以海藻酸三维支架构建骨髓问充质干细胞源性生物人工肝组织的实验研究  

The construction of bioartificial liver by BMSC and alginate scaffold

作　　者：林继宗[1] 汤照峰[1] 方和平[1] 林楠[1] 胡昆鹏[1] 杨军[2] 项鹏[3] 许瑞云[1] 

机构地区：[1]中山大学附属第三医院肝胆外科,广东广州市510630 [2]天津南开大学生物活性材料教育部重点实验室 [3]中山大学干细胞与组织工程研究中心

出　　处：《中华普通外科杂志》2009年第3期234-237,共4页Chinese Journal of General Surgery

基　　金：广东省科技厅重大科技专项基金资助项目（2005A30201007）;广东省自然科学基金资助项目（06021343）;教育部高等学校博士学科点专项科研基金资助项目（20070558259）.

摘　　要：目的以骨髓间质干细胞（bone mesenchymal stem cell，BMSC）为种子细胞，以海藻酸三维支架为载体，研究两者的相容性及其构建生物人工肝组织的可能性。方法将大鼠BMSC接种于海藻酸三维支架上，采用倒置相差显微镜、扫描电镜分别检测BMSC的黏附、生长与增殖情况，评价两者的相容性；同时以表皮生长因子（EGF）、肝细胞生长因子（HGF）、成纤维生长因子4（FGF-4）等细胞因子混合液诱导BMSC向肝细胞分化，检测培养液内尿素氮和白蛋白含量，RT-PCR检测培养细胞ALB和AFP基因的表达，糖原染色及免疫荧光法检测细胞糖原合成功能和CK-18的表达。结果大鼠BMSC在海藻酸三维支架上能正常地黏附、生长和增殖；于海藻酸三维支架内加入诱导液作用于BMSC后，培养液内尿素和ALB的含量逐渐上升，RT-PCR检测到培养细胞有AFP和ALB基因的表达，同时被诱导的细胞具有糖原储存的功能并表达CK-18。结论海藻酸三维支架与大鼠BMSC具有良好的相容性；以EGF、HGF、FGF-4等细胞因子混合液为诱导因子，能在海藻酸三维支架上成功诱导BMSC向肝细胞分化；海藻酸三维支架和BMSC可望成为肝脏组织工程理想的种子细胞和细胞载体。Objective To construct the bioartificial liver by bone mesenchymal stem cell （BMSC） and alginate scaffold. Method Alginate scaffold was used as the cell carrier for the cultivation of BMSC and the differentiation from BMSC into hepatic like cells was induced by the cell factors of HGF, EGF and FGF-4 in the scaffold in vitro. The compatibility of the cells and the scaffold was observed by microscopy and the function of the differentiatd cells was tested. The gene of AFP and ALB was detected by RT-PCR. The secretion of ALB and the urea synthesis of the ceils were tested by ALB kit and urea kit respectively. The glycogen synthesis and the CK-18 was tested by the glycogen stanning method and the immunofluorescence test. Results BMSC was able to attach, grow and proliferate well in the alginate scaffold, the well compatibility was observed by microscopy. ALB and urea were detected in the cultivating medium, the gene of ALB and AFP was identified by RT-PCR. The glycogen synthesis ability and the expression of CK-18 were induced during the differentiation. Conclusion The three dimensional alginate scaffold exhibited well compatibility with BMSC, BMSC could be differentiated into the hepatic like cell in the scaffold. BMSC and the alginate scaffold could be used to construct the bioartificial liver for the hepatic tissue engineering.

关 键 词：间质干细胞 肝 人工 细胞培养技术 海藻酸支架 

分 类 号：R686[医药卫生—骨科学]