大鼠骨髓间充质干细胞的分离鉴定及其在胶质瘤治疗中的作用

摘要

AIM:To establish a method of isolating,cultivating and purifying rat bone marrow mesenchymal stem cells (BMSCs) in vitro,to indentify cell morphology and cell surface markers,to investigate the tropism of BMSCs towards U87MG glioma and in-fection of lentiviral vector to BMSCs,to provide the experimental evidence for the gene therapy and medication of glioma with BM-SCs acting as vectors. METHODS: BMSCs keeping strong growth and stable morphology from rats were isolated,cultured and purified by the whole bone marrow adherence method com-bined with the different adherent capacities of kinds of cells and the cells morphology observations.Then the cell surface markers were assessed by flow cytometry.BMSCs were co-cultured with U87MG to investigate their tropism towards U87MG glioma and infected with the lentiviral vector to verify their potentiality for gene therapy.RESULTS:The isolated BMSCs were fibroblast-like,showing radial colony arrangement.Cells could keep strong growth and passage in continuous and stable manner over passa-ges.According to the FCM,BMSCs were positive for CD29 and CD90,but negative for CD11b /c and CD45.After being co-cul-tured with U87MG with the ritio of 1∶100,BMSCs had significant tropism towards U87MG and restrained the U87MG growth to some extent.Also,the lentiviral vector infected BMSCs successfully with high rate of infection.CONCLUSION:Our method is sim-ple and can isolate,purify and amplify BMSCs in vitro and BM-SCs have a tropism towards U87MG glioma and can be infected by the lentiviral vector.All these results provide adequate source of seed cells for further study of BMSCs and experimental evidence for gene therapy and medication of glioma with BMSCs as vectors.%目的：建立大鼠骨髓间充质干细胞（BMSCs）的分离、培养、纯化方法，并对其进行形态学和细胞表面分子标记鉴定，确定其对胶质瘤细胞的趋向性和慢病毒对其的可感染性，为其作为载体携带目的基因或药物对胶质瘤进行治疗提供实验依据。方法：通过全骨髓培养法、差速贴壁的原理和形态学观察分离、纯化出生长旺盛、形态单一的细胞，并通过流式细胞仪对分离细胞进行细胞表面标记分子的鉴定；确定分离细胞为 BMSCs 后将其与胶质瘤 U87MG 细胞共培养观察其对胶质瘤细胞的趋向性，并用慢病毒载体对其进行可感染性的确定。结果：分离出的 BMSCs 以成纤维细胞样为主，生长旺盛，呈放射状排列的细胞集落，可稳定传代。流式细胞仪结果显示，所分离的细胞均呈 CD29和 CD90的阳性表达，而CD11b ／c 和 CD45的表达均呈阴性；按1∶100的比例将 BMSCs和 U87MG 共培养后发现，BMSCs 有靶向 U87MG 的特性并且在一定程度上可以抑制 U87MG 的生长；慢病毒可感染我们分离出的 BMSCs 且感染率较高。结论：我们采用的方法操作简单，可在体外分离、鉴定出大量纯度高的 BMSCs，并证明了其在体外可以靶向胶质瘤细胞生长，且易于被慢病毒载体感染。这些结果均为后续研究 BMSCs 提供充足的种子细胞，将其作为载体携带目的基因或药物治疗胶质瘤提供了前期的实验依据。