摘要
目的:探讨硫化氢对Val30Ala突变型家族性淀粉样多发性神经病NIH3T3细胞模型中转甲状腺素蛋白(transthyretin,TTR)单体形成的调节作用。方法:分别构建pc DNA 3.1/myc-His-TTR-WT(TTR野生组)和pc DNA3.1/myc-His-TTR-Val30A(Val30Ala突变组)重组表达载体,并采用脂质体介导的方法分别将其转染入NIH3T3细胞,用MTT染色方法检测Na HS处理前后的细胞活力;用Western blot检测Na HS处理前后的自噬标志蛋白LC3-Ⅱ、LC3-Ⅰ、Beclin-1,以及TTR四聚体、TTR单体的表达情况。结果:(1)培育24 h后,Val30Ala突变组较TTR野生组中TTR单体水平升高、LC3-Ⅱ/LC3-Ⅰ比值下降(P<0.05);(2)与Na HS共培养24 h后,Val30Ala突变组LC3-Ⅱ/LC3-Ⅰ比值上升、TTR单体与TTR四聚体的比值降低(P<0.05);而在TTR野生组中无明显改变(P>0.05);(3)与Na HS共培养24 h后,各组中Beclin-1表达水平无明显改变(P>0.05);提前加入3-MA,可逆转Ha HS对自噬的诱导作用和TTR单体形成的抑制作用。结论:硫化氢可改善自噬障碍,并降低Val30Ala突变型TTR单体的形成。
Objective：To investigate the regulatory effect of hydrogen sulfide on the formation of TTR monomer in Val30Ala mutant familial amyloid polyneuropa- thy NIH3T3 cell model. Methods：pcDNA 3.1/myc -His -TTR -WT and pcDNA3.1/myc -His -TTR - Val30A recombinant expression vectors were constructed and transfected into NIH3T3 cells respectively using lipofectamine mediated method. MTT staining method was used to detect the cell viability before and after NariS treatment. The expressions of autophagy marker protein of LC3 - Ⅱ, LC3 - Ⅰ, Beclin - 1, tetrameric TTR and monomeric ITR were detected by Western blot analy- sis before and after NariS treatment. Results ：① After 24. hours of incubation, the level of TTR monomer in Val30Ala mutant group was higher than that in TrR wild type, and the ratio of LC3 - Ⅱ/ Ⅰ decreased obviously （ P 〈 0.05） ;②After 24 hours of coculture with NariS, the ratio of LC3 - Ⅱ / Ⅰ in Val30Ala mutant group increased obviously, and the ratio of TrR monomer to tetramers decreased obviously （ P 〈 0.05）, but there was no obvious change in TI＇R wild type （ P 〉 0.05） ; ③ After 24 hours of coculture with NariS, the level of Beclin - l in each group showed no significant change （ P 〉 0.05） ; the early addition of 3 - MA could reverse the induction of autophagy by Halls and the inhibition of ＇fTR monomer formation. Conclusion： Hydrogen sulfide can improve autophagy and reduce the formation of Val30Ala mutant TTR monomers.
出处
《包头医学院学报》
CAS
2017年第12期69-71,120,共4页
Journal of Baotou Medical College
基金
2016年湖南省医药卫生科研计划项目(编号C2016087)
