A Study About The Damage Effects And Mechanism Of The Solid Tumor Cell By Hypo-irradiation

Objectives:Hypofractionated radiotherapy,because treatment time is short,is wieldy used in treatment for tumors in body.Especially for early stage primary lung cancer and primary or metastases liver cancer,have got an excellent local control rate.The present study showed that:compared with the conventional radiotherapy,hypo-radiotherapy has more obvious damage effect on tumor cells,but large signal dose irradiation caused the death of tumor cells is unclear.After irradiated cells can undergo apoptosis and necrosis,and present study showed that:when cell undergo necrosis,it can be mediated by RIP1/RIP3 signaling pathway.So the main subjects of this study are as following:1.We will collect the patients' materials that have received hypo-radiotherapy in Cyberknife center of Tianjin medical university affiliated hospital.And analysis the local control of liver metastases treated with Cyberknife.2.We use non-small cell lung cancer cells as agents to research after different dose irradiated the styles of cell death and its mediate mechanism.We hope to provide a theoretical basis of radiation biology for hypofractionated radiotherapy in clinical practice.Methods:1.We collected and analysis 57 patients' materials that received hypofractionated radiotherapy in Cyberknife center of Tianjin medical university affiliated hospital,and used SPSS software to calculate the local control rate.2.We used Western Blotting to detect the expression level of endogenous RIP1 and RIP3 in two non-small lung cancer cell lines.3.We used 293T cells to generate RIP3 down-regulation lentivirus and this was used to establish stable A549 and LTEP-A2 cell lines.Then theRIP3 expression level was identified by Western Blotting analysis.4.We selected different dose to irradiate non-small lung cancer cell and then implement cell colony formation assy to detect the colony foration ability.5.After different dose(10Gy×1F?2Gy×1F?2Gy×9F)irradiated,we used Western blotting to detect the expression level of caspase 3 and cleaved caspase 3,the molecular marker of aopotosis,and HMGB1 expresses level,the molecular marker of necroptosis at different times(0.5h?1h?2h?4h?6h?8h?16h?24h).6.We used immunofluorescence to detect the necroptosis molecular marker HMGB1 expression level of A549 after large single dose irradiation.Results:1.The first year and second year local control rate of liver metastasis after Cyberknifer treatment was 94.4%and89.7%,respectively.2.RIP1 and RIP3 expressed in non-small lung cancer cell A549 and LTEP-A2 cell lines,but the expression level is different.3.The results of colony formation assay showed that:with the irradiation dose increased,the ubiquitous inhibitor of RIP1 can increase the colony formation of A549 and LTEP-A2,compared to DMSO.4.Western blotting dected the expression lebel of caspase 3,cleaved caspase 3 and HMGB1 in A549 and LTEP-A2,at different times after different dose irradiated.The results showed that:when extracted whole cell protein,the expression level of caspase 3,cleaved caspased 3 and HMGB1 did not change according to times.5.Immunofluorescence results showed that:After signal large dose(10Gy)irradiated for A549,nuclear HMGB1 would release from the nucleus to the cytoplasm with time.Conclusions:1.Cyberknife stereotatic body radiotherapy treats liver metastasis patients that with one to four metastases is safe and can get excellent local control.2.The RIP1 and RIP3 express in the normal non-small lung cancer cell A549 and LTEP-A2,so these two cell lines can provide agents for research necroptosi.3.HMGB1 is non-histone proteins present in the nucleus,so extracted the whole cell protein can not detect cells undergo necrosis level after irradiated.But the cleaved caspase 3 expression level increased with the irradiation dose escalated,suggest that according to the dose escalated cells undergo apoptosis increased.4.After different dose irradiated for A549,cytoplasmic HMGB1 gradually increased with time.Nec-1 can prevent HMGB1 releaseed frome nucleus to cytoplasmic.5.After different dose irradiated for two non-small lung caner cell lines,Nec-1 inhibitor of necroptosis can improve the colony formation ability compared to DMSO.These results suggest that after large dose irradiated the cells will occur necroptosis.