辐射诱导人骨髓间充质干细胞自噬反应的研究

目的 观察辐射条件下人骨髓间充质干细胞(hBMMSC)的自噬反应。方法 应用Annexin- Ⅴ/PI双标法检测0、2、4、8、10Gy X线照射后4 h hBMMSC凋亡率和坏死率的变化;通过透射电子显微镜观察0、8、10Gy X线照射后4h的hBMMSC自噬形态学变化,以经典的自噬诱导剂雷帕霉素处理组作为阳性对照,并应用RT-PCR技术检测自噬相关基因Beclin1和微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,MAPLC3,简称为LC3)mRNA表达。结果 在低于8 Gy的剂量范围内,hBMMSC的凋亡率随照射剂量的增加而降低,坏死率和死亡(凋亡+坏死)率随照射剂量的增加而升高,但升高幅度较小。当剂量增至10 Gy时,凋亡率升至最高,而坏死率和死亡率的升高幅度增大。并且整体凋亡率变化比坏死率明显。透射电镜观察显示正常对照组中偶见自噬泡(自噬体和自噬溶酶体合称为自噬泡)出现,而照射组及阳性对照组中均可见明显自噬泡聚集,8 Gy照射组的自噬泡阳性细胞比例为(71.67 +7.64)％,高于10 Gy照射组的(56.67±7.64)％。RT-PCR检测结果显示正常对照组中Beclin1 mRNA和LC3 mRNA表达水平均很低,而照射组及阳性对照组中两个基因的表达明显升高,且8 Gy照射组(0.518±0.005、0.408±0.006)表达水平高于10 G照射组(0.441±0.002、0.360±0.019)％。结论 辐射应激可以诱导hBMMSC的自噬反应,在一定照射剂量范围内,自噬可能对hBMMSC有放射保护作用。

X-ray induces autophagy in human mesenchymal stem cells

Objective To investigate the autophagy in human bone marrow mesenchymal stem cells (hBMMSC) exposed to irradiation. Methods The apoptosis and necrosis rate were assessed by Annexin Ⅴ and propidium (PI) staining in hBMMSC at 4h after irradiated with X-ray at 0, 2, 4, 8 and 10 Gy. The autophagy was observed by transmission electron microscopy. The mRNA expression of Beclin1 and microtubuleassociated protein 1 light chain 3( MAPLC3 or LC3) was analyzed by RT-PCR in hBMMSC at 4h after X-ray irradiation at 0, 8 and 10 Gy. Results The apoptosis rate of hBMMSC was markedly decreased while the necrosis and death rate were slowly increased with the increase of irradiation dose when under 8 Gy. The apoptosis rate was significantly increased and reached a peak while the necrosis and whole death rate were obviously increased when irradiated with 10 Gy X-rays. In addition, the change of apoptosis rate was more significant than that of necrosis rate. By electron microscopy, a mass of autophagic vacuoles ( autophagosome and autolysosome) were observed in irradiation and positive control groups, but were only occasionally seen in normal control group. The proportion of hBMMSC with autophagic vacuoles in 8 Gy irradiation group was higher than that in 10 Gy one. The mRNA expression of Beclinl and LC3 in irradiation groups and positive control group was significantly higher than in normal control group, and so did in 8 Gy irradiation group than that in 10 Gy group. Conclusion Irradiation may induce the autophagy in hBMMSC, and autophagy could protect hBMMSC from irradiation injury in a certain dose range.