摘要
目的:探讨神经调节蛋白1(NRG1)基因修饰的大鼠骨髓间充质干细胞(BMSCs)对体外氧糖剥夺/复氧(OGD/R)损伤少突胶质细胞(CG4细胞)的保护作用及可能机制。方法:采用全骨髓贴壁法分离培养大鼠BMSCs,取第3代BMSCs转染NRG1基因。体外培养CG4细胞,分为常规培养(control)组、OGD/R组、OGD/R处理CG4细胞与BMSCs共培养组(BMSCs组)和OGD/R处理CG4细胞与NRG1-BMSCs共培养组(NRG1-BMSCs组)。采用倒置相差显微镜观察CG4细胞形态学变化;MTT法和细胞划痕实验检测CG4细胞活力和迁移能力;二氢乙啶(DHE)荧光探针检测CG4细胞活性氧(ROS)水平;比色法检测CG4细胞总抗氧化能力(T-AOC)和丙二醛(MDA)含量;衰老相关β-半乳糖苷酶染色检测CG4细胞衰老变化;乳酸脱氢酶(LDH)法和calcein/PI双染法检测CG4细胞损伤和死亡情况;Western blot检测CG4细胞衰老相关蛋白p21和p53,以及磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(PKB/AKT)信号通路相关蛋白表达。结果:成功分离培养BMSCs,并构建NRG1-BMSCs。与control组相比,OGD/R组CG4细胞突起短小、分枝少;细胞活力、迁移能力及T-AOC水平均显著降低(P<0.01);CG4细胞内ROS和MDA含量均显著升高(P<0.01);LDH漏出率、细胞衰老阳性率及死亡率均显著升高(P<0.01);衰老相关蛋白p21和p53表达显著增加(P<0.01);p-PI3K/PI3K和p-AKT/AKT比值显著降低(P<0.01)。与OGD/R组相比,NRG1-BMSCs组CG4细胞突起细长、分枝增多;细胞活力、迁移能力及T-AOC水平均显著增高(P<0.05);ROS和MDA含量显著降低(P<0.01);LDH漏出率、细胞衰老阳性率及死亡率均显著降低(P<0.01);p-PI3K/PI3K和p-AKT/AKT比值显著增加(P<0.01),衰老相关蛋白p21和p53表达均显著降低(P<0.01)。与OGD/R组相比,BMSCs组结果均呈现改善趋势,但差异无统计学意义(P>0.05)。结论:NRG1基因修饰的大鼠BMSCs在体外能够抑制OGD/R诱导的CG4细胞衰老及死亡,其作用机制可能与调节PI3K/AKT信号通路,减轻细胞氧化应激有关。
AIM:To investigate the effect of neuregulin 1(NRG1)gene-modified rat bone marrow mesenchymal stem cells(BMSCs)on oligodendrocytes(CG4 cells)with oxygen-glucose deprivation/reoxygenation(OGD/R)-induced injury and its possible mechanism.METHODS:Rat BMSCs were isolated and cultured by the whole bone marrow adherence method,and the 3rd-generation BMSCs were transfected with NRG1 gene(NRG1-BMSCs).The CG4 cells were cultured in vitro and divided into control group,OGD/R group,OGD/R-treated CG4 cells co-cultured with BMSCs group(BMSCs group),and OGD/R-treated CG4 cells co-cultured with NRG1-BMSCs group(NRG1-BMSCs group).The morphological changes of CG4 cells were observed by inverted phase-contrast microscopy.The viability and migration capacity of CG4 cells were detected by MTT assay and cell scratch test.The level of reactive oxygen species(ROS),total antioxidant capacity(T-AOC)and malondialdehyde(MDA)content in CG4 cells were detected by dihydroethidium(DHE)fluorescent probe and colorimetry.The senescence of CG4 cells was detected by senescence-associatedβ-galactosidase staining,and the injury and death of CG4 cells were detected by measurement of the release of lactate dehydrogenase(LDH)and calcein/PI double staining.The levels of senescence-related proteins p21 and p53,and phosphatidylinositol 3-kinase(PI3K)/protein kinase B(PKB/AKT)signaling pathway-related proteins in CG4 cells were evaluated by Western blot.RESULTS:Rat BMSCs were successfully isolated and cultured,and NRG1-BMSCs were constructed.Compared with control group,the CG4 cells in OGD/R group had shorter protrusions and fewer branches,and cell viability,migration ability and T-AOC level were significantly decreased(P<0.01).In addition,ROS and MDA content,LDH leakage rate,cell senescence positive and death rates,and expression of p21 and p53 were increased significantly(P<0.01),while the ratios of p-PI3K/PI3K and p-AKT/AKT were decreased in OGD/R group(P<0.01).Compared with OGD/R group,the CG4 cells in NRG1-BMSCs group had slender processes and more branches,and cell viability,migration ability and T-AOC level were significantly increased(P<0.05),while ROS and MDA content,LDH leakage rate,and cell senescence positive and death rates were significantly decreased(P<0.01).Moreover,p-PI3K/PI3K and p-AKT/AKT ratios were increased(P<0.01),while p21 and p53 protein expression was significantly decreased in NRG1-BMSCs group(P<0.01).Compared with OGD/R group,the results in BMSCs group showed a trend of improvement,but there was no statistical difference(P>0.05).CONCLUSION:NRG1 gene-modified rat BMSCs can inhibit OGD/R-induced senescence and death of CG4 cells in vitro,and the mechanism might be related to regulation of PI3K/AKT signaling pathway and reduction of oxidative stress in cells.
作者
赵富生
陈君
杨国宏
符禹玄
张可心
李媛媛
武庚
ZHAO Fu-sheng;CHEN Jun;YANG Guo-hong;FU Yu-xuan;ZHANG Ke-xin;LI Yuan-yuan;WU Geng(Mudanjiang Medical University,Mudanjiang 157000,China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2022年第11期1962-1971,共10页
Chinese Journal of Pathophysiology
基金
黑龙江省卫生健康委员会科研项目(No.2019-383)
黑龙江省自然科学基金资助项目(No.LH2020H073)
黑龙江省中医药管理局中医药项目(No.ZHY18-167)。
