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目的前期发现Drp1(Dynamin-related protein 1)可能参与小鼠颗粒酶F(Granzyme F,Gzm F)诱导的细胞凋亡通路,本文试图证明Drp1是颗粒酶F新的作用底物,并且参与诱导Bax/Bak非依赖性细胞死亡。方法表达活性Gzm F以及无活性点突变的颗粒酶F(Ser195Ala Gzm F,S-AGzm F),通过不同浓度梯度以及不同时间处理全细胞裂解液,Western blot鉴定颗粒酶F对Drp1的切割情况;模拟生理情况下的Loading条件,通过腺病毒Ad将颗粒酶F转导进入细胞,进一步验证Drp1为颗粒酶F的生理底物;构建Drp1高表达质粒pc DNA-Drp1,在细胞中高表达Drp1,Western blot验证并通过Ad将颗粒酶F导入此细胞,流式细胞术检测Drp1高表达对凋亡影响情况。结果颗粒酶F能够在不同浓度和时间切割Drp1,并且产生Mr约60 000的切割片段。随着颗粒酶F作用时间和浓度的提高,被切割的Drp1量也依次提高;模拟生理情况下,Gzm F/Ad转导进入细胞后,Drp1被切割,证明Drp1是颗粒酶F的生理底物;成功构建Drp1高表达载体,将颗粒酶F导入细胞,流式细胞术检测到Drp1高表达细胞的凋亡明显降低。结论 Drp1是颗粒酶F的底物且可能在颗粒酶F诱导细胞凋亡通路中扮演重要角色。
OBJECTIVE: It was found that Drp1 (Dynamin-related protein 1) may be involved in the apoptosis pathway induced by Granzyme F (Gzm F) in mice. This paper attempts to prove that Drp1 is a novel substrate of granzyme F and participates in the induction of Bax / Bak-independent cell death. METHODS: GzmF and Ser195Ala GzmF and S-AGzmF were used to detect the cleavage of Drp1 by Western blotting. The whole cell lysates were treated with different concentrations of GzmF and different time points. Under the condition of loading, the granzyme F was transduced into the cells by adenovirus Ad to further verify that Drp1 was the physiological substrate of granzyme F. The high expression plasmid pcDNA-Drp1 was constructed to express Drp1 and Western blot was used to verify that Granzyme F was transfected into this cell by Ad. Flow cytometry was used to detect the effect of Drp1 overexpression on apoptosis. Results Granzyme F was able to cleave Drp1 at different concentrations and times and produce a Mr fragment of about 60,000. As the time and concentration of granzyme F increased, the amount of Drp1 cleaved gradually increased. Under simulated physiological conditions, Drp1 was cleaved after transduction of Gzm F / Ad into cells, demonstrating that Drp1 is the physiological substrate of granzyme F The Drp1 high expression vector was successfully constructed and the granzyme F was introduced into the cells. The apoptosis of Drp1 overexpression cells was significantly reduced by flow cytometry. Conclusion Drp1 is a substrate of granzyme F and may play an important role in granzyme F-induced apoptosis.