放射诱导神经元细胞发生RIP3介导的程序性坏死

RIP3-mediated necroptosis induced by radiation injury in neuronal cells

目的观察PC12细胞X线照射后有无程序性坏死,检测受体相互作用蛋白3(RIP3)的表达变化及其对程序性坏死的调控作用。方法PC12细胞经不同剂量照射后在不同时间点通过乳酸脱氢酶(LDH)释放检测其坏死。程序性坏死特异性抑制剂Nec-1预处理后,通过LDH检测细胞坏死变化。免疫印迹(WB)检测照射干预后RIP3的表达情况。RIP3特异性抑制剂GSK’872预处理后通过LDH检测细胞坏死变化。通过WB筛选RIP3敲低效果最佳的转染序列。将细胞分为对照组、照射组、溶媒对照组、空载对照组和预处理组,采用WB、免疫荧光染色、MTT、LDH和AnnexV-FITC/PI流式检测分析。结果细胞经4 Gy照射后培养3 h细胞坏死程度相对最大,RIP3蛋白表达增加。Nec-1、GSK’872和RIP3基因敲低预处理后,细胞坏死减少。结论4 Gy照射可诱导PC12细胞出现程序性坏死,照射后培养3 h作用最明显;RIP3参与放射诱导PC12细胞的程序性坏死过程,并且发挥重要的正向调控作用。

Objective To observe the presence or absence of necroptosis in PC12 cells after radiation injury,and to detect the expression of receptor-interacting protein 3(RIP3)and evaluate its regulatory effect on necroptosis.Methods PC12 cells were treated with different doses of irradiation and their necroptosis was detected by lactate dehydrogenase(LDH)release at different time points.After pretreatment with necroptosis inhibitor Necrostatin-1(Nec-1),the changes of cell necroptosis were detected by LDH.The expression level of RIP3 after irradiation intervention was detected by Western blot(WB).After pretreatment with the RIP3-specific inhibitor GSK'872,the changes of cell necroptosis were detected by LDH.The best transfection sequence of RIP3 knockout was screened by WB.The cells were divided into the control group,irradiation group,solvent control group,no-load control group and pretreatment group.WB,immunofluorescence staining,MTT,LDH and Annex V-fluorescein Isothiocyanate/Propidium Iodide(AnnexV-FITC/PI)flow cytometry were used for detection and analysis.Results After 4 Gy irradiation,the degree of cell necrosis was the highest after 3 hours of culture,and the expression level of RIP3 protein was up-regulated.The cell necrosis was decreased after Nec-1,GSK'872 and RIP3 gene knockdown pretreatment.Conclusions The radiation injury of 4Gy can induce the necroptosis of PC12 cells,and the most significant effect can be observed when cultured for 3 hours after irradiation.RIP3 is involved in the process of necroptosis of PC12 cells induced by radiation injury,and plays a pivotal positive regulatory role.