RIP3对BCG感染后小鼠巨噬细胞凋亡的调控作用研究

目的:探讨受体相互作用蛋白3(RIP3)在卡介苗(BCG)诱导小鼠巨噬细胞RAW264.7凋亡过程中的调控作用。方法:构建RIP3腺病毒干扰载体并感染巨噬细胞,并用BCG进行感染。采用噻唑蓝(MTT)比色法检测细胞活力;利用流式细胞术对细胞凋亡率、线粒体膜电位及活性氧含量进行检测;通过Western blot检测RIP3及凋亡相关蛋白的表达水平。结果:BCG感染RAW264.7细胞后,细胞活力下降且RIP3蛋白表达量显著上调(P<0.01)。而与BCG单独感染组相比,BCG感染结合RIP3干扰处理组细胞凋亡率及活性氧含量降低,线粒体膜电位升高,同时促凋亡蛋白Bax与cleaved caspase-3的蛋白水平显著升高,抑凋亡蛋白Bcl-2的表达量显著降低(P<0.01)。结论:在BCG感染小鼠巨噬细胞RAW264.7的过程中,RIP3参与了BCG诱导的RAW264.7细胞的凋亡,且该过程可能是通过线粒体途径实现的。

Effect of RIP3 expression on apoptosis of mouse macrophages infected with BCG

AIM: To investigate the function of receptor-interacting proteins 3(RIP3) in regulating Bacillus Calmette-Guérin(BCG)-induced apoptosis of mouse macrophages(RAW264.7 cells). METHODS: The RIP3 adenovirus interference vector was constructed and used to infect the RAW264.7 cells, and then the RAW264.7 cells were infected with BCG. The cell viability was measured by MTT assay. The apoptotic rate, mitochondrial membrane potential and production of reactive oxygen species(ROS) were determined by flow cytometry analysis. The protein levels of RIP3 and apoptosis-associated proteins were examined by Western blot. RESULTS: The viability of RAW264.7 cells was decreased after BCG infection. In the meantime, the expression of RIP3 was up-regulated significantly(P<0.01). Compared with BCG infection group, the apoptotic rate and ROS level in BCG and RIP3 adenovirus interference vector co-infection group were significantly decreased(P<0.01). Importantly, RIP3 was able to further promote apoptosis in BCG-infected RAW264.7 cells in part by increasing mitochondrial membrane potential(P<0.01). In addition, Western blot analysis further demonstrated that RIP3 was involved in BCG-induced apoptosis partly through down-regulation of anti-apoptotic protein Bcl-2, and up-regulation of Bax and cleaved caspase-3(P<0.01). CONCLUSION: RIP3 is involved in BCG-induced apoptosis of RAW264.7 cells, and this process may be achieved by the mitochondrial pathway.