血红素氧化酶1在ADSCs增殖、凋亡和成骨分化的作用

Effect of proliferation, apoptosis and osteogenic differentiation of ADSCs transduced with heme oxygenase-1

目的:探讨应用慢病毒介导血红素氧化酶1(HO-1)基因转染诱导脂肪来源基质干细胞(ADSCs)后对细胞增殖、凋亡和成骨分化的影响。方法:取健康10周龄SD大鼠双侧腹股沟脂肪,分离、培养ADSCs,观察第3代细胞形态并进行成骨和成脂分化研究,流式分析细胞表面标记物,确立分离细胞为ADSCs。利用基因重组技术构建HO-1基因重组慢病毒载体,并以Polybrene(8μg/m L)介导慢病毒转染ADSCs,倒置显微镜下观察转染细胞荧光表达优化转染复数,Western blot检测HO-1蛋白表达。设HO-1转染组(A组)、空载体转染组(B组)和未转染组(C组);MTT,流式分析和茜素红钙结节染色分别检测各组增殖、凋亡和成骨分化情况。结果:分离获得的ADSCs具有多向分化潜能:CD29(+)、CD44(+)、CD90(+)、CD31(-)、CD45(-);经菌液PCR和Western blot鉴定HO-1基因重组慢病毒载体构建成功,与B、C组相比,A组具有较高的细胞活性(P<0.05),无血清培养基中较低的凋亡率(P<0.05),并增加细胞钙化基质形成的量(P<0.05)。结论:成功构建HO-1基因重组慢病毒表达载体并转染大鼠ADSCs,HO-1基因转染入ADSCs后表达HO-1蛋白成功,HO-1转染后可以发挥对ADSCs促增殖、抗凋亡和促成骨分化的作用。

Objective： To investigate the proliferation, apoptosis and osteogenic differentiation effect of heme oxygenase-1 （HO-1） overexpression mediated by lentivirus on adipose-derived stromal cells （ADSCs）. Methods： Fat tissue was harvested from inguinal area of 10-week-old SD rats, after which ADSCs were isolated and cultured. The morphology of third passages were observed, the multi-differentiation effect and surface markers were investigated to identify ADSCs. The lentivirus vector containing HO-1 gene was constructed through genetic recombination. ADSCs were transfected by lenvirus with polybrene （8μg/mL）. The multiplicity of infection was optimized and HO-1 expression in ADSCs was tested by Western blot. Cells were divided into three groups：ADSCs transfected with Lenti-HO-1 （group A）, ADSCs transfected with empty vector （group B） and ADSCs （C group）. The proliferation, apoptosis and osteogenic differentiation effect were investigated by MTT assay, flow cytometry and alizarin red staining. Results： ADSCs had a multi-differentiation character, CD29（＋）, CD44（＋）, CD90（＋）, CD31（-）, CD45（-）. The lentivirus containing HO-1 was constructed successfully evidenced by bacteria PCR and Western blot. Compared with group B and C, cells of group A had a better viability （P＜0.05）, exhibited anti-apoptotic effect （P＜0.05） and more osteogenic differentiation （P＜0.05）. Conclusion： The lenvirus vector containing HO-1 gene is successfully constructed, and HO-1 could express in ADSCs transfected with Lenti-HO-1. HO-1 expression could enhance pro-viability and anti-apoptotic effect, and it could stimulate osteogenic differentiation of ADSCs.