伽玛刀对脑胶质瘤干细胞增殖和凋亡影响的研究

Proliferation and apoptosis of glioma stem cells after Gamma knife treatment

目的探讨伽玛刀治疗对胶质瘤干细胞增殖和凋亡的影响。方法无血清悬浮培养胶质瘤干细胞，RT-PCR检测CD133的表达，荧光染色检测胶质瘤干细胞分化后细胞巢蛋白（nestin）、胶质纤维酸性蛋白（GFAP）、β-微管蛋白的表达。10Gy伽玛射线处理胶质瘤干细胞、U87细胞48h后显微镜下观察细胞存活数：15Cy伽玛射线处理胶质瘤干细胞8～10h后免疫荧光染色检测5-溴脱氧尿嘧啶核苷（BrdU）阳性细胞；流式细胞仪检测10、15Cy伽玛射线处理前后胶质瘤干细胞凋亡的变化。结果胶质瘤干细胞呈球形悬浮生长于培养基中。表达CD133和nestin,能够分化为星形胶质细胞和神经元，说明获得的胶质瘤干细胞具有分化能力。10Gy伽玛射线处理同等密度的胶质瘤干细胞、U87细胞后U87细胞大量死亡，胶质瘤干细胞大部分存活，比较差异有统计学意义（86±3 vs 22±2，P〈0．05）；15Gy伽玛射线照射后，胶质瘤干细胞的BrdU阳性细胞率低于照射前，差异具有统计学意义[（35±0．8）％vs（22±0．8）％，P〈0．05）]；用不同剂量的伽玛射线处理后细胞均发生凋亡，但凋亡率很低，高剂量组凋亡率（15Gy，0.312±0．011）高于低剂量组（10Gy，0．112±0．014），差异具有统计学意义（P〈0．05）。结论在无血清培养基培养条件下，人体胶质瘤组织能够培养出胶质瘤干细胞。伽玛刀治疗能够抑制胶质瘤干细胞的增殖，引起其凋亡，并呈剂量相关性。和胶质瘤细胞相比较，胶质瘤干细胞对伽玛刀不敏感，存在放疗抵抗性。

Objective To research the proliferation and apoptosis of glioma stem cells after Gamma knife treatment. Methods The glioma stem cells were cultured in serum-free suspension; real time-PCR was used to detect the CD133 expression; fluorescence staining was employed to observe the expressions of nestin, glial fibrillary acidic protein （GFAP） and β-tubulin after stem cell differentiation. After 10 Gy Gamma knife treatment for 48 h, the glioma U87 cell and stem cell survival was detected under microscope; after 15 Gy Gamma knife treatments for 8-10 h, immunofluorescent staining was performed to detect the 5-bromodeoxyuridine （BrdU）-positive cells; flow cytometry was employed to compare the changes of cell apoptosis before and after 10 and 15 Gy Gamma knife treatment. Results Under the culture conditions of serum-free medium, glioma stem cells became spherical suspended growth and had proliferation and self-renewal capacity, expressing CD133 and nestin, and containing the ability to differentiate into astrocytes and neural elements. After 10 Gy gamma knife treatments, the cell survival in the glioma stem cells was significantly higher than that in the U87 cells （86±3 vs. 22±2, P〈0. 05）. About 35% glioma stem cells showed positive BrdU staining before 15 Gy gamma knife treatment and it was about 22% after treatment with statistical difference （P〈0.05）. The apoptosis rate in the glioma stem cells was low; that in cells with 15 Gy gamma knife treatment （0.312±0.011） was significantly higher than that in cells with 10 Gy gamma knife treatment （0.112±0.014, P〈0.05）. Conclusions Under the culture conditions of serum-free medium, glioma stem cells can be derived from human glioma tissue. Treatment of gamma knife could inhibit the proliferation of glioma stem cells, causing their apoptosis. As compared with glioma cells, glioma stem cells are not sensitive to gamma knife radiotherapy and radio-resistant.