Repair Effect Of Hypoxia On IEC-6 Cell Acute Damaged By Irradiation And Its Possible Mechanisms

With the development of science and technology, the nuclear energy and the nuclear technique are applied abroad in medicine, industry, agriculture and research. The contiguous chance of radioactive ray became increasingly and the radiation damage increases at the same time. Damage concealment, condition complexity, progress quick, cure difficulty are the characteristics of acute radiate disease. Therefore, the acute radiate disease is an important tache for treating the radiate accident at peacetime and at wartime. More and more domestic and international scholars are attaching importance to it. Many factors might affect the effect of radiation, such as the kind and dose of radiation, the sensitivity of economy, the condition of surroundings and so on. Environment factors are the investigative key in radiation medical science and oxygen as the important environment factor has been researched for many years.In 1921 Holthusen found the phenomenon that the radiation effect of biosystem and molecular increased with the oxygen density increasing in medium. This phenomenon was called oxygen effect. There are two hypotheses for its mechanism one is the oxygen fixed hypothesis and the other is the electron transport hypothesis. The protection of hypoxia on radiation damage became the investigative point in recent years. Particularly the protection of hypoxic preconditioning produced the interests to many scholars. Findings indicates that HIF-1 Gene and Survivin Gene induced by hypoxic preconditioning can restrain apoptosis produced by radiation. Hypoxic microenvironment in tumor tissue is the main abortive reason of radiotherapy. To increase the sensitivity of anoxic cell and decrease the radiation damage of normal tissue is the research topic of clinic radiotherapy. Now hypoxic radiotherapy has been applied for many years.The research of the effect of hypoxic circumstance on irradiation effect main concentrated in the effect of hypoxia on radiation damage and the research of the effect of hypoxia on radiation repair is very little. This investigate will concentrate on the effect of hypoxia on the repair and its possible mechanism based on the formerly research. Taking ratjejunal epithelium cell (IEC-6) as the study object, after irradiation by 60 Co- Y rays treated with hypoxia, we will observe the effect of hypoxia on the repair and approach its possible mechanisms.Experiment study contains tow partsFirst part: Effect of Hypoxia on the Repair of the Acute Radiation Damage.Taking rat jejunal epithelium cell (IEC-6) as the study object, taking survival rate as detection target and taking hyperxia as contrast we will observe the effect of hypoxia on the repair of the radiation damage from organism level and cellular level. Select the radiation dose, hypoxic density and hypoxic time; establish the experiment platform for approaching its possible mechanisms.Experimental method:1. Observe the effect of different density oxygen on the survival rate of mouse after irradiation.2. Observe the effect of different density oxygen on the survival rate of IEC-6 cell after irradiation.(1) . Detect the survival rate of IEC-6 cell after different dose of 60 Co— y rays only one time with MTT experiment and definite the radiation dose.(2) . Fixed the radiation dose, we detect the effect of different density oxygen on the survival rate of IEC-6 cell after irradiation and select the best hypoxic density and the best hypoxic time.Experimental result1. After one time irradiation of 8 Gy 60Co— y rays, the survival rate of the hypoxic group (put the mouse in the altitude chamber for 6 hours imitating the environment of 5500m plateau.) increased 13% (pO.Ol) on the seventh day contrast to the control group. The hyperxic group (continue aspiration oxygen with the low flow rate 2L/min for 6 hours imitating Clinical.) cut down 7% (p<0.01) .2. IEC-6 is a radiation sensitive cell. Following the increase of the dose, the growth inhibiting became more serious. The average survival is 40% after 12Gy(LD6o.3. Aftr one time irradiation of 12 Gy 60Co— y rays, the survival rate of the hypoxic group increased 10% (pO.Ol) contrast to the control group, the hyperxic group (put the IEC-6 cell in the 50% oxygen for 6 hours) cut down 11% (pO.Ol)Conclusion.Taking mouse and IEC-6 cell as the experiment object, after irradiation with the dose( LD60)of 60Co— yrays and treated with different density oxygen, the survival rate changed very obviously .The survival rate of the radiation hypoxic group increased obviously, but the radiation hyperxic group decreased obviously. The results indicated that hypoxia can protect the radiation effect, promote the repair and increase the survival rate but hyperxia is on the contrary. This experiment selected the radiation dose (12Gy), the best hypoxic density (5%) and the best hypoxic time (6 hour).Second part: Research of the possible mechanism that hypoxia may promote IEC-6 cell to repair the acute radiation injuryThe results of the first part indicated that hypoxia could