KIF1A过表达对氧糖剥夺-再灌注损伤PC12细胞的作用机制研究

The effects of KIF1A overexpression on oxygen-glucose deprivation and reperfusion induced PC12 cell injury

中文关键词:  Kinesin-3家族成员蛋白1A  PC12细胞  氧糖剥夺  凋亡  哺乳动物雷帕霉素靶蛋白

英文关键词:Kinesin-3 family member 1A  PC12 cell  Oxygen-glucose deprivation and reperfusion  Apoptosis  Mammalian target of rapamycin

中文摘要:

  【摘要】 目的：研究Kinesin-3家族成员蛋白（Kinesin-3 family member1A，KIF1A）对氧糖剥夺-再灌注诱导的PC12细胞活力、自噬和凋亡的影响，为进一步研究KIF1A在脊髓缺血再灌注损伤治疗方面提供理论依据。方法：PC12细胞（美国ATCC公司）分为四组：A组，对照组，无处理；B组，氧糖剥夺再灌注（oxygen glucose deprivation/reperfusion，OGD/R）组，PC12细胞用无糖DMEM培养基，于含混合气体（95% N2和5% CO2）的37℃恒温箱内密闭缺氧培养4h；C组，pcDNA3.1空质粒组，PC12细胞转染pcDNA3.1空质粒48h，进行OGD/R处理；D组，pcDNA3.1-KIF1A质粒组，PC12细胞转染pcDNA3.1-KIF1A质粒48h，进行OGD/R处理。B、C、D组经OGD/R处理后，更换常规培养基，正常孵育24h，收集细胞总RNA及蛋白质，进行实时定量PCR（quantitative real-time PCR，qRT PCR）和Western blot检测KIF1A mRNA和蛋白的表达情况；CCK8检测细胞存活率变化；凋亡ELISA及Caspase-3活性检测试剂盒检测细胞凋亡及Caspase-3活性；Western blot检测各组自噬相关基因LC3-Ⅰ、LC3-Ⅱ、P62以及哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin，mTOR）信号通路的蛋白表达变化。结果：与A组（1.00±0.00）相比， B组细胞KIF1A mRNA（0.41±0.05）和蛋白表达水平（0.52±0.07，P＜0.05）显著下调；细胞活力[（51.60±7.35）%，P＜0.05]显著降低。与B组（1.00±0.00）相比，C组空质粒对KIF1A mRNA（0.91±0.13）及蛋白质（1.08±0.08）表达，细胞活力[（51.60±7.35）% vs （47.30±4.16）%]，细胞凋亡（1.95±0.18 vs 2.08±0.16，P>0.05）等无显著影响。而D组KIF1A过表达后能显著上调KIF1A mRNA（2.63±0.16）以及蛋白表达（2.51±0.18，P＜0.05），显著缓解OGD/R引起的细胞存活率下降[（51.60±7.35）% vs （86.40±9.03）%]及凋亡[1.95±0.18 vs 1.36±0.12，P＜0.05]；与B组相比，D组自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ的比值（1.68±0.14 vs 1.19±0.09，P＜0.05）及pmTOR的表达（1.00±0.00 vs 1.26±0.02，P＜0.05）显著受抑制，P62表达显著升高（0.53±0.05 vs 0.89±0.09，P<0.05）。结论：KIF1A过表达可促进缺血再灌注损伤诱导的PC12细胞存活、抑制细胞自噬与凋亡，其机制可能与抑制mTOR通路相关。

英文摘要:

  【Abstract】 Objectives: To investigate the effects of the Kinesin-3 family member, Kinesin-3 family member 1A(KIF1A) on cell survival, autophagy and cell apoptosis capacity of PC12 cells after oxygen-glucose deprivation and reperfusion, and to provide theoretical basis for further study of KIF1A in the treatment of spinal cord ischemia reperfusion injury. Methods: PC12 cells were divided into four groups as follows: group A, Control, no treatment; group B: oxygen glucose deprivation/reperfusion (OGD/R), PC12 cells were cultured in a glucose-free DMEM medium in a 37℃ incubator containing mixed gases (95% N2 and 5% CO2) for 4h; group C: cells were transfected with pcDNA3.1 plasmid for 48h and then underwent OGD/R; group D: cells were transfected with pcDNA3.1-KIF1A plasmid for 48h and then underwent OGD/R. For group B, C, D, after OGD/R, normal complete medium were changed for 24h incubation. Total RNA and proteins were isolated for quantitative real-time PCR (QRT-PCR) and Western blot analysis. CCK8 was used to observe cell viability. Cell death ELISA kit and Caspase-3 activity detection kit were used to detect apoptosis and caspase-3 activity. The expression levels of autophagy related proteins and mammalian target of rapamycin (mTOR) signaling were investigated by Western blot. Results: Compared with group A(1.00±0.00), OGD/R treatment (group B) could significantly decrease mRNA (0.41±0.05) and protein expression levels (0.52±0.07) of KIF1A in PC12 cells(P<0.05) and inhibit cell viability [(1.00±0.00)% vs (51.60±7.35)%; P＜0.05]. Compared with group B, pcDNA3.1transfection in group C had no significant influence on KIF1A mRNA (0.91±0.13) and protein expression (1.08±0.08), cell viability [(51.60±7.35)% vs (47.30±4.16)%] and apoptosis [1.95±0.18 vs 2.08±0.16, P>0.05]. However, compared with group B, KIF1A overexpression in group D significantly up-regulated KIF1A mRNA (2.63±0.16) and protein (2.51±0.18, P<0.05) expression levels, and promoted the survival rate [(51.60±7.35)% vs (86.40±9.03)%, P<0.05] and apoptosis (1.95±0.18 vs 1.36±0.12, P＜0.05). KIF1A overexpression inhibited the ratio of autophagy-related protein LC3-Ⅱ/ LC3-Ⅰ(1.68±0.14 vs 1.19±0.09, P<0.05) and expression of pmTOR (1.00±0.00 vs 1.26±0.02, P＜0.05), and promoted P62 expression (0.53±0.05 vs 0.89±0.09, P<0.05). Conclusions: Overexpression of KIF1A in PC12 cells could significantly promote OGD/R induced cell survival and inhibited OGD/R induced autophagy and apoptosis. mTOR pathway may be involved in the protective mechanism of KIF1A.