沉默膜联蛋白A1基因影响兔骨髓间充质干细胞的增殖及成骨分化能力

摘要

背景:膜联蛋白A1蛋白参与细胞的增殖和分化的调控.激素性股骨头坏死与骨髓间充质干细胞增殖和分化能力的改变相关.目的:观察沉默膜联蛋白A1基因对兔骨髓间充质干细胞增殖及成骨分化能力的影响.方法:将新西兰大白兔的骨髓间充质干细胞分离培养后分为RNA干扰组,用携带针对膜联蛋白A1基因的短发夹RNA慢病毒载体LV-shANXA1感染骨髓间充质干细胞下调膜联蛋白A1基因的表达;阴性对照组,用携带无义基因序列的慢病毒载体LV-shANXA1-NC感染骨髓间充质干细胞;空白对照组,不加任何干预措施.结果与结论:纯化后的骨髓间充质干细胞CD44和CD105表达阳性率分别为97.2%和95.8%,而CD45阳性率为2.5%.经嘌呤霉素筛选后,感染复数为50时,LV-shANXA1对骨髓间充质干细胞感染效率为80%;感染复数为100时,感染效率为95%.实时 PCR 和Western blot检测显示感染复数为50时,LV-shANXA1对膜联蛋白A1基因的沉默效率效率可达72.4%以上.膜联蛋白A1表达下调后,骨髓间充质干细胞出现明显生长抑制,在随后成骨分化诱导后,细胞的碱性磷酸酶染色阳性率和碱性磷酸酶活性明显降低,且茜素红染色呈阴性反应.表明沉默膜联蛋白A1基因降低骨髓间充质干细胞增殖及成骨分化能力,这可能是激素性股骨头坏死发病机制之一.%BACKGROUND: Annexin A1 (ANXA1) protein is involved in regulation of cell proliferation and differentiation. Steroid-induced osteonecrosis of femoral head is related to altered proliferation and osteogeneic differentiation of bone marrow mesenchymal stem cells (BMSCs).OBJECTIVE: To investigate the effects of silencing ANXA1 gene on proliferation and osteogenic differentiation of rabbit BMSCs. METHODS: New Zealand white rabbit BMSCs were cultured in vitro and divided into three groups. In the RNA interference group, BMSCs were transfected with lentiviral vectors carrying short hairpin RNA (LV-shANXA1) to down regulate the expression of ANXA1 gene. In the negative control group, BMSCs were transfected with lentiviral vectors carrying nonsense gene sequences (LV-shANXA1-NC). In the blank control group, there was no intervention.RESULTS AND CONCLUSION: The positive rate of CD44, CD105 and CD45 in BMSCs after purification was 97.2%, 95.8 % and 2.5%, respectively. The infection efficiency of lentivirus was 80% at multiplicity of infection (MOI) of 50 and 95% at MOI of 100 in BMSCs after selection by puromycin. Real-time PCR and western blot results showed that the silencing efficiency was above 72.4% at MOI of 50. After down regulating the expression of ANXA1 gene, significant growth inhibition could be found in BMSCs; the alkaline phosphatase positive rate and alkaline phosphatase activity decreased, and the alizarin red staining results showed negative reaction in the subsequent induction of osteogenic differentiation in the cells. The experimental results show that silencing ANXA1 gene reduces the proliferation and osteogenic differentiation capacity of BMSCs, which may be one of mechanisms underlying steroid-induced avascular necrosis of femoral head.