Ad-Gax转染对缺氧性大鼠肺动脉平滑肌细胞凋亡及其相关基因表达的影响  

The effects of Ad-Gax transfection on apoptosis and related gene expression in pulmonary arterial smooth muscle cells in hypoxic rat model

作　　者：夏世金[1] 董竞成[1] 白莉[2] 胡明冬[2] 钱桂生[2] 邰先桃[1] 谢瑾玉[3] 

机构地区：[1]复旦大学附属华山医院中西医结合研究所细胞分子生物学实验室,上海200040 [2]第三军医大学附属新桥医院全军呼吸疾病研究所 [3]云南中医学院第一附属医院

出　　处：《中华内科杂志》2007年第9期755-759,共5页Chinese Journal of Internal Medicine

基　　金：中国博士后科学基金(20060400578)

摘　　要：目的观察 Gax 基因转染对缺氧性肺动脉平滑肌细胞(PASMCs)凋亡及其相关基因表达的影响。方法腺病毒介导 Gax 转染体外原代培养的 PASMCs。透射电镜观察细胞凋亡形态;原位细胞凋亡检测法观察 Ad-Gax 转染前后在常氧和缺氧处理2h、6h、12h、24h、48h 大鼠 PASMCs凋亡情况;免疫细胞化学法测 PASMCs Bcl-2、Bax 蛋白表达。结果透射电镜观察到 Ad-Gax 转染后PASMCs 产生凋亡现象。未转染缺氧刺激前后,均无或可见极少量的阳性细胞;Ad-Gax 转染后,如无缺氧刺激,也无或仅可见少量的阳性细胞,予缺氧刺激后,阳性细胞显著增多,尤其在转染后24～48h更明显。转染组常氧、缺氧2h、6h、12h、24h、48h 细胞凋亡百分率均显著高于未转染组(P<0.01)。Ad-Gax 转染前,与常氧时比较,缺氧刺激 PASMCs 后,Bax 蛋白表达略为升高但无统计学意义;而 Bcl-2蛋白表达显著升高,差异有统计学意义(P<0.05)。Ad-Gax 转染 PASMCs 后,缺氧刺激使 Bcl-2蛋白表达显著降低(P<0.01),Bax 蛋白表达显著增高(P<0.01)。转染组细胞凋亡率与 Bcl-2/Bax 比值呈负相关(r=-0.53,P<0.01)。结论 Ad-Gax 转染可诱导缺氧性 PASMCs 凋亡,其机制可能是通过上调 Bax 蛋白和下调 Bcl-2蛋白的表达,尤其是通过降低 Bcl-2/Bax 比值实现的。Objective To explore the effects of growth arrest-specific homeobox （Gax）transfection on apoptosis and expressions of Bcl-2 and Bax proteins of rat pulmonary arterial smooth muscle cells （PASMCs） exposed to hypoxia. Methods PASMCs were transfected with Gax gene by a replicationdeficient adenovirus expressing the hemagglutinin-tagged Gax cDNA （Ad-Gax）. After exposure to normal oxygenation （21%O2） or hypoxia（2. 5%O2） for 2 h,6 h,12 h,24 h and 48 h, apoptosis was observed by transmission electronic microscope and TUNEL-positive staining. The expressions of Bcl-2 and Bax proteins in PASMCs were detected by immunocytochemistry. Results Before Ad-Gax transfection, none or few of TUNEL-positive PASMCs were detected. After Ad-Gax transfection, the PASMCs unexposed to hypoxia displayed none or few TUNEL-positive stain, while the PASMCs exposed to hypoxia displayed a marked increase in TUNEL-positive stain, especially in cells exposed to hypoxia for 24 h to 48 h. The ratio of apoptosis of PASMCs at normoxic, hypoxia 2 h,6 h,12 h,24 h and48 h were（9. 11 ±1.21）% ,（34. 13 ± 1.02）% ,（39. 12 ±0.43）% ,（50.09 ±0. 13）% ,（60.04 ±1.12）% and （55.47 ±0.03）% （P 〈0.01）, respectively. Before Ad-Gax transfection, the level of Bax protein showed no significant increase in PASMCs exposed to hypoxia, while that of Bcl-2 protein increased significantly,as compared to that in PASMCs under normoxic condition. The average optical density （ AOD ） of Bcl-2 was 2. 31 ± 0. 12,2. 35 ± 0. 23,2. 49 ± 0. 27,2. 51 ± 0. 19,2. 54 ± 0. 25 and 2. 53 ± 0. 20 at normoxic, hypoxia for 2 h, 6 h, 12 h, 24 h and 48 h before Ad-Gax transfection, respectively （P 〈 0. 05, P 〈 0. 01 ）. After Ad-Gax transfection to PASMCs exposed to hypoxia, Bax protein increased; the AOD of Bax was 3.82 ± 0. 38,3.12 ± 0. 42,3. 53 ± 0. 61,4.52 ± 0.23,4.25±0. 76 and 4. 03± 0. 38 at normoxie, hypoxia for 2 h, 6 h, 12 h, 24 h and 48 h （P 〈 O. 01 ） ,respectively. But Bcl-2 protein decreased significantly;

关 键 词：GAX基因 低氧 肺动脉平滑肌细胞 脱噬作用 凋亡相关基因 

分 类 号：R543.2[医药卫生—心血管疾病]