Objective: To establish a simple method of separating and culturing fibroblasts from newborn mice skin. Methods: Firstly, skin was taken from newborn Balb/c mice and made into 1 mm2 tissues with scissors sterilely. Secondly, tissues were explanted into culture dishes in equilibrium manner with DMEM (including 10% fetal bovine serum) medium. Thirdly, the purification of fibroblasts was completed by digesting cells with trypsin and re -explanting cells into dishes. Lastly, the vimentin expression of fibroblasts was identified by immunofluorescence, flow cytometry and Western blot. Results: Several cells near the tissues could be visible as thin spindle or polygons 3 days after tissues were explanted. More and more thin spindle or polygons cells were visible with the extension of tissues culture 5 days after tissues were explant -ed. The cells were growing in swirling arrangement, crisscross or radiate manner when tissues were cultured for 7 days. After cells were digested with trypsin and re-explanted, the cells were grown as layer of cell rapidly. Both of fibroblasts and NIH3T3 cells were found to express vimentin by immunofluorescence, flow cytometry and Western blot respectively. Flow cytometry assays showed that 13.33% and 28.11% cells were vimentin positive in cultures fibroblasts and NIH3T3 cells respectively. When geometric mean fluorescence was considerred to asscess vimentin expression, the geometric mean fluorescence of vimentin in fibroblasts and NIH3T3 cells were 9.89 and 14.16 respectively. Conclusion: Fibroblasts can be separated and cultured by economic and convenient tissues explantation method from newborn mice. The cells have typical fibroblasts morphology and have actually vimentin expression which may be useful to establish and mimic tumor microenvi -ronment in vitro.%目的:建立一种简单易行的自新生小鼠皮肤分离培养成纤维细胞的方法,并对分离细胞进行鉴定.方法:无菌取新生Balb/c小鼠背部皮肤,剪碎呈1 mm2大小,组织块均匀放置于培养皿并以DMEM(含10%胎牛血清)培养基对组织块进行培养;以胰蛋白酶消化分离细胞-重新贴壁法对分离培养细胞进行纯化;以NIH3T3细胞为对照,免疫荧光、流式细胞术、Western blot检测波形蛋白表达情况以对培养的成纤维细胞进行鉴定.结果:新生小鼠背部皮肤组织培养3 d时可见细胞呈细长梭形或呈多角形,组织培养5 d后可见大量细胞自组织块游离生长,组织培养7 d后可见细胞呈单层漩涡状排列或纵横交错,贴壁细胞团呈放射状生长.免疫荧光、流式细胞术、Western blot检测发现分离细胞和NIH3T3细胞均表达波形蛋白;流式细胞术证实分离的成纤维细胞波形蛋白表达阳性率和平均荧光密度分别为13.33%和9.89,而NIH3T3细胞波形蛋白阳性率和平均荧光密度分别为28.11%和14.16.结论:分离培养的细胞具有成纤维细胞的形态学特征,并表达成纤维细胞的特征分子波形蛋白.以组织块直接培养法成功自新生小鼠皮肤分离培养成纤维细胞,为建立肿瘤体外模拟微环境提供实验材料和研究基础.
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