肝肺综合征大鼠肺微血管内皮细胞肌样分化时转化生长因子β1表达的变化

Changes in expression of transforming growth factor beta-1 during myogenic differentiation of pulmonary microvascular endothelial cells in rats with hepato-pnimonary syndrome

目的评价肝肺综合征（HPS）大鼠肺微血管内皮细胞（PMVECs）肌样分化时转化生长因子β1（TGF-β1）表达的变化。方法健康SD大鼠40只，雌雄不拘，3～4月龄。采用随机数字表法，随机取20只大鼠，采用慢性胆总管结扎法制备HPS模型，剩余大鼠行假手术，取腹主动脉血样4ml，离心后取血清。另取健康成年SD大鼠10只，原代培养、纯化及鉴定PMVECs。PMVECs接种于低糖DMEM培养基（10^6个／cm2），采用随机数字表法，将细胞随机分为2组：对照组（C组）和HPS组，每组24皿。C组给予正常大鼠血清，HPS组加入HPS大鼠血清，血清终浓度为10％。于细胞孵育24h（T1）、48h（T2）和72h（T1）时分别采用RT-PCR法和Westernblot法测定PMVECs内平滑肌肌球蛋白重链（SM—MHC）、平滑肌肌动蛋白a（SM-α-actin）、肌钙蛋白、TGF-β1 mRNA及其蛋白的表达。结果C组PMVECs内SM-α-actin、肌钙蛋白表达阴性，可见少量SM．MHC表达，HPS组PMVECs内SM-MHC、SM-α—actin、肌钙蛋白表达阳性。与C组比较，HPS组SM—MHC、TGF-β1mRNA及其蛋白的表达上调（P〈0．05）；与T1时比较，T2，3时HPS组SM-MHC、SM-α-actin、肌钙蛋白、TGF—p1mRNA及其蛋白的表达上调（P〈0．05）；与T2时比较，L时HPS组SM-MHC、SM-α-actin、肌钙蛋白、TGF-β1mRNA及其蛋白的表达上调（P〈0．05）。结论TGF-β1在HPS大鼠PMVECs肌样分化时可能起到重要的调控作用。

Objective To investigate the changes in the expression of transforming growth factor beta-1 （TGF-β1） during differentiation of pulmonary microvascular endothelial cells （PMVECs） into smooth muscle cells in rats with hepato-pulmonary syndrome （HPS）. Methods Primary PMVECs were harvested from healthy adult SD rats of both sexes aged 3-4 months and inoculated in low-glucose DMEM culture medium （10^6/cm2） and randomly divided into 2 groups （n = 24 dishes each）： control group （group C） and HPS group. HPS was produced by chronic ligation of common bile duct. In group C serum obtained from normal rats was added to PMVECs, while in HPS group serum obtained from rats with HPS was added. The final concentration of serum was 10%. After being incubated for 24, 48 and 72 h, the expression of SM-MHC, SM-α-actin and calponin protein and TGF-β1 mRNA and protein in PMVECs was determined by RT-PCR and Western blot analysis. Results The expression of SM- MHC, SM-α-actin and calponin protein was positive in HPS group whereas the expression of SM-α-actin and calpo- nin protein was negative and the expression of SM-MHC protein was barely detectable in group C. The expression of SM-MHC,TGF-β1 mRNA and protein was significantly higher in HPS group than in group C. The expression of SM-MHC, SM-α-actin and calponin protein and TGF-β1 mRNA and protein was increasing with duration of incuba- tion from T1 to T3 in group HPS. Conclusion TGF-β1 plays an important role in the myogenic differentiation of PMVECs in rats with HPS.