不同年龄段大鼠血清对大鼠骨髓内皮祖细胞活力的影响

Serum from rats at different ages modulates the functional activities of rat bone marrow-derived endothelial progenitor cells

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。

AIM ： To investigate the effects of serum from rats at different ages on the functional activities of rat bone marrow - derived endothelial progenitor cells （EPCs）. METHODS： Mononuclear cells were obtained from bone marrow of young （ 1 to 2 month - old） and aged （ 19 to 26 month - old） Sprague - Dawley rats by Ficoll density gradient centrifugation and cultured with medium DMEM/F12 （containing 10% fetal bovine serum, endothelial cell growth supplement ECGs 100 mg/L, 1 × 10^5 U/L of penicillin and streptomycin, respectively）, 48 h later, the suspending cells were translocated to be cultured in new flasks coated with fibronectin, the secondary attached cells were used to perform the further experiments. EPCs were characterized as double positive for Dil - ac - LDL uptake and lectin binding. The cells were further identified by CD31 and vWF expression. Serum from young （ 1 to 2 month - old） and aged （ 19 to 26 month - old） rats was collected and used to culture EPCs. The experiments were divided into four groups ： A = aged rat EPCs ＋ aged rat serum; B = aged rat EPCs ＋ young rat serum; C = young rat EPCs ＋ aged rat serum and D = young rat EPCs ＋ young rat serum. After cultured in DMEM/F12 supplemented with 10% serum from rats at different age （ no fetal bovine serum addition in this medium）, the average fluorescence intensities of EPCs stained with Dil - ac - LDL were tested by laser scanning co - focal microscopy. Migration and proliferation were assayed by modified Boyden chamber and MTT, respectively. Cell adhesion was performed by replacing EPCs onto cultured DAPI - labeled confluent smooth muscle cell monolayer and the adherent cells were counted. RESULTS： Young rat serum significantly improved the ability of aged rat EPCs for uptake of Dil - ac - LDL （P 〈 0. 01 ）, increased the migration （P 〈 0. 01 ）, adhesion （P 〈 0. 05 ） and proliferation activity （ P 〈 0. 01 ） of aged rat EPCs, whereas aged rat serum obviously decreased the migration （ P 〈 0. 05 ） and adhesion （ P 〈 0. 05 ） activity of the young rat EPCs. CONCLUSION： Young rat serum significantly improves the activity of aged rat EPCs. On the contrary, aged rat serum partially inhibits the activity of young rat EPCs.