大鼠骨髓间充质干细胞的纯化、冻存及成瘤性分析

Purification,cryopreservation and tumorigenicity of rat bone marrow mesenchymal stem cells

背景:骨髓间充质干细胞在骨髓中的含量极低,体外的长期培养过程中易丧失干细胞潜能以及体内移植后安全性等问题的存在,限制了骨髓间充质干细胞在临床上的广泛应用。目的:探索体外分离纯化、冻存大鼠骨髓间充质干细胞的最适方法,并观察以此方法培养的骨髓间充质干细胞移植体内后是否具有成瘤性。方法:分别采用差速贴壁结合24h首次换液、24h首次换液、48h首次换液的方法纯化培养骨髓间充质干细胞,筛选最适纯化方法进行后续实验。配制含体积分数为10%,20%,30%,40%,50%胎牛血清的细胞冻存液冻存细胞,复苏后计算细胞存活率,测定复苏后细胞的生长曲线及成脂诱导能力。将第3,15代骨髓间充质干细胞进行裸鼠肌肉、肝脏局部注射体内移植,45d后取注射部位行病理组织标本检查。结果与结论:差速贴壁结合24h首次换液法所获得骨髓间充质干细胞纯度最高,而细胞增殖能力与其他两组无明显差别,因此选用该方法纯化骨髓间充质干细胞,然后进行传代培养;含体积分数为30%血清冻存液既可保证细胞活性与增殖能力,又可保证干细胞特性及多向分化潜能。骨髓间充质干细胞传至15代仍保持间充质干细胞特性,骨髓间充质干细胞移植入裸鼠肝脏45d后仍可在肝脏局部存活,生长状态与体外培养相似,无异型性及向周围浸润生长,提示体外长时间培养15代以内的骨髓间充质干细胞可在裸鼠体内存活且无成瘤性。

BACKGROUND：Bone marrow mesenchymal stem cells are restricted in clinical application due to low contents in bone marrow,easy to lose stem cell potential during long-term in vitro culture,and unclear safety after in vivo transplantation.OBJECTIVE：To explore an optimal isolation,purification and cytopreservation method of rat bone marrow mesenchymal stem cells and observe the tumorigenicity after implanting the in vitro cultured cells into nude mice.METHODS：Bone marrow mesenchymal stem cells were obtained from bone marrow cavity,purified and cutured by three different methods.The culture-expanded bone marrow mesenchymal stem cells were cryopreserved with cytoprotectant solution containing different serum concentrations at-80 ℃.Following recovery from cryopreservation,cell growth capacity and adiogenic capacity were determined.Passage 3 and 15 bone marrow mesenchymal stem cells were injected into the muscle and liver of nude mice to observe the neoplasia in local site.RESULTS AND CONCLUTION：The purity of bone marrow mesenchymal stem cells obtained by differential adhesion combined with 24-hour first exchange method was higher compared to other control groups,and there was no significant difference in cell proliferation capacity between groups.Bone marrow mesenchymal stem cells cryopreserved with 30% serum could ensure the viability,proliferation ability and stem cell characterisitics and multi-differentiation potential.Bone marrow mesenchymal stem cells might keep stem cell properties until to the 15th generation.After 45 days of transplantation to nude mice,no neoplasia was found in the local site.The muscle tissue had normal structure.Passage 15 bone marrow mesenchymal stem cells could survive and proliferate in the liver with no hetermorphism and invasion to the surrounding liver tissue.