摘要目的 观察人脂肪间充质干细胞(human adipose-derived mesenchymal stem cell,hAMSC)在不同比例海藻酸钠-胶原支架材料表面及内部的增殖、分化情况,探讨适合骨组织工程的材料比例,为骨组织缺损修复提供基础.方法 制备含10％海藻酸钠-胶原的支架材料,按海藻酸钠与胶原比例分为A(4:1)、B(2:1)和C组(1:1);各组分别制备冻干薄膜和凝胶小球两种形式;冻干薄膜为冻干后灭菌,将hAMSC覆于薄膜表面;凝胶小球制备时与hAMSC混合.细胞检测试剂盒检测hAMSC在各组冻干薄膜表面的增殖情况;活细胞-死细胞染色检测各组凝胶小球内部hAMSC的细胞存活率;实时荧光定量PCR检测各组冻干薄膜表面和凝胶小球内部hAMSC骨钙蛋白和Runt相关转录因子2(Runt-related transcription factor-2,RUNX2)基因的表达差异;免疫荧光染色观察各组hAMSC骨钙蛋白的表达差异.结果 随胶原比例增加,细胞增殖逐步增多,C组细胞增殖量最多.hAMSC在3组凝胶小球内部均可增殖,其中B组细胞存活率[(87.50±2.65)％]最高,与A组[(78.20±2.81)％]及C组[(74.76±1.98)％]相比差异均有统计学意义(P＜0.05).3组冻干薄膜表面hAMSC骨钙蛋白和RUNX2基因表达差异均有统计学意义(P＜0.05),其中C组骨钙蛋白和RUNX2基因表达量最高;而B组凝胶小球内部骨钙蛋白基因表达量显著高于其他组(P＜0.05);骨钙蛋白免疫荧光染色结果与基因检测结果一致.结论 海藻酸钠-胶原支架材料hAMSC增殖、成骨向分化无不利影响,10％水凝胶材料海藻酸钠与胶原比例为2:1时,可利于细胞分化和增殖.

abstractsObjective To build scaffold materials with different concentrations of alginate and collagen,and to observe the effects of alginate/collagen ratio on the proliferation of human adipose-derived mesenchymal stem cells (hAMSC) and osteogenic differentiation.The optimal concentration of alginate/collagen will be chosen for constructing hydrogel that will be used for bone tissue engineering.Methods Soluble hydrogel scaffold materials containing alginate/collagen were prepared,and the following groups were established based on different alginate/collagen ratio:4:1 (group A),2:1 (group B),and 1:1 (group C).Cell proliferation on the material surface was observed using the cell counting kit-8 (CCK-8) assay,while cell viability in each material group were observed using live/dead staining.Quantitative real-time PCR (qPCR) was used to measure the differential expression of osteogenesis-related genes on and in the materials.Immunofluorescence staining was used to measure the differential gene expression of osteogenesis-related proteins in each group.Results The results from the CCK-8 assay showed increasing cell proliferation rate on the lyophilized hydrogel material surface as the collagen concentration increased,and the highest cell proliferation was observed in group C.Live/dead staining assay indicated that cells were able to proliferate in all three types of hydrogel materials,and the highest cell viability was found in material from group B ([87.50±2.65]％).qPCR showed that the expression of osteogenesis-related genes in group C was the highest,among the three groups,while the expression of osteocalcin in group B was significantly higher than those in the other two groups (P＜0.05).Immunofluorescence staining was carried out for osteocalcin on and in the hydrogel material and the results were consistent with that of qPCR.Conclusions The alginate/collagen scaffold materials did not show adverse effects on the cell proliferation of hAMSC and osteogenenic differentiation.Bone tissue engineering can use 10％ hydrogel material,and when the sodium alginate and collagen have a ratio of 2:1,the hydrogel can be conducive to cell differentiation and proliferation.