摘要
目的:分离培养人脐血间充质干细胞,观察其冻存复苏后向肝细胞定向分化的能力。方法:实验于2004-01/2006-01在四川大学华西医院感染性疾病中心生物治疗国家重点实验室进行。采用密度梯度离心与直接贴壁相结合的方法,1×107L-1和1×108L-1两种接种密度进行人脐带血间充质干细胞的分离,观察细胞生长及检测其表面抗原。将第6代的人脐带血间充质干细胞采用梯度冷冻技术冻存6个月,复苏后培养至第8代时,加入肝细胞生长因子和成纤维细胞生长因子-4联合诱导28d。将第8代人脐带血间充质干细胞爬片与诱导28d后的类肝细胞爬片共培养,第8代人脐带血间充质干细胞爬片与未诱导培养28d的人脐带血间充质干细胞爬片共培养作为阴性对照。检测加入肝细胞生长因子和成纤维细胞生长因子4后不同时间点及共培养28d后的人脐带血间充质干细胞中CK8&18、白蛋白和糖原的表达。结果:①密度梯度离心收获的单个核细胞培养传代后,能获得均一贴壁的间充质干细胞,第5代细胞中,CD44阳性的细胞达97.9%。②人脐血间充质干细胞冻存复苏后,活细胞约为70%,复苏后接种密度为1×107L-1的生长状态优于1×108L-1。③添加肝细胞生长因子和成纤维细胞生长因子4后,可以在细胞内检测到CK8&18、白蛋白及糖原的表达。④与类肝细胞共培养28d后,人脐血间充质干细胞中小部分细胞中同时表达CK8&18和白蛋白,并有糖原的表达。结论:人脐血间充质干细胞可采用密度梯度离心法结合直接贴壁法分离获得,并可冷冻保存;复苏后在肝细胞生长因和成纤维细胞生长因子4的联合诱导下,能定向分化为表达CK8&18,并合成白蛋白和糖原的类肝细胞,类肝细胞可能具有诱导人脐血间充质干细胞向肝系细胞定向分化的作用。
AIM： To isolate and culture the human umbilical cord blood-derived mesenchymal stem cells （UCBMSCs） and study the potential to differentiate into hepatocyte cells after they ware frozen and thawed. METHODS： The experiment was performed at the State Key Laboratory of Biotherapy, Center for Infectious Disease, West China Hospital, Sichuan University from January 2004 to January 2006. The UCBMSCs ware isolated by means of density gradient centrffugation combined with directly adherence growth at 1×10^7L^-1 and 1×10^8L^-1. The cell growth was observed and its surface antigen was detected. The six-passage UCBMSCs ware preserved in liquid nitrogen by reducing the temperature gradually. Six months later, cells ware thawed. After having been frozen and thawed, the eight-passage UCBMSCs ware treated with differentiation medium, which was supplemented with hepatocyte growth factor （HGF） and fibroblast growth factor （FGF）-4 for 28 days. Then the UCBMSCs ware co-cultured with the hepatocyte-like cells for 28 days. The 8-passage UCBMSCs was co-cultured with non-induced UCBMSCs for negative control. The expressions of cytokeratin-8＆18 （CK8＆18）, albumin and glycogen in cytoplasm by different days and 28 days after co-culturing ware determined after adding HGF and FGF-4. RESULTS： （1）The adherent homogeneous UCBMSCs ware obtained by combination density gradient centrifugation and directly adherence growth and 97.9% of UCBMSCs expressed CD44 at passage 5. （2）The living cells of UCBMSCs ware 70% after having been frozen and cells after thawing at the density of 1×10^8L^-1 grew apparently better than the group of the 1×10^7L^-1. （3）After been induced, some cells expressed human CK8＆18, human albumin and glycogen. （4）28 days later, a small part of cells which ware co-cultured with the hepaotocyte-like cells expressed CK8＆18 and albumin together and glycogen in cytoplasm. CONCLUSION： UCBMSCs can be obtained by means of density gradient centrffugation combined with directly adherence growth and preserved by cryopreservation. They can differentiate into hepatocyte-like cells, which express CK8＆18, albumin and glycogen by HGF and FGF-4 together. The hepatocyte-like cells possibly induce differentiation of UCBMSCs into hepatocyte cells.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第7期1252-1255,I0003,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
