胆红素对大鼠肺微血管内皮细胞增殖的影响

Effect of bilirubin on proliferation of pulmonary microvascular endothelial cells of rats

目的 评价胆红素对大鼠肺微血管内皮细胞（PMVECs）增殖的影响.方法 健康雄性SD大鼠10只,体重120 ～ 150 g,2～3月龄,原代培养、纯化及鉴定PMVECs.PMVECs接种于低糖DMEM培养基或96孔培养板,采用随机数字表法将其分为5组：对照组（C组）和不同浓度胆红素组（B1-4组）,每组30孔或48瓶.C组加入低糖DMEM培养基1ml,B11-4组分别加入5、10、20和50 μmol/L胆红素1ml.于孵育24、48和72 h时采用CCK-8法和3 H-TdR掺入法检测PMVECs增殖水平,采用RT-PCR法测定Akt1 mRNA和Smad3 mRNA的表达水平,采用Westen blot法测定磷酸化Akt1 （p-Akt1）蛋白和Smad3蛋白的表达水平.结果 与C组比较,B1组和B2组孵育各时点PMVECs增殖水平、Akt1mRNA和p-Akt1蛋白表达、Smad3 mRNA及其蛋白表达差异无统计学意义（P＞0.05）,B3组孵育48 h时PMVECs增殖减弱,Akt1 mRNA和p-Akt1蛋白表达、Smad3 mRNA及其蛋白表达下调,B4组孵育48和72 h时PMVECs增殖减弱,Akt1 mRNA和p-Akt1蛋白表达、Smad3 mRNA及其蛋白表达下调（P＜0.05）;与B3组比较,B4组孵育72 h时PMVECs增殖减弱,Akt1 mRNA和p-Akt1蛋白表达、Smad3 mRNA及其蛋白表达下调（P＜0.05）.结论 高浓度胆红素可抑制PMVECs的增殖,其机制与下调Akt1和Smad3蛋白的表达有关.

Objective To evaluate the effect of bilirubin on proliferation of pulmonary microvascular endothelial cells （PMVECs） of rats.Methods Primary PMVECs harvested from 10 healthy male Sprague-Dawleyrats,weighing 120-150 g,aged 2-3 months,were cultured,purified and identified.PMVECs were seeded in low-glucose DMEM culture medium or 96-well culture plates,and divided into 5 groups according to the random number table：control group （group C） and different concentrations of bilirubin groups （B1-4 groups） with 30 wells or48 flasks in each group.In group C,low-glucose DMEM 1 ml was added.In B1-4 groups,5,10,20 and 50 μmol/L bilirubin 1 ml were added,respectively.At 24,48 and 72 h of incubation,proliferation of PMVECs was measured using CCK-8 assay and 3 H-TdR incorporation assay,Akt1 mRNA and Smad3 mRNA expression was measured by RT-PCR,and phosphor-Akt1 （p-Akt1） protein and Smad3 protein expression was detected using Western blot.Results Compared with group C,no significant changes were found in each parameter mentioned above at each time point in B1 and B2 groups,the proliferation of PMVECs was weakened,and Akt1 mRNA,p-Akt1 protein and Smad3 protein and mRNA expression was down-regulated at 48 h of incubation in B3 group,and the proliferation of PMVECs was weakened,and the expression of Akt1 mRNA,p-Akt1 protein and Smad3 mRNA and protein was down-regulated at 48 and 72 h of incubation in B4 group.Compared with group B3,the proliferation of PMVECs was weakened,and the expression of Akt1 mRNA,p-Akt1 protein and Smad3 mRNA and protein was down-regulated at 72 h of incubation in B4 group.Conclusion High concentration of bilirubin can inhibit proliferation of PMVECs and down-regulated expression of Akt1 and Smad3 is involved in the mechanism.