| With the continuous development of modern science and technology,nuclear energy has been widely used in military,medical,energy,agriculture and other fields.Coupled with the complex and changeable international situation,nuclear leakage accidents occasionally occur,and radiation damage caused by ionizing radiation(IR)has attracted more and more attention.Intestine is highly sensitive to radiation due to its rapid proliferation and is one of the main target organ for radiation damage.At present,radiotherapy is still an important adjuvant therapy for many tumors.The intestines of patients who have received abdominal,pelvic or rectal radiotherapy will suffer from acute and chronic intestinal dysfunction to a certain extent.The main clinical symptoms of radiation enteritis are diarrhea,blood in the stool,dehydration and malnutrition due to malabsorption,which greatly reduces the quality of life of radiotherapy patients.Therefore,radiation enteritis has become the limiting factor of radiotherapy dose for many tumors,and there is no effective means of prevention and treatment.Therefore,in-depth study of the mechanism of radiation intestinal injury and search for new targets are of great significance to overcome the difficulties in the treatment of radiation intestinal injury and improve intestinal tolerance to ionizing radiation.Ionizing radiation can cause different degrees of damage,among which the most core biological effect is DNA damage,including single strand break and double strand break.In response to DNA damage caused by ionizing radiation,organisms have evolved a whole set of complex systems,namely DNA damage response(DDR),including DNA damage recognition,cell cycle stop for DNA repair,and finally programmed cell death or inactivation when repair fails.Proliferating cells are more sensitive to ionizing radiation than resting cells.Cell cycle checkpoints are regulated by activating DDR in proliferating cells that have been damaged by radiation,giving them more time for DNA repair.Double strand DNA breaks or single strand DNA breaks caused by ionizing radiation rapidly activate ATM and ATR,respectively.ATM and ATR then transmit signals to Chk2 and Chk1,respectively,and activate Cdc25 A,Cdc25C and Wee1,thereby inhibiting(cyclin dependent kinase 1,CDK1)and cyclin dependent kinase2(CDK2)to delay the progression of cell cycle.In the process of DDR,it is difficult for proliferating cells to effectively repair DNA once they enter the M phase,so delaying the entry of cells with DNA damage can give them more time for DNA repair.Such cycle arrest mainly occurs in G1-S,S phase and G2/M phase.If the cells have incomplete DNA repair,proliferative death,known as mitotic cell death,can occur.Although the repair of irradiated mice intestinal epithelial cells has been studied for many years,the time window for the associated cell cycle arrest of intestinal epithelial cells is not clear in the existing model.The key to this marker is to look at the rate of DNA synthesis and cell division in cells that are in the S phase at different times and those that are not in the S phase.Therefore,in this study,the nucleoside analog 5-acetyne-2-deoxyuridine(2’-Deoxy-5-ethynyluridine,Ed U)was used to label and distinguish cells in S phase and G0/G1 phase in advance,and flow cytometry and other techniques were used at different times.Changes in DNA content of S phase cells and G0/G1 phase cells at previous observation sites can be observed,and the cell cycle operation pattern can be mapped,so as to more accurately describe the dynamic changes of intestinal epithelial cell cycle,which can be described by accurate time Windows for a variety of damage factors,especially radiation-induced cell cycle arrest and cell division death.It also provides a more accurate functional evaluation of some small drug molecules that affect the cell cycle.The main research results and conclusions are as follows:1.The G0/G1,S and G2/M phases of CCD 841 Co N cells were 6 hours,14 hours and 6hours,respectively.2.In the early stage of radiation,intestinal epithelial cells can produce S-phase and G2/M phase arrest,and with the increase of ionizing radiation dose,the start time and duration of the arrest are longer,and it takes longer time to repair DNA.However,2 Gy,4 Gy and 6 Gy radiation doses did not lead to significant G1-S phase arrest in the early stage.3.Inhibition of m TOR can induce G1-S,S,G2/M block in normal intestinal epithelial cells,and make G1-S block and S phase block more obvious in irradiated intestinal epithelial cells.4.Inhibition of CBS can inhibit the proliferation of crypt cells,and arrest the G1-S,S and G2/M phases of CCD 841 Co N cells under normal conditions,thus delaying the cell cycle of intestinal epithelial cells.5.Inhibition of ARPC2 can promote or inhibit crypt cell proliferation in a dose-tolerant manner,accelerating the G1-S and S phases and slowing down the G2/M phases of CCD 841 Co N cells under normal conditions.6.It takes 4 hours,4 hours,and 4 hours respectively to detect the progenitor cells in the cell cycle in the mouse recess by nucleoside analogitic advanced labeling method in the homeostasis of G0/G1 stage to early S stage,early S stage to late S stage,and late S stage to complete division.S phase and G2/M phase block were detected 4 hours after radiation.S phase block ended at 12 hours,and G2/M phase block reached its peak at 12 hours.7.The crypt cells were further subdivided by the combination of CD24,CD44 and GRP78 markers as well as the level of CD24 expression,which provided a new idea for further research on the effect of radiation on the cycle of intestinal stem cells.In conclusion,in this study,Ed U was used for early labeling of cells to distinguish the cell heterogeneity before intervention factors,and combined with surface markers,the cycle operation and arrest of intestinal epithelial cells in vitro and in vivo could be effectively observed.The durations of human intestinal epithelial cell lines and mouse crypt precursor proliferating cells in different cell cycle stages under homeostasis and cell cycle arrest time after radiation were determined.The effects of three cell cycle-related small molecules,m TOR,CBS and ARPC2,on different cell cycle stages of different intestinal epithelial cells were clarified,and the cell cycle operation and arrest could be detected which could not be easily detected by conventional methods.This model can be used to conduct corresponding studies on other proliferative cells,providing new research methods and strategies for the study of various types of cell cycle arrest caused by radiation and drug screening targeting cell cycle regulation. |
