Research On Radiation Biological Effects For Reproductive Cell Death In The Vulval Tissue Model Of Caenorhabditis Elegans

Reproductive cell death(RCD)also known as clonogenic death,is a form of cell loss of regenerative capacity and death that occurs after one or more cell divisions following the stress of physicochemical factors,RCD is usually investigated using in vitro clonogenic assay.The model organism Caenorhabditis elegans(C.elegans)which is the only in vivo tissue model of radiation induced RCD has a clear lineage of the vulva cells development,and cells death during its division would cause vulva morphogenesis which is visibled.Some radiation biology studies related to reproductive cell death,including radio-adaptive response(RAR),radiation-induced bystander effects(RIBE)and heavy-ion radiation effects,are mainly determined by clonogenic assay in cell-culture models in vitro,and little is known about experiment and mechanism in vivo.Therefore,this research mainly based on the vulva tissue model to investigate the radiation induced reproductive cell death,and studies on the radio-adaptive response for RCD,radiation-induced bystander effects(RIBE)and heavy-ion radiation effects.The main results are as follows:1.The radio-adaptive response induced by reproductive cell death in vivo and the role of the DNA damage response(DDR)were clarified.First,the early larval worms(L1/L2)were irradiated with low-dose adaptive radiation(AR)of gamma-rays,2 hours later coutinuely irradiated with high-dose challenge radiation(CR),and the radtio of protruding vulvas was examined in the adult C.elegans..The results showed that the 5 Gy AR significantly reduced the ratio of vulva morphological aberration induced by subsequent 50 Gy,75 Gy and 100 Gy CR,and demonstrated the existence of radio-adaptive response for RCD in vivo.The study found that the radio-adaptive response for RCD showed a strong time dependence and could only be induced on 14 h and 16 h nematodes with a very narrow time window(2 h).Single gene knockout mutant wan employed in futher study to investigate the the role of the DDR pathway in the vulval tissue of C.elegans RCD.The results showed that the function deletion of DNA damage checkpoint genes atm-1,mrt-2,hus-1,clk-2 and the non-homologous end joining(NHEJ)gene lig-4 can effectively inhibit the induction of radio-adaptive response,indicating that the DDR pathway is involved in the radio-adaptive response of the vulva cells.However,unlike the cell experimental system,the cep-1/p53 and the homologous recombination(HR)gene brc-1 did not participate in this process,indicating that there is a special process and mechanism for the induction of radio-adaptive response in the vulva cells.2.The coupling between the RAR and RIBE in vivo.First,the posterior pharynx bulbs of larval worms were first locally targeted by a proton microbeam.After 2 hours,the whole-body of worms were exposed of high dose gamma rays,and the ratio of protruding vulva was examined in the adult C.elegans.The results showed that the irradiation of posterior pharynx bulb by 2000 protons could significantly suppress the induction of protruding vulva induced by ?-irradiation(75 Gy and 100 Gy).2000 protons irradiated the rectal valves of C.elegans can also inhibited protruding vulva caused by ?-irradiation(100 Gy).These results suggest contribution of RIBE to RAR in vivo,and the induction of radio-adaptive response of vulva cells has a remote mode.DNA damage checkpoint(atm-1 and hus-1)deficiency can inhibit the RAR of vulva that is initiated by microbeam irradiation of the pharynx bulk.While,only the hus-1 mutation can inhibit the RAR of vulva that is initiated by microbeam irradiation of by rectal valve.The above results indicate that the DNA damage checkpoint mechanism in the upstream of DDR in the worm system involves in the remote induction RAR of vulva,and its function is in a tissue-dependent manner.3.To determine the range-distribution of heavy-ions in the C.elegans vulva RCD.At present,RCD induced by heavy-ion irradiation has been demonstrated using clonogenic assay in vitro,little is known at the level of whole organisms.This research based on the characteristic of worms individual small,a polymethyl methacrylate wedge was designed to provide a gradually varying thickness of shielding,so worms could be exposed to the entire range(divided into nine chambers)of 80 mev/u carbon-ion irradiation.After irradiated the and the radtio of protruding vulvas was examined in the adult of the all nine chambers.The results show that the carbon-ion irradiation led to a significant induction of RCD over the entire range in a dose-dependent manner,and the biological peak did not correspond to the physical Bragg peak,which is the fate replacement model of vulval precursor cells resulted in the abnormal biological peak position moved forward with the radiation dose increase.The degree and shape of the range-distribution of RCD were also affected by the developmental stages of the worms,and the sensitivity of Bragg peak is L2>L3>L1>L4.The gene mutations in DNA-damage checkpoints(atm-1,mrt-2 and hus-1)did not affect the positions of biological peaks,but decreased radio-sensitivity in the entrance region,while the functional deletion of cep-1 reduced the radiation sensitivity.Different from gamma radiation experiments,NHEJ is the main repair mechnism of DNA damage,HR repair plays a crucial role in vulval cells of C.elegans in dealing with the carbon-ion-induced DNA damage.