| Objective:The rapid development of radiotherapy technology significantly increases the overall survival of patients with brain tumors after radiotherapy.However,the brain injury caused by ionizing radiation such as cognitive dysfunction may seriously reduce the long-term quality of life of cancer patients after treatment,thus has attracted many researchers’attention.Clinical studies have shown that the incidence of cognitive dysfunction can be as high as 50%-90%in patients who survive longer than six months after receiving whole brain radiation therapy.Previous studies have found that the inhibition of neurogenesis by ionizing radiation plays an important role in the occurrence of radiation-induced cognitive impairment.Therefore,neurogenesis inhibition is considered to be one of the important mechanisms of radiation-induced cognitive impairment.Neural stem/progenitor cells(NSC/NPCs)existing in a microenvironment composed of many types of cells are vulnerable to the influence from other cells.In the scenario of cranial radiation therapy,cancer cells are the target,but they may affected unirradiated neural stem cells.Therefore,this study mainly aims to explore whether irradiated cancer cells can cause biological changes in unirradiated neural stem cells through radiation-induced bystander effects,as well as the contribution to neurogenesis and cognition.Clarifying the role and the mechanisms of bystander effects in radiation-induced cognitive impairment may have an important practical role in reducing the incidence of complications for cancer patients after cranial radiotherapy.Methods:In the first part of this study,X-ray irradiated glioma cells were co-cultured with unexposed neural stem cells.Flow cytometry was used to detect the changes in the levels of reactive oxygen species(ROS)in neural stem cells.Flow cytometry was also used to detect the apoptosis of neural stem cell.Proliferation changes of neural stem cells were detected by cell counting method.Changes in neurosphere formation were evaluated by measuring the diameter of neurospheres after taking pictures.Immunofluorescent staining experiments were performed to assess the stemness and differentiation of neural stem cells.In the second part of this study,the size of exosomes extracted from cancer cells was measured,the exosomes were lebeled with fluorescent probe and their uptake by NSC was observed under a fluorescent microscope.The exosomes extracted from U251 cells 3 h after irradiation were added to the culture of neural stem cells,then the proliferation and the ability of neurosphere formation were detected.In the third part of this study,the method of stereotactic injection was used to inject the exosomes secreted by unirradiated and irradiated GL261 cells in the hippocampus of mice.Then Morris water maze was used to detect changes in cognitive ability of mice,and immunofluorescent staining was used to measure the changes in the number of neonatal neurons in the hippocampal DG region of mice which reflected the effect of radiation-induced bystander response on neurogenesis.Result:1.By using flow cytometry,it was found that the irradiated cancer cells had no obvious effect on the apoptosis of NSC and indicated that irradiated glioma cells did not induce intracellular oxidative stress in NSC co-cultured with them.2.It was found that compared with untreated NSC the number of NSC decreased by 15.04%after co-culture with unirradiated U251 cells for 48 h,and the number of NSC decreased by 25.83%after co-cultured with irradiated U251(10 Gy)for 48 h;The number of NSC decreased by 11.08%after co-culture with unirradiated GL261 cells for 48h,and the number of NSC decreased by 14.16%after co-culture with irradiated GL261 cells(10 Gy)for 48h;After 48 hours of co-culture with unirradiated and irradiated H460 cells(5 Gy),the number of NSC decreased by 47.42%and 55.17%,respectively.3.Compared with NSC cultured alone,the ability of NSC to form neurospheres decreased significantly after co-culture with unirradiated U251,GL261 and H460 cells for 48 hours.After co-culturing with the irradiated U251,GL261 and H460 cells for 48 hours,the ability of NSC to form neurospheres decreased even more significantly,and the diameter of the neurospheres was much smaller,indicating that U251,GL261 and H460 cells can affect the NSC and decrease their ability to form neurospheres,and the irradiated U251,GL261 and H460 cells had a more significant effects on the ability of NSC to form neurospheres through medium-mediated mechanism.4.Immunofluorescence staining was used to detect the changes in the ability of NSC to maintain the stemness.Compared with NSC cultured alone,the ability of NSC to maintain the stemness decreased after co-culture with U251 and GL261 cells for 48 h.The stemness maintenance ability of NSC decreased by 2.99%after co-culture with U251 cells,and that of NSC decreased by 14.23%after co-culture with U251 cells irradiated with 10 Gy X-rays.The stemness maintenance ability of NSC decreased by 12.78%after co-culture with GL261 cells,and that of NSC decreased by 28.07%after co-cultured with GL261 cells irradiated with 10 Gy X-rays.5.Immunofluorescence staining was used to examine the changes in the differentiation ability of NSC.Quantitative results showed that compared with the control group,the positive rate of NSC differentiating into neuronal cells decreased by 48.55%and 73.08%after co-culture with unirradiated and irradiated U251 cells,respectively;Compared with the control group,the positive rate of NSC differentiating into neuronal cells decreased by 33.33%and 65.31%after co-culture with unirradiated and irradiated GL261 cells,respectively,indicating that glioma cells decreased the ability of NSC to differentiate into neuronal cells through medium-mediated mechanism,and irradiation of glioma cells aggravated the reduction in the ability of NSC to differentiate into neuronal cells.6.It was found that compared with the control NSC group the expression of glial cell marker protein GFAP decreased in the NSC group co-culturing with cancer cells.Compared with the control group,the positive rate of NSC differentiating into glial cells decreased by 26.06%and 60.81%after co-culture with unirradiated and irradiated U251 cells,respectively;Similarly,compared with the control group,the positive rate of NSC differentiating into glial cells decreased by 6.21%and 17.62%after co-culture with unirradiated and irradiated GL261 cells,respectively.7.The exosomes of U251 cells were extracted and co-cultured with NSC.It was found that exosomes could enter NSC within 2 hours.In addition,compared with NSC cultured alone,the proliferation and the neurosphere-forming ability of NSC in both groups were decreased.Exosomes secreted by irradiated glioma cells caused greater reduction in the proliferation and the neurosphere-forming ability of bystander NSC.8.The exosomes secreted by unirradiated and irradiated GL261 glioma cells were stereotactically injected into the hippocampus of mice.Compared to the control mice injected with PBS,in the 0 Gy exosome group,the number of newborn cells in the hippocampus of mice decreased by 25.71%,the number of newborn neurons decreased by 32.05%,the cognitive function of the mice also decreased;In the 10 Gy exosome group,and the number of newborn cells in the hippocampal of mice decreased by 51.82%,the number of newborn neurons decreased by 54.38%,and the cognitive function of the mice decreased even more seriously.Conclusion:This study confirmed that glioma cells exposed to X-ray irradiation could affect NSC through bystander signals mediated by medium.Irradiated glioma cells did not significantly induce apoptosis and oxidative stress in NSC,but rather caused an inhibition of the proliferation,stemness and the neurosphere-forming ability of NSC.In addition,the differentiation ability of NSC was significantly reduced,not only the positive rate of differentiation was reduced,but also the degree of differentiation of NSC was reduced.Further studies have confirmed that the exosomes secreted by irradiated glioma cells significantly reduced the proliferation and the neurosphere-forming ability of NSC.Finally,animal experiments have confirmed that ionizing radiation-induced bystander effects could cause significant cognitive decline in mice.And there was a positive correlation between cognitive changes in mice and the number of neonatal neurons in the hippocampus. |
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