诱导人骨髓间充质干细胞向肝系细胞分化过程中肝细胞生长因子和碱性成纤维细胞生长因子的作用

背景:骨髓间充质干细胞是一种存在于骨髓内的非造血干细胞,因其具有自我更新能力及多向分化潜能,且分离培养方法成熟,在组织器官修复及再生方面显示出良好的应用前景,将为终末期肝病的治疗如生物人工肝及肝细胞移植提供新的细胞来源,但目前人骨髓间充质干细胞向肝系细胞诱导分化培养体系尚不成熟。目的:观察肝细胞生长因子和碱性成纤维细胞生长因子联合应用诱导人骨髓间充质干细胞在体外向肝系细胞分化的可能性。设计:开放性实验。单位:南昌大学第一附属医院传染科与泌尿外科研究所。材料:实验于2004-07/2005-03在南昌大学第一附属医院泌尿外科研究所完成。所用骨髓由成年健康志愿者提供(均对本实验知情同意)。DMEM/F12培养基(Gibco公司);表皮细胞生长因子,肝细胞生长因子,胰岛素,转铁蛋白,鼠抗人甲胎蛋白单克隆抗体,FITC-兔抗鼠IgG(Sig-ma公司);碱性成纤维细胞生长因子(Invitrogen公司);鼠抗人角蛋白18,19单克隆抗体(Chemicon公司);胎牛血清(杭州四季青)。方法:以骨髓腔穿刺方式抽取成年健康志愿者髂后上棘处骨髓10mL,采用密度梯度离心法分离和反复贴壁法纯化骨髓间充质干细胞。取培养至4～8代细胞,消化后以107L-1接种到放置20mm×20mm爬片的预先铺被有0.1%明胶的24孔板中,次日换液,改用无血清DMEM/F12培养基(含20μg/L的表皮细胞生长因子和10μg/L的碱性成纤维细胞生长因子)培养2d后,换成无血清DMEM/F12培养基(含20μg/L的肝细胞生长因子,10μg/L的碱性成纤维细胞生长因子,5mg/L的胰岛素,5mg/L的转铁蛋白)进行诱导分化,每3d换液1次,以未加入肝细胞生长因子和碱性成纤维细胞生长因子的骨髓间充质干细胞作为对照组。骨髓间充质干细胞诱导培养过程中,放射免疫荧光法测甲胎蛋白浓度。诱导当天及第7,14,21,28天,取诱导细胞爬片,细胞免疫荧光法检测肝细胞表面标志物甲胎蛋白、角蛋白18和角蛋白19的表达,并进行糖原染色检测诱导细胞的糖原合成情况。主要观察指标:①细胞形态学观察。②培养上清液中甲胎蛋白表达量的测定。③肝细胞表面标志检测结果。④诱导细胞的糖原合成情况。结果:①诱导第14天可观察到细胞变成短梭形和多角形,随培养时间的延长多角形细胞增多,并可形成肝细胞样的细胞集落。②诱导第14天培养上清液内可检测到甲胎蛋白的分泌,每孔浓度为0.1μg/L;第17天达高峰0.4μg/L;第21天下降至0.3μg/L。③诱导第14天甲胎蛋白、角蛋白18表达呈阳性,第28天角蛋白19表达呈阳性。④诱导第21天糖原染色呈阳性反应。结论:肝细胞生长因子和碱性成纤维细胞生长因子能够诱导人骨髓间充质干细胞在体外向肝细胞分化,表达肝细胞特异性表面标志,并具有合成糖原、分泌甲胎蛋白的功能。

Hepatocyte growth factor and basic fibroblast growth factor induce differentiation of human bone marrow mesenchymal stem cells into hepatic lineage cells

BACKGROUND: Within the bone marrow stroma there exists a subset of non-hematopoietic stem cells referred to as marrow stromal cells or mesenchymal stem cells. Mesenchymal stem cells (MSCs) are a group of cells with highly capability of self-renew and potential of multilineage differentiation, these properties make them present a promising prospect for clinical practice. Of particular concern is hepatogenic potential that can be used for liver-directed stem cell therapy and transplantation. However, the culture system has not been developed.OBJECTIVE: To explore whether human MSCs are able to differentiate into functional hepatocyte-like cells with hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF) in vitro.DESIGN : Open study.SETTING: Department of Infectious Disease and Institute of Urology Surgery, First Affiliated Hospital, Nanchang University.MATERIALS: The study was performed in the Institute of Urolgoy Surgery, the First Affiliated Hospital of Nanchang University from July 2004 to March 2005. Bone marrow was donated by healthy adult with informed consent. DMEM/F12 medium (Gibco); insulin, transferrin, human epidermal growth factor (EGF); human HGF; monoclonal antibodies against human AFP; FITC-conjugated rabbit anti-mouse IgG (Sigma); human bFGF (Invitrogen); monoclonal antibodies against human CK18 and CK19 (Chemicon); fetal bovine serum (Si jiqin, Hangzhou).METHODS: Bone marrow (10 mL) in this study was aspirated from the iliac crest of healthy donors. MSCs were isolated by density gradient centrifugation in combination with plastic adherence. For hepatic differentiation, the 4th- to 8th-passage human MSCs seeded on 24-well tissue culture plates coated with 0.1% gelatin, at 1×104 MSCs/mL, were serum deprived for 2 days, in DMEM/F12 supplemented with 10 μg/L EGF, 10 μg/L bFGF, 5 mg/L insulin and 5 mg/L transferrin. Differentiation was induced by treating MSCs with differentiation medium, consisting of DMEWF12 supplemented with 10 μg/L bFGF, 20 μg/L HGF, 5 mg/L insulin, 5 mg/L transferrin. Medium changes were performed every three days. MSCs without HGF and bFGF in medium served as the control. In the differentiating period, the concentration of AFP in the suernatant was determined dynamically by radioimmunoassay (RIA). The hepatic surface phenotype including AFP, CK18 and CK19 were identified by immunofluorescent staining at day 0, 7, 14, 21 and 28. Glycogen storage was detected by Periodic Acid-Schiff (PAS) staining.MAIN OUTCOME MEASURES: ① the morphological changes of induced MSCs; ② the concentration of AFP in the supernatant; ③ the hepatic surface phenotype; ④ glycogen storage.RESULTS: ① After 14 days ofinduction, the fibroblast-like morphology of human MSCs was lost and cells became broadened and fiattened. After prolonged culture, polygonal cells were seen and further matured hepatocyte-like colonies were seen by day 28. ② The concentration of AFP in the supernatant was first detected on day 14, at a concentration of 0.1 μg/mL, and increased to 0.4 μg/mL by day 17, then decreased to 0.3 μg/mL by day 21. ③ Immunofiuorescent staining showed the expression of AFP and CK18 until day 14. The expression of CK19 was detected by day 28. ④ Glycogen storage could be detected by day 21.CONCLUSION: Human bone marrow MSCs are able to differentiate into functional hepatocyte-like cells and may sere as a new source of cells for cell therapy of hepatic diseases.