protect the irradiation effect and increase the survival rate. This part will take IEC-6 as the research object and detect cell cycle, DNA damage and repair, apoptosis and associated protein; search the possible mechanisms that hypoxia promotes the repair of acute radiation injury.Experimental method1. Cell proliferation detected with 3H—TdR incorporation.2. DNA repair detected with Single Sell Gel Electrophoresis (SCGE).3. DNA damage detected with Unscheduled DNA Synthesis (UDS).4. Cell cycle and apoptosis detected with Flow Cytometry (FCM).5. Associated protein with repair (p53, GADD45^ CDC25C^ XRCC1 and NF-kBp65) detected with Western—Blot.Experimental results1. Effect of hypoxia after irradiation on BEC-6 cell proliferation: The results of 3H~ TdR incorporation indicated that the incorporation efficiency presented progressive tendency in 24 hours, the hypoxic group increased obviously contrast to the control group, average increased 10% (p<0.01) .2. Effect of hypoxia after irradiation on IEC-6 cell damage: The results of SCGE indicated that the drag tail rate and tail length increased first then decreased in 24 hours after irradiation. The hypoxic group decreased 26% (p<0.01) in drag tail rate and 15%(p<0.01) in tail length contrast to the control group.3. Effect of hypoxia after irradiation on the repair of IEC-6 cell: The results of UDSindicated that 3H—TdR incorporation efficiency presented the early fast repair stage (0~6h) and the late slow repair stage (6~24h). In the fast stage the 3H—TdR incorporation efficiency of hypoxic group increased 15% (p<0.01) contrast to the control group.4. Effect of hypoxia after irradiation on apoptosis of IEC-6 cell: The results of FCM indicated that two groups presented two apoptotic peaks (6h and 18h), but the hypoxic group decreased 3% contrast to the control group.5. Effect of hypoxia after irradiation on cycles of IEC-6 cell:(1) Gj stage: The cell number of control group dropt to the lowest at 3h after irradiation then went up at 12h and arrived the peak at 18h; the hypoxic group dropt to the lowest at 6h late 3 hours (p<0.01) than the control group and then became as the control.(2) S stage: The cell number of S stage rose first and then dropt after irradiation, the control group arrived the peak at 3h then dropt and got the lowest at 9h, at 18h rose again. The hypoxic group arrived the peak at 6h late 3 hours (p<0.01) than the control group and then became as the control.(3) G2 stage: The cell number of the control group began to blockage at 3h after irradiation and arrived the peak at 9h, then dropt and got to normal; the hypoxic group began to blockage slowly at Oh then bcame rapid at 6h .It arrived the peak at 12h but super the control group ang late 3 hours (p<0.01) then became as the control.6. Effect of hypoxia after irradiation on the proteins associated with repair(1) p53 protein: The amount of p53 protein in nuclear increased first and then decreased within 12 hours after irradiation, the control group arrived the peak at 6h then decrease to normal at 12h; the hypoxic group arrived the peak at the same time but it continued to 9h and then became as the control group.(2) GADD45 protein: The amount of GADD45 protein in nuclear increased first and then decreased within 12 hours after irradiation, the control group arrived the peak at 3h then decreased to the lowest at 9h but more than normal, at 12h increased again; the hypoxic group arrived the peak at 6h late 3 hours (p<0.01) than the control group and lower the normal at 12h.(3) CDC25C protein: The amount of CDC25C protein in nuclear decreased progressively within 12 hours after irradiation, the control group arrived the lowest at 9h then rose again at 12h but lower than the normal; the hypoxic group decreased to 12h andlower the control group, but it super the control group at the other time.(4) XRCC1 protein The amount of XRCC1 protein in nuclear increased first then decreased within 12 hours after irradiation, the control group arrived the peak at 6h then decreased to normal at 12h; the hypoxic group arrived the peak at 9h late 3 hours than the control group then decreased and super the normal at 12h.(5) NF-kBp65 protein: The amount of NF-kBp65 protein in nuclear increased first then decreased within 12 hours after irradiation, the control group arrived the peak at 9h then decreased to normal at 12h; the hypoxic group arrived the peak at the same time but it super the normal at 12h.At each point the hypoxia super the control group (pO.Ol) .ConclusionG2 stage blockage is the main change of cell cycle; the control group arrived the peak at 9h after irradiation, but the hypoxic group late 3 hours and got to the top at 12h. Not repaired cell exited from cell cycle by apoptosis so that lead the second DNA damage (at 12h after irradiation) and the second apoptotic peak (at 18h) Hypoxia affected the protein associated with repair and prolonged the time (3 hours) of G2 stage blockage, provided more time for repairing the DNA damage. At the same time repair incorporation efficiency increased 15%, apoptosis decreased 3% and the survival rate increased 10%.