H_(2)O_(2)诱导小鼠睾丸间质TM3细胞铁死亡模型的构建

Construction and Identification of Mice TM3 Cell Ferroptosis Model Induced by H_(2)O_(2)

目的 构建并鉴定H_(2)O_(2)诱导小鼠睾丸间质TM3细胞铁死亡模型。方法 常规培养TM3细胞,采用MTS法选择H_(2)O_(2)的处理浓度,设置TM3细胞常规培养组(T组)、2 mmol·L^(-1)H_(2)O_(2)处理TM3细胞1 h组(H_(2)O_(2)-T组)、1μmol·L^(-1)GSH过氧化物酶4抑制剂(RSL3)处理TM3细胞1 h组(RSL3-T组)和3μmol·L^(-1)i FSP1处理TM3细胞24 h组(i FSP1-T组);采用流式细胞术检测活性氧(ROS)水平;检测试剂盒检测细胞的丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性和脂质过氧化物(LPO)含量,Phen-Green-SK荧光探针经激光共聚焦显微镜检测Fe2+含量;采用透射电镜观察线粒体形态结构。结果 根据MTS法选择2 mmol·L^(-1)H_(2)O_(2)处理TM3细胞;与T组相比,H_(2)O_(2)-T组ROS水平、MDA和LPO含量均上升(P均<0.001)、SOD活性下降(P <0.001);细胞内Fe2+含量上升(P<0.01),与RSL3-T组和i FSP1-T组相比,H_(2)O_(2)-T组细胞内LPO、Fe2+含量差异均无统计学意义(P均>0.05);透射电镜结果显示,H_(2)O_(2)-T组细胞线粒体变小皱缩,嵴减少,外膜破裂,与RSL3-T组和i FSP1-T组相似。结论 H_(2)O_(2)可诱导小鼠睾丸间质细胞TM3细胞发生铁死亡。

Objective To construct and identify the ferroptosis model of mice leydig cells(TM3 cells)induced by H_(2)O_(2).Methods TM3 cells were routinely cultured.The treatment concentration of H_(2)O_(2) was selected by MTS method.Four groups were set.There were TM3 cells routinely cultured(group T),TM3 cells treated with 2 mmol·L^(-1) H_(2)O_(2) for 1 h(group H_(2)O_(2)-T),TM3 cells treated with 1μmol·L^(-1) RSL3 for 1 h(group RSL3-T),and TM3 cells treated with 3μmol·L^(-1) iFSP1 for 24 h(group iFSP1-T).The reactive oxygen species(ROS)level was detected by flow cytometry;the malondialdehyde(MDA)content,superoxide dismutase(SOD)activity and lipid peroxide(LPO)content of the cells were detected by detection kits respectively.The content of Fe2+was detected by laser confocal using the cell membrane permeable dye Phen-Green-SK;the morphological structure of mitochondria was observed by transmission electron microscope was showed that.Results TM3 cells were treated with 2 mmol·L^(-1) H_(2)O_(2) according to the MTS method,compared with group T,the ROS level,MDA content,and LPO level of group H_(2)O_(2)-T were increased respectively(P all<0.001),SOD activity decreased(P<0.001),and intracellular Fe2+content increased(P<0.01).There were no significant difference in LPO level and intracellular Fe2+content in group H_(2)O_(2)-T compared with group RSL3-T and group iFSP1-T(P all>0.05).Similar to the RSL3-T group and the iFSP1-T groups,it was showed that the sizes of mitochondria shrunken,the cristaes decreased,and transmission electron microscope was showed that outer membrane ruptured in group H_(2)O_(2)-T.Conclusion The H_(2)O_(2)-induced mice TM3 cell ferroptosis model was constructed successfully.