p53和Parkin通过调节线粒体自噬抵抗凋亡和衰老促进干细胞修复股骨头坏死

p53 and parkin promote stem cells to repair osteonecrosis of the femoral head by regulating mitophagy to resist apoptosis and aging

目的观察肿瘤蛋白p53和帕金森相关蛋白(Parkin)调节线粒体自噬对骨髓间充质干细胞(BMSCs)应激性凋亡和衰老的影响,以及对BMSCs修复早期激素性股骨头坏死(SONFH)的作用。方法2017年10月至2019年11月,将p53干扰慢病毒和Parkin过表达慢病毒(Lv-Shp53和Lv-Parkin)转染兔BMSCs,用高浓度H2O2处理BMSCs 24 h,将其分5组:BMSCs、BMSCs/绿色荧光蛋白(EGFP)、BMSCs/Shp53、BMSCs/Parkin和BMSCs/Parkin/Shp53,检测线粒体自噬、凋亡、β-半乳糖甘酶(β-gal)活性等指标,探明p53和Parkin调节线粒体自噬对BMSCs应激性凋亡和衰老的影响。然后将BMSCs移植修复早期SONFH,分6组:空白对照、XACB、XACB/BMSCs、XACB/BMSCs/Parkin、XACB/BMSCs/Shp53、XACB/BMSCs/Parkin/Shp53,术后4、8和12周,通过苏木精-伊红(HE)、马松(Masson)染色及成骨标志物水平,评估早期SONFH的修复效果。采用单因素方差分析(ANOVA)数据。结果在氧化应激条件下,BMSCs、BMSCs/EGFP、BMSCs/Shp53、BMSCs/Parkin和BMSCs/Parkin/Shp53组细胞凋亡率分别为(56.900±4.371)、(58.680±4.926)、(28.683±3.767)、(40.223±4.402)、(11.283±3.945)%,β-gal阳性细胞比率分别为(74.133±5.350)、(81.993±4.012)、(43.453±3.503)、(45.310±5.605)、(19.913±4.224)%,其中BMSCs/Parkin/Shp53组线粒体自噬显著增强,细胞凋亡率和β-gal阳性细胞比率最低,差异均有统计学意义(F=64.204和89.383,P<0.05)。术后4周和8周,XACB/BMSCs/Parkin/Shp53组与空白对照、XACB、XACB/BMSCs、XACB/BMSCs/Parkin、XACB/BMSCs/Shp53组比较,骨坏死区的新骨组织和骨钙素水平显著增加,但骨坏死区尚未完全修复。术后12周,空白对照、XACB、XACB/BMSCs、XACB/BMSCs/Parkin、XACB/BMSCs/Shp53和XACB/BMSCs/Parkin/Shp53组骨钙素表达量分别为0.060±0.040、0.097±0.031、0.093±0.040、0.213±0.041、0.267±0.059、0.367±0.040,其中XACB/BMSCs/Parkin/Shp53组,骨坏死区已完全修复,可见成熟骨组织,骨钙素表达最高,差异有统计学意义(F=23.944,P<0.05)。结论p53和Parkin调节线粒体自噬可有效抵抗BMSCs应激性凋亡和衰老,能提高BMSCs对早期SONFH的修复作用。

Objective To investigate the effects of tumor protein p53(p53)and PD-related protein(Parkin)regulation of mitophagy on stress-induced apoptosis and aging of bone marrow mesenchymal stem cells(BMSCs),and its role in repairing early steroid-induced osteonecrosis of the femoral head(SONFH)by BMSCs transplantation.Methods In this study,from October 2017 to November 2019,Rabbit BMSCs were transfected with p53 interfering lentivirus and Parkin overexpression lentivirus(Lv-Shp53 and Lv-Parkin),and BMSCs were treated with high concentration H2O2 for 24 hours.The experiment was divided into 5 groups:BMSCs,BMSCs/Enhanced green fluorescent protein(EGFP),BMSCs/Shp53,BMSCs/Parkin,BMSCs/Parkin/Shp53.The effects of p53 and parkin on stress-induced apoptosis and aging of BMSCs were investigated by detecting mitophagy,apoptosis andβ-galactosylglycase(β-gal)activity.Then BMSCs were transplanted to repair the early SONFH,the experiment was divided into 6 groups:Control,XACB,XACB/BMSCs,XACB/BMSCs/Parkin,XACB/BMSCs/Shp53,XACB/BMSCs/Parkin/Shp53.At 4,8 and 12 weeks after surgery,the repair effect of early SONFH was evaluated by hematoxylin-eosin(HE),Masson staining and osteogenic marker levels.ANOVA was used to analyze data,P<0.05 was statistically significant.Results Under oxidative stress,the apoptosis rates of BMSCs,BMSCs/EGFP,BMSCs/Shp53,BMSCs/Parkin and BMSCs/Parkin/Shp53 groups were(56.900±4.371),(58.680±4.926),(28.683±3.767),(40.223±4.402),(11.283±3.945)%,and the ratio ofβ-gal positive cells were(74.133±5.350),(81.993±4.012),(43.453±3.503),(45.310±5.605),(19.913±4.224)%,respectively.Among them,the BMSCs/Parkin/Shp53 group had enhanced mitophagy,the rate of apoptosis andβ-gal positive cells were the lowest,and the differences were statistically significant(F=64.204 and 89.383,P<0.05).At 4w and 8w after operation,the levels of new bone tissue and osteocalcin in osteonecrosis area of XACB/BMSCs/Parkin/Shp53 group were significantly increased compared with those of Control,XACB,XACB/BMSCs,XACB/BMSCs/Parkin and XACB/BMSCs/Shp53 groups,but the osteonecrosis area was not completely repaired.At 12 weeks after operation,the expression of osteocalcin in Control,XACB,XACB/BMSCs,XACB/BMSCs/Parkin and XACB/BMSCs/Shp53 and XACB/BMSCs/Parkin/Shp53 groups were 0.060±0.040,0.097±0.031,0.093±0.040,0.213±0.041,0.267±0.059,0.367±0.040,respectively.Among them,in the XACB/BMSCs/Parkin/Shp53 group,the osteonecrotic area was completely repaired,the bone tissue was mature,and the expression of osteocalcin was the highest,the difference was statistically significant(F=23.944,P<0.05).Conclusion p53 and Parkin regulation of mitophagy can effectively resist the stress-induced apoptosis and senescence of BMSCs,and can improve the repair effect of BMSCs on early SONFH.