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目的:研究不同硬度的聚丙烯酰胺(PA)水凝胶基底对人成骨细胞系MG63生长及分化的影响.方法:制备不同硬度的PA水凝胶,检测其吸水膨胀、润湿性、弹性模量等性能;将MG63细胞接种在包被有Sulfo-SANPAH和Ⅰ型胶原蛋白的不同硬度PA水凝胶表面,光镜和扫描电镜观察细胞生长情况,实时定量RT-PCR和细胞免疫荧光染色检测成骨相关和细胞干性相关基因和蛋白的表达.结果:制备出硬度分别为(31.32±1.04)kPa和(147.62±1.30)kPa的PA水凝胶(PA1和PA2),均具有良好的亲水性能.细胞在PA水凝胶上呈团块生长.相比于传统培养皿,PA水凝胶上培养的细胞性别决定相关基因簇2(SOX2)、八聚体结合转录因子4(OCT4)基因和蛋白表达显著升高(P<0.05),Runt相关转录因子2(RUNX2)、骨钙素(OCN)基因和蛋白表达显著降低(P0.05),PA2组细胞OCT4及SOX2基因表达高于PA1(P<0.05).结论:较高硬度的PA水凝胶有利于成骨细胞生长和维持细胞干性.“,”Objective: To investigate the effects of polyacrylamide (PA) hydrogel substrates with different stiffness on the growth and differentiation of cell line of osteoblats (MG63). Methods: PA hydrogels with different stiffness was prepared and its properties such as water absorption, wettability and elastic; modulus were tested. Then we inoculated MG63 cells on different PA hydrogels coated with Sulfo-SANPAH and collagen I, and we observed the cell growth by light microscope and scanning electron microscope, then the real-time quantitative RT-PCR and cell immunofluorescence staining were used to detect the expression of genes and proteins about osteogenesis and stemness of MG63. Results: PA hydragels (PA1 and PA2) with stiffness of(31.32±1.04) kPa and(147.62±1.30) kPa were prepared with good hydrophilic; properties. The cells grew in clumps on PA hydrogels. Compared with traditional culture dishes，the expression of sex-determining region Y-box 2(SOX2)，octamer-binding transcription factor 4(OCT4) genes and protein in cells cultured on PA hydrogels were significantly increased(P 0.05)，while the expression of OCT4 and SOX2 genes in the cells of the PA2 group was higher than that of PA1 (P< 0.05). Conclusions: The stiffer PA hydrogel is beneficial to the growth and maintenance of the stemness of osteoblast.