Beclin 1基因突变对成骨细胞自噬-凋亡表达的影响
Effect of Beclin1 gene mutation on autophagy and apoptosis expression in osteoblasts
  
DOI:10.3969/j.issn.1006-7108.2024.06.002
中文关键词:  Beclin 1  骨代谢  成骨细胞  自噬  细胞凋亡
英文关键词:beclin1  bone metabolism  osteoblast  autophagy  apoptosis
基金项目:国家自然科学基金(81973886,82174395);广州中医药大学“双一流”与高水平大学学科协同创新团队重点项目(2021XK21);黄宏兴广东省名中医传承工作室[粤中医办函(2018)5号];广东省研究生教育创新计划项目(2022XSLT013)
作者单位
赵瑞1 林燕平1 黄佳纯1 杨昊霖1 黄宏兴2* 张志海2 万雷2 李颖2 杨志杰1 吴建军1 陈桐莹1 1.广州中医药大学第三临床医学院广东 广州 510405 2.广州中医药大学第三附属医院广东 广州 510378 
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中文摘要:
      目的 观察 Beclin 1野生型、突变型质粒对成骨细胞自噬-凋亡表达的影响。方法 采用原代分离培养的大鼠颅骨成骨细胞,构建Beclin 1突变型质粒转染成骨细胞(MUT)和野生型质粒转染成骨细胞(WT),使用自噬激活剂rapamycin(Rapa)、自噬抑制剂Baf-A1(Baf)和Beclin1/Bcl2 结合抑制剂mifepristone(Mif)干预细胞,分为NC组、Beclin1 WT组、Beclin1 WT+Baf-A1组、Beclin1 WT+mif组、Beclin1 Mut组、Beclin1 Mut+Rapa组。 ,使用免疫共沉淀检测Beclin1-Bcl2复合物结合水平,通过MDC染色、溶酶体染色、流式检测、线粒体膜电位检测、Hoechest染色、免疫荧光、WB分别检测成骨细胞的自噬和凋亡表达情况。结果 免疫共沉淀检测:与NC组相比,MUT组中Beclin1-Bcl2复合物水平显著降低(P<0.05);MDC染色显示:相对于NC组,WT细胞中溶酶体染色明显增加;细胞凋亡方面:与NC组相比,WT组、WT+Mif组细胞凋亡率显著上升,MUT细胞凋亡率低于WT细胞,比较差异具有统计学意义;JC-1检测显示:与NC组相比,WT细胞的线粒体跨膜电位异常改变,而MUT细胞样本的跨膜电位表达活跃。结论 ①Beclin 1的过表达可以抑制Beclin 1-Bcl2复合物的生成;②Beclin 1是参与自噬的重要基因,适度的Bcilin 1表达有利于维持细胞群的代谢平衡,Beclin 1的过表达对细胞自噬无明显影响;③Beclin 1过表达能抑制成骨细胞凋亡。
英文摘要:
      Objective To observe the effect of Beclin1 wild type and mutant plasmid on the expression of autophagy and apoptosis in osteoblasts. Methods Beclin1 mutant plasmid transfected osteoblast (MUT) and wild-type plasmid transfected osteoblast (WT) were constructed from rat skull osteoblasts isolated and cultured in primary culture. Cells were treated with autophagy activator rapamycin (Rapa), autophagy inhibitor BAF-A1 (Baf) and Beclin1/Bcl2 binding inhibitor mifepristone (Mif), respectively. Cells were divided into Beclin1 WT group and Beclin1 WT+mif group, Beclin1 WT+ Baf-A1 group, Beclin1 Mut group, and Beclin1 Mut+Rapa group. The Beclin1-BCL2 complex binding level was detected with immunoprecipitation. The autophagy and apoptosis of osteoblasts were detected with MDC, lysosome, flow detection, mitochondrial membrane potential, Hoechest, immunofluorescence, and WB, respectively. Results Immunoprecipitation detection: compared to that in NC group, Beclin1-Bcl2 complex level in MUT group significantly (p<0.05). MDC staining showed that compared to that in blank group, lysosome staining in WT cells increased significantly. Apoptosis: compared to those in NC group and WT group, the apoptosis rate in WT+Mif group increased significantly. The apoptosis rate in MUT cells was lower than that in WT cells, and the results were statistically different. JC-1 detection showed that compared to that in NC group, the mitochondrial transmembrane potential of WT cells changed abnormally, while the expression of transmembrane potential of MUT cell samples was active. Conclusions (1) Beclin1 overexpression inhibits the formation of Beclin1-BCl2 complex. (2) Beclin1 is an important gene involved in autophagy. Moderate expression of Beclin1 is conducive to maintaining the metabolic balance of cell population. Beclin1 overexpression has no significant effect on autophagy. (3) Beclin1 overexpression inhibits osteoblast apoptosis.
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