脂质体介导的重组质粒pIRES-hBMP-2-hVEGF_(165)体外转染对hBMSCs成骨能力的影响

INFLUENCE OF LIPOSOME-MEDIATED RECOMBINANT PLASMID pIRES-hBMP-2-hVEGF_(165) ON OSTEOGENIC ACTIVITY OF hBMSCs IN VITRO

目的探讨重组质粒pIRES-hBMP-2-hVEGF165体外转染对hBMSCs成骨诱导分化的影响。方法构建hBMP-2和hVEGF165真核共表达载体。取健康成人自愿捐献的骨髓分离培养hBMSCs,采用阳离子脂质体将构建的质粒pIRES-hBMP-2-hVEGF165、pIRES-hBMP-2、pIRES-hVEGF165和空质粒载体(pIRES-neo)转染至hBMSCs,作为A、B、C、D组;另取相同数量的未转染细胞作为对照组(E组)。培养4周,采用RT-PCR检测hBMP-2、hVEGF165以及骨钙mRNA表达,Western blot检测细胞hBMP-2和hVEGF165表达,定量检测ALP活性,免疫组织化学方法检测ColⅠ表达。结果与E组比较,A组hBMSCs高表达hBMP-2、hVEGF165、ColⅠ及骨钙素,B组大量表达骨钙素及ColⅠ,C组高表达hVEGF165,而B组hVEGF165及C组hBMP-2和ColⅠ的表达与D、E组相似,呈阴性或仅有痕量表达。A、B、C、D及E组ALP活性分别为0.91±0.03、0.90±0.02、0.64±0.03、0.67±0.01、0.66±0.02,A、B组与E组比较差异有统计学意义(P<0.05),C、D、E组差异无统计学意义(P>0.05)。结论重组质粒通过阳离子脂质体能成功转染hBMSCs,外源性hBMP-2和hVEGF165基因在转染细胞中能持续表达,并能增强hBMSCs的成骨能力。

Objective To investigate the effects of the recombinant plasmid pIRES-hBMP-2-hVEGF165 on differentiation and maturation of hBMSCs in vitro. Methods The co-expressing vector of hBMP-2 and hVEGF165 was constructed. The BMSCs were isolated and cultured from health adult human denoted marrow. By the lipofection method, the reconstructed plasmids pIRES-hBMP-2-hVEGF165, pIRES-hBMP-2, pIRES-hVEGF165 and pIRES neo empty vector, were transfected to hBMSCs （groups A, B, C and D）. The untransfected cells were harvested as control group （group E）. After 4 weeks of culture, RT-PCR was employed to assay the hBMP-2, hVEGFj65 and osteocalcin mRNA expression in hBMSCs. The expressions of hBMP-2 and hVEGF165 of BMSCs were assayed by Western blot. The level of ALP activities of BMSCs was determined. Col Ⅰ was also determined by immunohistochemical staining. Results Compared to group E, the hBMSCs in group A secreted high level of hBMP-2, hVEGF165, Col Ⅰ and osteocalcin; osteocalcin and Col Ⅰ expressed at high level in group B, and hVEGF165 expressed at high level in group C. Otherwise, the expression of hVEGF165 in group B and the expressions of hBMP-2 and ColⅠ in group C resemble to that of groups D and E, no expression or few expression was observed. The activities of ALP in groups A, B, C, D and E were 0.91± 0.03, 0.90 ±0.02, 0.64 ±0.03, 0.67 ± 0.01 and 0.66 ± 0.02, respectively. The activity of ALP of groups A and B were significantly increased compared with that of group E （P 〈 0.05）; there was no significant difference among groups C, D and E （P 〉 0.05）. Conclusion The recombinant plasmid plRES-hBMP-2-hVEGF165 can be successfully transfected into BMSCs with cation liposome-mediated transfection method, the exogenous hBMP-2 and hVEGF165 genes can be expressed constitutively in the transfected BMSCs, and it can enhance the differentiation abilities of BMSCs.