美托洛尔对小型猪缺血再灌注损伤后移植内皮祖细胞的保护效应

Protective effect of betaloc on transplanted endothelial progenitor cells in minipigs with ischemia/reperfusion injury

目的:观察美托洛尔对小型猪骨髓内皮祖细胞作用的有效剂量和对缺血再灌注损伤后移植内皮祖细胞的保护作用。方法:实验于2004-12/2005-07在解放军第四军医大学放射医学教研室完成。①选用健康雄性中国小型猪6只,抽取骨髓细胞,经密度梯度离心法分离,差速贴壁纯化,诱导生成猪内皮祖细胞,用荧光双标、激光共聚焦法进行生物学标志物的鉴定。②将内皮祖细胞设立1×10-3mol/L,1×10-4mol/L,1×10-5mol/L,1×10-6mol/L,1×10-7mol/L的美托洛尔组,观察美托洛尔对内皮祖细胞作用的有效剂量。③另设立美托洛尔预处理后缺血再灌注损伤组、缺血再灌注损伤后加美托洛尔组、美托洛尔处理组、缺血再灌注损伤组和正常对照组。用光镜和四甲基偶氮唑盐法观察细胞生长情况;用生化法测定乳酸脱氢酶和一氧化氮合酶的活性,观察内皮祖细胞的分泌功能;用流式细胞仪检测内皮祖细胞凋亡情况;应用WesternBlotting法测定内皮祖细胞表面Flk-1的表达。结果:①经5个不同剂量组筛查,显示1×10-6mol/L的美托洛尔对内皮祖细胞的增殖作用最强。②美托洛尔能上调内皮祖细胞表面Flk-1的表达水平。③美托洛尔预处理后缺血再灌注损伤组内皮祖细胞的凋亡率低于缺血再灌注损伤组和缺血再灌注损伤后加美托洛尔组(6.8%,13.9%,7.4%,P<0.05),但高于正常对照组和美托洛尔组(3.7%,2.3%;P<0.05)。④美托洛尔预处理后缺血再灌注损伤组细胞的乳酸脱氢酶、诱导型一氧化氮合酶活性低于缺血再灌注损伤组和缺血再灌注损伤后加美托洛尔组(P<0.05),但高于正常对照组和美托洛尔组(P<0.05)。结论:美托洛尔促进内皮祖细胞增殖的有效剂量是1×10-6mol/L;美托洛尔可能通过上调内皮祖细胞的Flk-1水平,减少内皮祖细胞凋亡及有害的诱导型一氧化氮合酶生成,促进细胞增殖能力等细胞功能的恢复。

AIM： To observe the effective dosage of betaloc on marrow-derived endothelial progenitor cells （EPCs） of the minipigs and the protective effect of betaloc on transplanted EPCs after ischemia/reperfusion injury. METHODS： The experiment was conducted at Department of Radiation Medicine, Fourth Military Medical University of Chinese PLA from December 2004 to July 2005. ①Totally 6 healthy male Chinese minipigs were selected. Bone marrow cells were extracted and then isolated by density gradient centrifugation and purified by differential adherent technique, and induced into minipig EPCs. Biological markers were identified by fluorescent double-labeling method and laser confocal scanning microscope. ②EPCs were assigned into 1×10^-3 mol/L, 1×10^-4 mol/L, 1×10^-5 mol/L, 1×10^-6 mol/L, 1×10^-7 mol/L betaloc groups. The effective dosage of betaloc on marrow-derived EPCs was recorded. ③lschemia/reperfusion injury after betaloc pretreatment group, betaloc administration after ischemia/reperfusion injury group, betaloc management group, ischemia/reperfusion injury group and normal control group were set. Cell growth was observed by MTF method and light microscope. Activities of lactate dehydrogenase （LDH） and nitricoxide synthase （NOS） were measured by biochemistry method to observe the secretion function of EPCs. EPCs apoptosis and FIk-1 expression on the surface of EPCs were identified by flowcytometry and Western Blotting, respectively. RESULTS：①The concentration of betaloc in 1×10^-6 mol/L had the most extreme proliferative promotion to EPCs. ②The expression of FIk-1 was up-regulated in the groups pretreated with betaloc. ③The apoptotic rate of EPCs in the ischemia/reperfusion injury after betaloc pretreatment group was lower than that in the ischemia/reperfusion injury group and betaloc administration after ischemia/reperfusion injury group （6.8%, 13.9%, 7.4%,P〈0.05）, but higher than normal control group and betaloc management group （3.7%,2.3% ;P〈0.05）. ④Activities of LDH and inducible nitric oxide synthase （iNOS） in the ischemia/reperfusion injury after betaloc pretreatment group were lower than those in the fschemia/reperfusion injury group and betaloc administration after ischemia/reperfusion injury group （P 〈 0.05）, but higher than normal control group and betaloc management group （P 〈 0.05）. CONCLUSION： The most effective concentration of betaloc to promote the proliferation of EPCs is 1×10^-6 mol/L. Betaloc can reduce EPCs apoptosis and deleterious iNOS formation and promote cell function recovery such as proliferative ability by up-regulating FIk-1 expression of EPCs.