| Objective:The most importan member HSP70 in the HSPs family was selected to study as the represent,while its expression regularity and survival in lymphocyte was studied under 42℃heat shock treatment for 90min.The effect of 42℃heat shock treatment for 90 min on lymphocyte survival,apoptosis and hprt gene mutation was observed.The influence of HSP70 that was induced by heat shock treatment on the lymphocyte apoptosis and hprt gene mutation which were induced by X-ray radiation was researched as the point.This study aims at approaching the mechanism of HSP70 protecting cells and reducing the radiation damiage and providing basis study data for developing anti-radiation damage medicine and for searching some drugs to elevate the effect of radiation therapy on tumor.Methods:(1) The Ficoll method was applied to separate the lymphocytes from peripheral blood in rats.The comb-scraping method was used to prepare spleen lymphocytes suspension.The typan-blue staining method,flow cytometry and polynucleation cell method were respectively applied to detect the effect of 42℃heat shock treatment for 90 min on the peripheral blood and spleen lymphocytes survival, apoptosis and hprt gene,and so the heat shock condition for inducing HSP70 expression was determined.At the same time,the flow cytometry was used to detected the HSP70 expression regularity in lymphocytes inducing by 42℃heat shock treatment for 90 min and so the preparation for studying HSP70's radiation protection was made.(2) The 42℃heat shock treatment for 90min was done to the peripheral blood and spleen lymphocytes in rats and such lymphocytes recovered for different time(3h,6h,10h) under normal culture conditions,and then irradiated for different X-ray dose(0GY,0.5Gy, 1.0Gy,1.5Gy,2.0Gy,2.5Gy,3.0Gy),after that,the flow cytometry was used to detected the changes of lymphocytes apoptosis.And so whether the HPS70 could reduce apoptosis inducing by radiation was researched.(3) The 42℃heat shock treatment for 90 min was done to the peripheral blood and spleen lymphocytes in rats and such lymphocytes recovered for different time(3h,6h,10h) under normal culture conditions,and then irradiated for different X-ray dose(0Gy,0.5Gy, 1.0Gy,1.5Gy,2.0Gy,2.5Gy,3.0Gy),after that,the polynucleation cell method was used to detected the changes of hprt gene mutation in lymphocytes.And so whether the HPS70 could weaken the hprt mutation inducing by radiation was studied.Results:(1) The 42℃heat shock treatment for 90min had no influence to the lymphocyte survival,apoptosis or hprt mutation.(2) After 42℃heat shock treatment for 90min,the expression level of HSP70 in lymphocytes increased immediately and reached max during 2-4h(P＜0.01),and then decreased gradually,and could keep on for 24h at least. It is a sound to stimulate the lymphocytes.(3)Under the 0.5-3.0Gy X-ray radiation,the peripheral blood and spleen lymphocytes recovered from heat shock pretreatment for 6h or 10h,and the difference of the lymphocytes apoptosis between the combination group(heat shock and irradition) and the irradiation group had statistical significance(P＜0.01).But for 3h recovering time,the difference had no statistical significance(P＞0.01).After heat shock pretreatment,the lymphocytes recovered for 3h,6h or 10h,and the difference of the lymphocytes necrosis between the combination group(heat shock and irradiation) and the irradiation group had no statistical significance(P＞0.01).(4) Under the 0.5-3.0Gy X-ray radiation,the peripheral blood and spleen lymphocytes recovered from heat shock pretreatment for 3h,6h or 10h,and the difference of the lymphocytes hprt gene mutation between the combination group(heat shock and irradiation) and the irradiation group had no statistical significance(P＞0.01).The protection efficiency of HSP70 to peripheral blood and spleen lymphocytes was about 70% under 0.5Gy and about 30%under 3.0Gy.So the HSP70 protection efficiency was much more obivious under low dose irradiation. Conclusions:(1) There was no significan effect on the survival,apoptosis and hprt gene mutation in peripheral blood and spleen lymphocytes which were pretreated by 42℃heat shock for 90min.That's to say,the 42℃heat shock for 90min was the ideal condition to induce lymphocytes to highly express HSP70.(2) The HSP70 expression level in peripheral blood and spleen lymphocytes increased immediately after 42℃heat shock for 90min and reached max during 2-4h,and then decreased gradually and could keep on for 24h.Followed by a gradual decline in expression,24h,when the expression is still higher than normal levels.(3) When the irradiation dose was 0.5-3.0Gy,the lymphocytes in peripheral blood and spleen accepted 42℃heat shock for 90min and then recovered for 6h or 10h,and the inducing expressed HSP70 could obviously elevate the ability of anti-radiation apoptosis in lymphocytes.But for 3h recovering time,there was no pretection effect.Meanwhile, HSP70 had no protection effect on the lymphocytes necrosis inducing by ionizing radiation.(4) When the irradiation dose was 0.5-3.0Gy,the lymphocytes in peripheral blood and spleen accepted 42℃heat shock for 90min and then recoverd for 3h,6h,10h,and the inducing expressed HSP70 could produce protection effect on the lymphocytes hprt gene mutation which was induced by X-ray,what's more,such protection effect was much more obvious under low dose irradiation.
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