CD133+ U87人脑胶质瘤干细胞放射敏感性和DNA双链断裂损伤修复的实验研究

目的 探讨CD133+ U87人脑胶质瘤干细胞放射敏感性及DNA双链断裂损伤修复的情况。 方法 选择人脑胶质瘤U87细胞系，采用免疫流式分选技术分选出CD133+、CD133-细胞；采用克隆形成实验研究细胞的放射敏感性；采用中性单细胞凝胶电泳实验检测4 Gy X射线垂直照射后不同时间点的DNA双链断裂；采用间接免疫荧光技术检测不同时间点磷酸化组蛋白H2AX（γ-H2AX）荧光灶、Rad51（一种同源重组修复蛋白）荧光灶的表达。 结果 假照射条件下，CD133+细胞克隆的形成率明显高于CD133-细胞（t=3.66，P < 0.01）；CD133+细胞经4 Gy照射后的克隆形成率无明显变化（t=0.71，P > 0.05），而CD133-细胞经4 Gy照射后的克隆形成率下降（t=2.91，P < 0.05）。4 Gy照射后0.5 h，CD133+、CD133-细胞间尾力矩差异无统计学意义（t=1.44，P > 0.05），照射后6、24 h，CD133+细胞尾力距下降程度大于CD133-细胞（t=5.31和8.09，P < 0.01）；照射后0.5、6 h，CD133+、CD133-细胞间γ-H2AX灶的表达率差异均无统计学意义（t=0.12和0.99，P > 0.05），照射后24 h，CD133+细胞的γ-H2AX灶的表达率下降程度大于CD133-细胞（t=4.99，P < 0.01）；照射后0.5 h，CD133+、CD133-细胞间Rad51灶的表达率差异无统计学意义（t=1.12，P > 0.05），照射后6、24 h，CD133-细胞的Rad51灶的表达率与CD133+细胞相比明显下降（t=22.88和12.43，P < 0.01），而CD133+细胞无明显变化。 结论 CD133+ U87人脑胶质瘤干细胞具有放射抵抗性，可能与其照射后DNA双链的断裂修复能力较高有关。

CD133 positive U87 glioma stem cell radiosensitivity and DNA double-strand break repair

Objective To explore the radiosensitivity and DNA double-strand break repair of CD133+ U87 glioma stem cell. Methods CD133+ and CD133- cells were isolated from glioma U87 cell lines by flow cytometry sorter system. After irradiated vertically by 4 Gy X-rays, the radiosensitivity of cells was determined by clonogenic assay. The radiation-induced DNA double-strand break repair of CD133+ and CD133- cells was determined by the neutral comet assay, and the expression of phosphorylated histone H2AX (γ-H2AX) and Rad51 foci were measured by immunofluorescence. Results The clone forming rate of CD133+ cells was higher than CD133- cells (t=3.66, P < 0.01) with no radiation. The clone forming rate of CD133+ cells irradiated by 4 Gy X-rays has no significant changes compared to that of the non-irradiation cells(t=0.71, P > 0.05), but for CD133- cells, it decreased compared to non-irradiation cells(t=2.91, P < 0.05). The tailmoment between CD133+ cells and CD133- cells had no difference at 0.5 h after irradiation (t=1.44, P > 0.05); the tailmoment of CD133+ cells was lower than CD133- cells at 6 and 24 h after irradiation, respectively(t=5.31 and 8.09, P < 0.01). There was no significant difference in the expression of γ-H2AX foci between CD133+ and CD133- cells at 0.5 and 6 h after irradiation(t=0.12 and 0.99, P > 0.05), γ-H2AX foci of CD133+ cells was significantly decreased compared to CD133- cells at 24 h after irradiation(t=4.99, P < 0.01). For Rad 51 foci, there was no difference between CD133+ and CD133- cells at 0.5 h after irradiation(t=1.12, P > 0.05). The expression of Rad 51 foci of CD133- cells was decreased compared to that of CD133+ cells at 6 and 24 h after irradiation, respectively (t=22.88 and 12.43, P < 0.01). And the expression of Rad51 foci of CD133+ cells had no significant changes at 6-24 h after irradiation. Conclusions Glioma stem cells is more radioresistive than glioma non-stem cells. The probable mechanism is that the DNA double-strand break repair capacity of glioma stem cells is more powerful than non-stem cells.