肝肺综合征大鼠血清对肺微血管内皮细胞Akt表达的影响

Effect of serum obtained from rot with hepatopuimonary syndrome on Akt mRNA and protein expression in rot pulmonary microvascular endothelial cells

目的探讨肝肺综合征（HPS）大鼠血清对肺微血管内皮细胞（PMVECs）丝氨酸苏氨酸蛋白激酶（Akt）表达的影响。方法健康3～4月龄SD大鼠30只，雌雄不拘，采用慢性胆管结扎法制备HPS模型。另取正常大鼠，原代培养、纯化及鉴定PMVECs。PMVECs接种于低糖DMEM培养基（106，cm^2）或96孔培养板（200μl/孔），随机分为2组：对照组（C组）和HPS组，每组24皿或90孔，C组不予处理，HPS组加入HPS大鼠血清，血清终浓度为10％。于HPS大鼠血清中孵育24、48和72h（T1-3）时分别采用RT-PCR法和Western blot法检测Akt。mRNA、A地mRNA和AkhmRNA及其蛋白的表达，采用MTY法和^3H-Tdn掺人法检测PMVECs增殖情况。结果与c组比较，HPS组PMVECs增殖增强，Akt蛋白及其mRNA表达水平上调（P〈0．05）；与L时比较，T2．3时HPS组PMVECs增殖增强，Akt蛋白及其mRNA表达水平上调（P〈0．05）；与T2时比较，T3时HPS组PMVECs增殖增强，Akt蛋白及其mRNA表达水平上调（P〈0．05）。结论Akt可能参与了HPS大鼠PMVECs增殖的调节。

Objective To investigate the effect of the serum obtained from rat with hepatopulmonary syndrome （HPS） on Akt mRNA and protein expression in rat pulmonary microvascular endothelial cells （PMVECs） and the role of Akt signaling pathway in the proliferation of PMVECs in the HPS. Methods Healthy 3-4-month-old SD rats of both sexes were used in this study. HPS was produced by chronic ligation of common bile duct according to the method described by Fallon. Liver cirrhosis and pulmonary microvascular proliferation were verified by microscopic examination of the liver and lung tissue 2 weeks after bile duct ligation. Serum was obtained from blood taken from aorta of HPS rats. Primary PMVECs were cultured and randomly divided into 2 groups： control group and HPS group. In HPS group serum was added to cultured PMVECs （final concenlration was 10% ） and incubated. Akt mRNA and protein expression was determined at 24, 48 and 72 h of incubation by RT-PCR and Western blot. The proliferation of PMVECs was detected by MTI＂ and 3 H-TdR. Results The proliferation of PMVECs was significantly enhanced and the expression of Akt mRNA and protein was significantly increased in HPS group as compared with control group. Conclusion The Akt signaling pathway might play an important role in proliferation of PMVECs in the HPS.