目的 :克隆小鼠分泌型 Hemopexin(s PEX)编码区的 c DNA序列 ,构建含 Egr- 1启动子的 p Egr- s PEX真核表达载体 ,检测辐射诱导重组质粒在 B16 F10细胞中的表达。方法 :用 RT- PCR从 NIH3T3细胞中扩增出s PEX,经测序证实后 ,构建 p Egr-...目的 :克隆小鼠分泌型 Hemopexin(s PEX)编码区的 c DNA序列 ,构建含 Egr- 1启动子的 p Egr- s PEX真核表达载体 ,检测辐射诱导重组质粒在 B16 F10细胞中的表达。方法 :用 RT- PCR从 NIH3T3细胞中扩增出s PEX,经测序证实后 ,构建 p Egr- s PEX重组质粒 ,以脂质体转染 B16 F10细胞 ,用 Western blotting方法检测B16 F10细胞上清中辐射诱导 PEX的表达。结果 :测序表明扩增的 s PEX c DNA序列与 Gen Bank中登录的序列完全一致 ,s PEX c DNA正确插入表达载体 pc DNA3.1- Egr- 1,转染入 B16 F10细胞内 PEX基因在体外辐射诱导下成功获得表达。结论 :成功地克隆了小鼠 s PEX基因 ,并在体外证实了 p Egr- s PEX具有辐射诱导表达特性。展开更多
Carbon monoxide releasing molecules (CORMs) have been recently developed for research and pharmacological purposes. A considerable amount of studies demonstrated a wide spectrum of biological activities for lipophilic...Carbon monoxide releasing molecules (CORMs) have been recently developed for research and pharmacological purposes. A considerable amount of studies demonstrated a wide spectrum of biological activities for lipophilic CORM-2 (tricarbonyldichlororuthenium (II) dimer). It is generally accepted that the liberated gas provides the specific activities to CORMs, with a little attention paid to any possible effect of complementary core molecules. However, the versatile repertoire of actions attributed to CORM-2 is surprisingly wide for CO, a molecule with the sole chemical activity of binding to ferrous iron in protein prosthetic groups. The study was designed to analyze CORM-2 and its core molecule (“i”CORM) activities at a molecular level. With respect to the hydrophobic nature of the compounds, we followed their interactions with several amphipathic entities: the heme sites of hemoproteins, heme binding proteins and cell membranes. CORM-2/“i”CORM decreased the Soret optical density of hemoglobin and myoglobin, indicating that both compounds interact with the protein amphipathic site in the heme pocket. Pre-addition of CORM-2/“i”CORM to the apo-forms of the plasma heme binding proteins, hemopexin and albumin, partially abolished their heme binding capacity. In contrast, the compounds had no effect on the preformed heme-protein complexes. Addition of CORM-2/“i”- CORM to blood or isolated erythrocytes revealed aggregation of the cells or lysis, depending on the rea-gent-to-cells ratio. It was concluded that the ruthenium containing core molecule of CORM-2 may be physiologically active due to non-specific hydrophobic interactions. As each type of CORMs is expected to have a different mode of action beyond CO activity, their potential therapeutic uses will require clarification.展开更多
文摘目的 :克隆小鼠分泌型 Hemopexin(s PEX)编码区的 c DNA序列 ,构建含 Egr- 1启动子的 p Egr- s PEX真核表达载体 ,检测辐射诱导重组质粒在 B16 F10细胞中的表达。方法 :用 RT- PCR从 NIH3T3细胞中扩增出s PEX,经测序证实后 ,构建 p Egr- s PEX重组质粒 ,以脂质体转染 B16 F10细胞 ,用 Western blotting方法检测B16 F10细胞上清中辐射诱导 PEX的表达。结果 :测序表明扩增的 s PEX c DNA序列与 Gen Bank中登录的序列完全一致 ,s PEX c DNA正确插入表达载体 pc DNA3.1- Egr- 1,转染入 B16 F10细胞内 PEX基因在体外辐射诱导下成功获得表达。结论 :成功地克隆了小鼠 s PEX基因 ,并在体外证实了 p Egr- s PEX具有辐射诱导表达特性。
