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目的:观察不同年龄段大鼠血清对大鼠骨髓内皮祖细胞(endothelial progenitor cells,EPCs)活力的影响。方法:PBS冲洗出1-2月龄、19-26月龄SD大鼠股骨和胫骨骨髓,Ficoll密度梯度离心法分离单个核细胞(MNCs),应用含10%FBS的DMEM/F12培养基(含内皮细胞生长添加剂100mg/L、肝素100mg/L、青霉素1×105U/L、链霉素1×105U/L)差速贴壁法进行体外培养,以Dil-ac-LDL与FITC-UEA-1荧光双染,直接或间接荧光标记CD31及vWF分别通过流式和免疫组化进行鉴定。制备1-2月龄、19-26月龄大鼠血清,将培养的EPCs分A(老年大鼠EPCs+老年大鼠血清)、B(老年大鼠EPCs+年轻大鼠血清)、C(年轻大鼠EPCs+老年大鼠血清)、D(年轻大鼠EPCs+年轻大鼠血清)4组,EPCs经含10%不同年龄段大鼠血清的DMEM/F12培养基(不含胎牛血清)培养后,激光共聚焦检测EPCs吞噬Dil-ac-LDL后平均荧光强度,改良Boyden小室和黏附能力测定实验分别观察EPCs迁移和黏附能力,MTT法检测细胞增殖活性。结果:年轻大鼠血清显著促进老年大鼠EPCs吞噬Dil-ac-LDL能力(P<0.01)及增强其迁移(P<0.01)、黏附(P<0.05)和增殖能力(P<0.01),而老年大鼠血清显著抑制年轻大鼠EPCs迁移(P<0.05)和黏附能力(P<0.05)。结论:年轻大鼠血清显著增强老年大鼠EPCs的细胞活力,而老年大鼠血清可部分抑制年轻大鼠EPCs功能活性。
Objective: To observe the effects of serum of different age groups on the activity of rat bone marrow endothelial progenitor cells (EPCs). Methods: Bone marrow of femur and tibia of 1 to 2 month old and 19 to 26 month old SD rats were flushed with PBS. Mononuclear cells (MNCs) were separated by Ficoll density gradient centrifugation and cultured in DMEM / F12 medium containing 10% FBS The cells were cultured in vitro with Dil-ac-LDL and FITC-UEA-1 (100mg / L, 100mg / L heparin, 1 × 105U / L penicillin and 1 × 105U / Fluorescent double staining, direct or indirect fluorescent markers CD31 and vWF were identified by flow cytometry and immunohistochemistry. The serum of rats aged 1-2 months old and 19-26 months old were prepared. The EPCs of the rats were divided into A (old rat EPCs + senile rat serum), B (old rat EPCs + young rat serum), C (young rats EPCs + senile rat serum), D (young rat EPCs + young rat serum), EPCs were cultured in DMEM / F12 medium (without fetal bovine serum) containing 10% rat serum of different ages. EPCs phagocytosis Dil-ac-LDL phagocytosis average fluorescence intensity, modified Boyden chamber and adhesion assay were observed EPCs migration and adhesion capacity, MTT assay cell proliferation activity. RESULTS: Serum of young rats significantly promoted phagocytosis of Dil-ac-LDL (P <0.01) and enhanced migration (P <0.01), adhesion and proliferation of EPCs in aged rats Senile rat serum significantly inhibited EPCs migration and adhesion (P <0.05) in young rats. CONCLUSION: Serum from young rats can significantly increase the viability of EPCs in aged rats, whereas the serum from aged rats can partially inhibit the functional activity of EPCs in young rats.