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目的:分离培养脂肪基质细胞(ASCs),探讨其分泌的细胞因子及其生物活性作用。方法:人脂肪组织用胶原蛋白酶Ⅰ消化后培养获得ASCs,流式细胞术鉴定其表型,ELISA法测定其分泌的血管内皮生长因子(VEGF)、肝细胞生长因子(HGF)、基质细胞衍生因子-1α(SDF-1α)等,RT-PCR测定相应mRNA表达。ASCs培养液培养人脐静脉内皮细胞,细胞计数测定内皮细胞的增殖,AnexinⅤ/PI双染色法测定内皮细胞的凋亡情况。结果:培养获得ASCs,表型为CD34+、CD105+、CD31-、CD45-,VEGF、HGF、SDF-1α的含量分别为(75±16)、(637±157)、(482±113)ng/L,能促进内皮细胞的增殖(P<0·05),降低其凋亡(P<0·05),并能上调其bcl-2的基因表达(P<0·05)。结论:ASCs能分泌细胞因子,且对内皮细胞的增殖与凋亡有明显作用。
OBJECTIVE: To isolate and culture adipose-derived stromal cells (ASCs) and investigate the secretion of cytokines and their biological activity. Methods: Human adipose tissue was cultured with collagenase Ⅰ and then cultured to obtain ASCs. The phenotype of ASCs was identified by flow cytometry. The expression of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), stromal cell derived factor -1α (SDF-1α), etc. The corresponding mRNA expression was determined by RT-PCR. Cultured human umbilical vein endothelial cells with ASCs culture medium, the proliferation of endothelial cells was measured by cell counting, and the apoptosis of endothelial cells was determined by AnexinV / PI double staining. Results: The ASCs cultured in vitro had the highest expression of CD34 +, CD105 +, CD31-, CD45-, VEGF, HGF and SDF-1alpha in the culture medium as (75 ± 16), (637 ± 157) and (482 ± 113) ng / L , Promoted the proliferation of endothelial cells (P <0.05), decreased the apoptosis (P <0.05) and up-regulated the gene expression of bcl-2 (P <0.05). Conclusion: ASCs can secrete cytokines and have a significant effect on the proliferation and apoptosis of endothelial cells.