文摘目的探讨血红素结合蛋白（HPX）是否通过上调血红素氧合酶-1（HO-1）改善大鼠脑缺血再灌注（IR）损伤。方法 60只雄性SD大鼠,7~8周龄,体重250~280 g,采用随机数字表法分为五组：假手术（Sham）组、缺血再灌注（MCAO）组、溶剂（叠氮钠）（MCAO＋Vehicle）组、HPX（MCAO＋HPX）组和HO-1抑制剂组（MCAO＋HPX＋ZnppⅨ）组,每组12只。采用大脑中动脉栓塞（MCAO）法制备大鼠缺血再灌注模型,MCAO组、MCAO＋Vehicle组、MCAO＋HPX组、MCAO＋HPX＋ZnppⅨ组大鼠分别在IR即刻经侧脑室单次注射生理盐水（10μl,0.9%）、叠氮钠（10μl,0.1%）、HPX（10μl,1.86 g/L）和10μl HPX＋ZnppⅨ混合液（HPX 1.86 g/L＋叠氮钠0.1%）。IR后24 h、7 d分别采用Carcia评分法评价大鼠神经行为学评分（NBS）,采用实时荧光定量聚合酶链式反应（RT-PCR）法检测半暗带区脑组织中HO-1 m RNA水平。结果 IR后24 h Sham组、MCAO组、MCAO＋Vehicle组、MCAO＋HPX组、MCAO＋HPX＋ZnppⅨ组大鼠的NBS评分分别为（15.50±2.43）、（5.17±1.60）、（5.00±2.10）、（10.83±1.47）、（5.33±1.97）分,比较差异有统计学意义（F=34.819,P〈0.05）;IR后7 d Sham组、MCAO组、MCAO＋Vehicle组、MCAO＋HPX组、MCAO＋HPX＋ZnppⅨ组大鼠的NBS评分分别为（16.83±1.60）、（8.83±3.31）、（9.17±3.19）、（12.50±3.08）、（9.33±2.07）分,比较差异有统计学意义（F=13.113,P〈0.05）。与Sham组相比,MCAO组大鼠IR后24 h、7 d缺血半暗带区脑组织HO-1 m RNA水平显著增加（P〈0.05）;与MCAO组相比,MCAO＋Vehicle组大鼠IR后24 h、7 d缺血半暗带区脑组织HO-1 m RNA水平无明显变化（P〉0.05）;与MCAO＋Vehicle组大鼠相比,MCAO＋HPX组大鼠IR后24 h、7 d缺血半暗带区脑组织HO-1 m RNA水平显著升高（P〈0.05）。结论 HPX可通过上调HO-1表达改善大鼠脑缺血再灌注后神经行为学表现。
文摘Carbon monoxide releasing molecules (CORMs) have been recently developed for research and pharmacological purposes. A considerable amount of studies demonstrated a wide spectrum of biological activities for lipophilic CORM-2 (tricarbonyldichlororuthenium (II) dimer). It is generally accepted that the liberated gas provides the specific activities to CORMs, with a little attention paid to any possible effect of complementary core molecules. However, the versatile repertoire of actions attributed to CORM-2 is surprisingly wide for CO, a molecule with the sole chemical activity of binding to ferrous iron in protein prosthetic groups. The study was designed to analyze CORM-2 and its core molecule (“i”CORM) activities at a molecular level. With respect to the hydrophobic nature of the compounds, we followed their interactions with several amphipathic entities: the heme sites of hemoproteins, heme binding proteins and cell membranes. CORM-2/“i”CORM decreased the Soret optical density of hemoglobin and myoglobin, indicating that both compounds interact with the protein amphipathic site in the heme pocket. Pre-addition of CORM-2/“i”CORM to the apo-forms of the plasma heme binding proteins, hemopexin and albumin, partially abolished their heme binding capacity. In contrast, the compounds had no effect on the preformed heme-protein complexes. Addition of CORM-2/“i”- CORM to blood or isolated erythrocytes revealed aggregation of the cells or lysis, depending on the rea-gent-to-cells ratio. It was concluded that the ruthenium containing core molecule of CORM-2 may be physiologically active due to non-specific hydrophobic interactions. As each type of CORMs is expected to have a different mode of action beyond CO activity, their potential therapeutic uses will require clarification.
