静脉移植缺氧预处理骨髓间充质干细胞在大鼠早期激素性股骨头坏死中的作用

Effect of hypoxic preconditioning of bone marrow mesenchymal stem cells with intravenous transplantation in early steroid-induced femoral head necrosis in rats

目的探讨缺氧预处理对骨髓间充干细胞(BMSCs)的细胞基本功能的影响及静脉移植缺氧预处理BMSCs在大鼠早期激素性股骨头坏死(SONFH)中的防治作用。方法提取大鼠BMSCs分离培养,流式细胞仪鉴定表面抗原(CD34、CD44、CD45和CD90),通过细胞计数试剂盒(CCK-8)分析、划痕实验、茜素红及碱性磷酸酶染色对常氧组(No)和缺氧组(Hp)BMSCs细胞功能进行评估。32只SD大鼠随机分为4组,对照组(C组)、模型组(M组)、常氧组(N组)和缺氧预处理组(H组),M组使用脂多糖[2 mg/(kg·d)]联合甲基强的松龙[20 mg/(kg·d)]构建SONFH模型,N组和H组构建模型的同时分别通过尾静脉注射107单位的常氧培养和缺氧预处理大鼠骨髓间充质干细胞(rBMSCs),6周后取股骨头固定后进行Micro CT扫描及重建。股骨头脱钙切片后用苏木精-伊红(HE)染色观察并统计骨坏死发生率。两组间均数比较采用t检验,多组间比较采用单因素方差分析(组间两两比较采用LSD-t检验)。结果rBMSCs流式鉴定结果显示:rBMSCs高表达CD44(100%)和CD90(99.4%),低表达CD34(0.48%)和CD45(0.39%);CCK-8显示缺氧预处理能够增强BMSCs的增殖能力(t=7.199,P<0.05);划痕实验表明Hp组在12 h(t=2.462,P<0.05)、24 h(t=2.471,P<0.05)和36 h(t=3.434,P<0.01)均表现出增强的迁移能力;茜素红染色显示缺氧处理对rBMSCs的体外矿化能力有显著的增强作用(t=6.722,P<0.05)。碱性磷酸酶(ALP)染色表明缺氧预处理能够显著增强rBMSCs的体外成骨能力(t=13.37,P<0.05)。动物实验中C、M、N和H组的骨坏死发生率分别为0%(0/16)、87.5%(14/16)、25.0%(4/16)和12.5%(2/16),移植缺氧预处理rBMSCs能够明显减少SONFH的发生率;HE染色C、M、N和H组的空骨陷窝率分别为(2.765±0.423)%、(30.817±1.203)%、(14.724±1.312)%和(8.487±1.671)%,表明rBMSCs治疗组空骨陷窝的发生率明显下降(F=286.3,P<0.05);Micro CT扫描测量结果表明rBMSCs治疗后大鼠股骨头BV/TV、Tb.Th、Tb.N均明显增加(F=197.6、290.7、68.7,P<0.05),而Tb.Sp明显减少(F=123.8,P<0.05)。证明静脉移植rBMSCs能够改善股骨头的微观结构,并且缺氧预处理后的rBMSCs改善作用更加显著(P<0.01)。结论缺氧预处理能够明显改善rBMSCs的增殖、迁移和体外成骨能力,有利于增强rBMSCs对大鼠SONFH的防治作用。

Objective To investigate the effect of hypoxia preconditioning on the cell function of bone marrow mesenchymal stem cells(BMSCs)and the preventive and therapeutic effects of hypoxia preconditioning BMSCs in steroid-induced femoral head necrosis(SONFH)in rats after intravenous transplantation.Methods BMSCs were isolated from the bone marrow cavity of the femur of rats.Flow cytometry identification of surface antigens(CD34,CD44,CD45 and CD90).Evaluation of cell function of BMSCs in normoxia group(No)and hypoxia group(Hp)by cell counting kit-8(CCK-8)analysis,scratch test,alizarin red staining and alkaline phosphatase staining.A total of 32 SD rats were randomly divided into four groups(n=8):(a)a control group,(b)a model group,(c)a normoxic group,and(d)a hypoxic group.Model group used lipopolysaccharide[2 mg/(kg·d)]combined with methylprednisolone[20 mg/(kg·d)]to construct the SONFH model.After SONFH model was constructed,the normoxic-rat bone marrow mesenchymal stem cells(rBMSCs)group and hypoxic-rBMSCs group were injected with 107 normoxic or hypoxia preconditioned rBMSCs through the tail vein.All rats were sacrificed and the femoral heads were collected and evaluated by Micro CT.After femoral heads decalcification,the slices were stained with hematoxylin-eosin to observe and count the incidence of osteonecrosis.Results The results of flow cytometric identification of rBMSCs showed that rBMSCs expressed high expression of CD44(100%)and CD90(99.4%),and low expression of CD34(0.48%)and CD45(0.39%).The CCK-8 analysis showed that hypoxia preconditioning can enhance the proliferation ability of rBMSCs(t=7.199,P<0.05).The scratch test analysis showed that compared with No group,Hp group enhanced rBMSCs′migration ability at 12 h(t=2.462,P<0.05),24 h(t=2.471,P<0.05)and 36 h(t=3.434,P<0.01).The alizarin red staining showed hypoxia precondition significantly enhanced the mineralization ability of rBMSCs in vitro(t=6.722,P<0.05).The alkaline phosphatase(ALP)staining showed that hypoxia pretreatment can significantly enhance the osteogenic ability of rBMSCs in vitro(t=13.37,P<0.05).In animal experiments,the incidence of osteonecrosis in group C,group M,group N,and group H were 0%(0/16),87.5%(14/16),25.0%(4/16),12.5%(2/16).The hypoxia precondition of rBMSCs can reduce the incidence of SONFH.The empty bone lacuna rates of HE staining in group C,M,N,and H were(2.765±0.423)%,(30.817±1.203)%,(14.724±1.312)%and(8.487±1.671)%,indicated the incidence of empty bone lacuna in the rBMSCs treatment groups were significantly reduced(F=286.3,P<0.05);The Micro CT scan measurement showed that BV/TV,Tb.Th and Tb.N were significantly increased after rBMSCs treatment(F=197.6,290.7,68.7,P<0.05),while Tb.Sp was significantly reduced(F=123.8,P<0.05).It is proved that intravenous transplantation of rBMSCs can improve the microstructure of the femoral head,and the improvement effect of rBMSCs after hypoxia preconditioning is more significant(P<0.01).Conclusion Hypoxic precondition can significantly improve the proliferation,migration and osteogenic ability of rBMSCs in vitro,which is beneficial to enhance the preventive and therapeutic effects of rBMSCs on SONFH in rats.