骨髓间充质干细胞在C6脑胶质瘤细胞微环境中瘤性转化的生物学分析

Biological analysis of malignant transformation of bone marrow-derived mesenchymal stem cells in the microenvironment of C6 glioma cells

摘要:

目的：探讨骨髓间充质干细胞（bone marrow-derived mesenchymal stem cells，BMSCs）在 C6 脑胶质瘤细胞微环境中是否发生了生物学特性的改变。方法：全骨髓贴壁筛选法分离培养 SD 大鼠 BMSCs，免疫荧光（immunofluorescence，IF）法检测BMSCs表面标志分子CD90+、CD105+、CD45-；活细胞荧光染料 CM-Dil标记 BMSCs（CM-Dil+BMSCs），流式细胞术（flow cytome－try，FCM）检测标记效率，台盼蓝染色法连续监测 BMSCs 死亡率；CM-Dil+BMSCs与C6脑胶质瘤细胞直接接触共培养为直接共培养组，BMSCs与 C6 脑胶质瘤细胞非接触共培养为间接共培养组，阳性对照组为C6脑胶质瘤细胞单独培养，阴性对照组为 BMSCs单独培养，培养 7 d 后，IF法检测各组酸性胶质纤维蛋白（glial fibrillary acidic protein，GFAP）、CD90、CD105表达情况；FCM分选实验组CM-dil+BMSCs 和 C6 脑胶质瘤细胞，实时荧光定量聚合酶链反应（real time quantitative polymerase chain re－action，RT-qPCR）检测各组GFAP、PTEN、Bcl-xl、CyclinD1、CD90、CD105基因的表达。结果：①培养至第3代的BMSCs 90%以上表达CD90、CD105且不表达CD45；②IF结果显示直接共培养组、间接共培养组BMSCs细胞表达GFAP水平较阴性对照组 BMSCs均增高。③RT-qPCR 结果显示直接共培养组、间接共培养组BMSCs 细胞GFAP、PTEN、Bcl-xl、CyclinD1基因表达水平较阴性对照组BMSCs明显增高（P＜0.05），且直接共培养组与间接共培养组比较无明显差异（P ＞0.05）；同时，直接共培养组、间接共培养组BMSCs的CD90、CD105基因表达水平较阴性对照组BMSCs显著降低（P＜0.05）。结论：BMSCs 在C6脑胶质瘤细胞微环境中存在潜在瘤性转化的倾向。

Abstract:

Objective：To discuss the changes of the biological characteristics of bone marrow-derived mesenchymal stem cells（BMSCs） in the microenvironment of C6 glioma cells. Methods：Whole bone marrow adherent cultivation was used to isolate and culture SD rat BMSCs. Immunofluorescence（IF） was used to detect the surface makers of BMSCs（CD90+，CD105+，CD45-）. BMSCs was labeled with a fluorescent dye CM-Dil（CM-Dil+BMSCs） and the labeling efficiency was examined with flow cytometry（FCM）. The inhibitory rate of CM-Dil+BMSCs was continuously tested by trypan blue staining assay. In direct-co-culture group，CM-Dil+BMSCs was directly co-cultured with C6 glioma cells；in indirect-co-culture group，BMSCs was indirectly co-cultured with C6 glioma cells；in positive control group，C6 glioma cells was separately cultured and in negative control group，BMSCs was separately cultured. After 7 d，IF was applied to detect the expression of glial fibrillary acidic protein（GFAP） of each group. Gene expression of GFAP，PTEN，Bcl-xl，CyclinD1，CD90，CD105 of each group was detected by real time quantitative polymerase chain reaction（RT-qPCR）. Results：（1）It was showed by IF that more than 90% of the third generation of BMSCs expressed CD90，CD105，but not CD45. （2）Expression of GFAP of BMSCs in direct-co-culture group and indirect-co-culture group was increased compared with that in negative control group，while the expression of CD90 and CD105 were decreased. Gene expression of GFAP，PTEN，Bcl-xl，CyclinD1 was significantly increased in direct-co-culture group and indirect-co-culture group compared with that in negative control group（P＜0.05）. There was no difference between direct-co-culture group and indirect-co-culture group（P ＞0.05）. Gene expression of CD90，D105 was significantly decreased in direct-co-culture group and indirect-co-culture group compared with that in negative control group（P＜0.05）. Conclusion：BMSCs in the microenvironment of C6 glioma cells have the potential malignant transformation tendency.