| Objective:Ionizing radiation(IR)is widely used in modern society.Although it accelerates scientific development and human health,it inevitably damages or even threatens human life.The hematopoietic system is the most sensitive to IR in the body.Hematopoietic system injury is a common type of radiation damage caused by IR,which is also one of the main causes of death in nuclear accidents.The various types of hematopoietic cells in the presence of the hematopoietic system are greatly reduced by IR damage.The bone marrow hematopoietic stem and progenitor cells(HSPCs)replenish the hematopoietic cells through proliferation and differentiation.Therefore,the essential cause of the hematopoietic system damage lies in the damage of HSPCs upon radiation exposure.The purpose of the present study is to evaluate the damage of different doses of IR to HSPCs and explore its potential mechanisms,and to provide a theoretical basis for effective mitigation of radiation damage to the hematopoietic system.Methods:Acute and chronic bone marrow injury models of whole-body radiation at different doses of X-rays were established,the radiation doses for the acute injury model were 1.5 Gy,3 Gy,and 6 Gy,respectively,while the chronic injury model were1.5 Gy×4,3 Gy and 6 Gy,respectively.HE staining was performed to observe the morphological and quantitative changes of the acutely injured bone marrow tissue after radiation.The changes in the bone marrow cells after radiation were observed by cell counting.The clonogenic ability of bone marrow cells was detected by cobblestone area-forming cell(CAFC)assay.The proportion and number changes of bone marrow HSPCs after radiation were analyzed by flow cytometry.The proportion and numbers of HSPCs after radiation were analyzed by flow staining.The cell cycle of HSPCs after radiation were detected by Ki67 and 7AAD staining.The senescence of HSPCs after radiation was detected by using C12FDG probe.The apoptosis proportion of HSPCs after radiation were detected by Annexin V staining.The reactive oxygen species(ROS)product of HSPCs after radiation were detected by the DCFDA probe.Gene expression of P53,PUMA,CDK4,P27,P16,P19 and P21 in HSPCs after radiation were detected by q PCR.Various Taq Man probes were used to detect the changes of mi RNA expression in LSK cells after radiation.Results:1.Bone marrow cells were loosely and sparsely arranged at 3 days after radiation.The numbers of bone marrow cells were significantly reduced with the increase of radiation doses(P<0.05).The numbers of bone marrow cells at 2 months after radiation were comparable with the unirradiated group.However,the clone forming ability of bone marrow cells was significantly decreased after radiation exposure(P<0.01).2.At 3 days after different doses of radiation,the proportion of Lin~-cells decreased after 3 Gy radiation(P<0.05);no significant change in the proportion of HPCs and LSK after radiation;the proportion of HSCs significantly increased after 6 Gy radiation(P<0.01);in the MPPs population,except that the proportion of MPP3.4 had no significant change after 1.5 Gy radiation,the proportion of MPP1 and MPP3.4significantly decreased after different doses of radiation(all P<0.01),while the proportion of MPP2 significantly increased after 6 Gy radiation(P<0.05).At 2 months after different doses of radiation,the proportion of HPCs significantly increased after1.5 Gy×4 radiation(P<0.001);the proportion of LSK decreased after 3 Gy and 6 Gy radiation(both P<0.05);the proportion of HSCs significantly increased after 1.5 Gy×4radiation(P<0.01),the proportion of ST-HSCs significantly increased in both 1.5 Gy×4and 6 Gy radiation groups(P<0.01 and P<0.05),no significant change in the proportion of LT-HSCs after radiation;the proportion of MPP1,MPP3 and MMP4 decreased after radiation(all P<0.05),while the proportion of MPP2 increased after 1.5 Gy×4 radiation(P<0.05).3.The proportion of G0 phase in LSK cells,HSCs,LT-HSCs,ST-HSCs,MPP1and MPP3.4 cell populations was reduced at 2 months after different doses of radiation compared with the unirradiated group(all P<0.05),and most HSPCs showed a more significant decrease in the 3 Gy radiation groups compared to the 1.5 Gy×4 and 6 Gy radiation group,while the cell cycle was not altered in HPCs and MPP2.The cell cycle-related gene CDK4 was significantly up-regulated in irradiated LSK cells(P<0.05).The cell cycle inhibitory gene P27 was significantly down-regulated in irradiated LSK cells(P<0.001).4.The proportion of senescent cells was significantly increased(P<0.05)in HSPCs after 2 months by different doses of radiation,and the senescence proportion of LSK and HSCs increased more significantly in the 1.5 Gy×4 and 6 Gy radiation groups compared to the 3 Gy radiation group.The expression of senescence-related genes P16,P19 and P21 were significantly up-regulated in HSCs under 1.5 Gy×4Gy and 6 Gy exposure(all P<0.05).5.The apoptosis proportion of Lin~-cells,HPCs,LSK cells and HSCs in the bone marrow significantly increased at 3 days after radiation exposure(all P<0.05).The proportion of apoptosis increased along with the increase of radiation doses.The expression of P53 and Puma were significantly up-regulated in Lin~-cells after radiation(P<0.01).6.ROS product of HSPCs had significantly increased in the 6 Gy radiation group at 3 days or 2 months after radiation(both P<0.05).7.q PCR of mi RNA showed that the expression of mi R-10a and mi R-18a in LSK cells was significantly up-regulated(P<0.05),while mi R-302a expression was significantly down-regulated(P<0.05)at 2 months after radiation.Conclusion:1.Different doses of radiation can cause acute or chronic damage to bone marrow cells and HSPCs,resulting in a decrease in the numbers and function of bone marrow cells.The proportion and quantity of HSPCs,as well as the cell cycle,are negatively affected after radiation exposure.The proportion of cell senescence and apoptosis were increased after radiation exposure.2.IR-induced damage to HSPCs might be related to the increase of intracellular ROS and mi RNA regulation after radiation. |
