bFGF对犬隐静脉间质细胞体外培养细胞内钙及钙化的影响

Effect of bFGF on free Ca^(2+) intracellular and calcification of canine saphenous vein interstitial cells(SVICs) cultured in vitro

目的:观察碱性成纤维细胞生长因子(bFGF)对作为组织工程瓣间质种子细胞的犬隐静脉间质细胞(SVICs),体外培养细胞内钙及钙化的影响。方法:分离培养犬隐静脉间质细胞,以免疫组织化学方法鉴定其特征。将3~6代传代细胞分为2组:对照组,细胞在正常标准条件下培养;bFGF组,细胞在含5μg/L浓度bFGF条件下培养。2周后激光共聚焦显微镜检测细胞内钙,流式细胞仪检测细胞凋亡率,原子吸收法测定细胞层钙沉积及von kossa染色。结果:犬隐静脉间质细胞主要由平滑肌细胞、肌纤维母细胞和成纤维细胞组成。培养2周后,对照组有细胞结节形成,bFGF组细胞呈极性密集生长,无明显细胞结节形成。对照组von kossa染色呈弱阳性,bFGF组阴性。对照组细胞内游离钙浓度相对比值高于bFGF组(45±3vs19±7,P<0.01),bFGF组细胞凋亡率及细胞层钙沉积显著低于对照组[(7.6%±0.88%vs25.4%±1.68%,P<0.01;0.192±0.024μmol/gvs2.198±0.173μmol/g,P<0.01)]。结论:bFGF具有维持犬隐静脉间质细胞内游离钙的平衡,抑制细胞凋亡及钙化的作用。

Abstract： AIM：To observe the effect of bFGF on intracellular free Ca^2＋ and calcification of saphenous vein interstitial cells（SVICs） as seed cells of tissue engineering valve cultured in vitro. METHODS： Canine SVICs were obtained and identified with immunocytochemistry. Cells of passage 3 - 6 were devided into the control and bFGF groups. In the control group,cells were cultured in standard medium. In the bFGF group, cells were cultured in medium with 5μg/L bFGF. After 2 weeks, free Ca^2＋ and cellular apoptosis rate were determined by laser confocal microscope and flow cytometry, respectively. The cellular layer Calcium deposition was measured by atomic absorption spectrometry and stained by yon kossa. RESULTS： ① SVICs were mainly composed of smooth muscle cells, myofibroblasts and fibroblasts. ② In the control group, cellular nodules formed and calcification was mild positive by yon kossa staining. In the bFGF group, cells grew with dense polarity;cellular nodules did not form and calcification was negative. ③ Intracellular free Ca^2＋ concentration in the control group was higher than that in the bFGF group. Cellular apoptosis rate and calcifical deposition in the bFGF group were significantly lower than those in the control group. CONCLUSION： bFGF could maintain SVICs [ Ca^2＋]i homeostasis and inhibit cell apoptosis and calcification.