肝肿瘤；骨髓间充质干细胞；肝样细胞

杨屹，霍建华，屈波，张明宇，王作仁.荷瘤兔骨髓间充质干细胞在射频消融自体血清诱导下向肝样细胞的分化[J].中国组织工程研究与临床康复，2008，12(21):4039-4043 [www.zglckf.com/zglckf/ejournal/upfiles/08-21/21k-4039(ps).pdf]

Differentiation of mesenchymal stem cells into hepatocyte-like cells induced by autologous serum after radiofrequency ablation for liver tumor in rabbits

Abstract

AIM: It is an immature condition that induces mesenchymal stem cells (MSCs) cultured in vitro differentiate into hepatocyte-like cells, in particular by using serum-free culture. This study was designed to investigate the effect of rabbit serum after radiofrequency ablation (RFA) for the liver tumor on inducing MSCs differentiate into hepatocyte-like cells, and find an autologous cell source for liver injury reparation.
METHODS: The experiment was carried out in the Central Laboratory of Medical College, Xi'an Jiaotong University (Key Laboratory of Environment and Genes by the Ministry of Education) between September 2006 and September 2007. Healthy New Zealand white rabbits of either gender, 2-3 months old, and 1.5-2.0 kg weight, were offered by the animal testing center of Medical College, Xi'an Jiaotong University. All the experimental procedures were in accordance with the ethical standard. Tumor mass in rabbits was presented by Professor Liu Ya-min, the Intervention Center of the First Hospital of Xi'an Jiaotong University, as a gift. A piece of tumor was transplanted into a tunnel (2-3 mm diameter) at right liver to establish the model of liver tumor. The whole blood and bone marrow were collected from rabbits 72 hours after RFA was subjected to the tumor of liver and normal liver tissue within 1 cm margin. MSCs were isolated from rabbit bone marrow, cultured in vitro and purified. P2 cells were harvested and cultured in four different media (L-DMEM) respectively: control group: L-DMEM containing 0.1 volume fraction of fetal calf serum; serum after RFA group: serum-free L-DMEM containing rabbit autologous serum after RFA; serum after RFA with heat inactivation group: serum-free L-DMEM containing rabbit autologous serum after RFA with heat inactivation; growth factor group: L-DMEM containing 0.1 fetal calf serum with hepatocyte growth factor (HGF) and epidermal growth factor (EGF). Morphology of MSCs was observed under the inverted microscope. Flow cytometry was used to measure the cell cycle, proliferation and growth. The expression of hepatocyte marker albumin and CK18 was detected by using immunohistochemistry to identify the characters of differentiated cells.
RESULTS: The primary culture, passage and flow cytometry revealed that, rabbit autologous serum after RFA, HGF and EGF all had an obvious effect on facilitating the proliferation of MSCs by induction, and the cells at S+G2+M stage were remarkably increased. After induction for 2 weeks, the differentiated cells expressed albumin and CK18 by immunohistochemistry. Rabbit MSCs grew in the medium containing rabbit autologous serum with heat inactivation and fetal calf serum, which were still inferior to rabbit autologous serum after RFA, HGF and EGF (P < 0.01). Induced for 14 days, no changes were found in the morphology of cells, and no expression of CK18 and albumin could be seen.
CONCLUSION: MSCs can be activated and induced to differentiate into hepatocyte-like cells in the rabbit serum after RFA for the tumor of liver, providing a feasibility of applying hepatocyte-like cells transplantation for liver injury reparation.

Yang Y, Huo JH, Qu B, Zhang MY, Wang ZR.Differentiation of mesenchymal stem cells into hepatocyte-like cells induced by autologous serum after radiofrequency ablation for liver tumor in rabbits.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(21):4039-4043(China) [www.zglckf.com/zglckf/ejournal/upfiles/08-21/21k-4039(ps).pdf]