目的：评价细胞穿透肽PEP-1介导血红素加氧酶-1（HO-1）预处理对L02肝细胞缺氧复氧损伤的影响。方法利用基因工程手段表达和纯化融合蛋白PEP-1-HO-1（PEP-1-heme oxy-genase-1，PEP-1-HO-1）。使用人肝细胞株（HL-7702）建立培养人肝细胞缺氧复氧模型。按实验需要以10％新生牛血清的DMEM液静置培养L02肝细胞，将细胞进行随机分为7组：A组，正常对照组（常规培养）；B组，缺氧复氧组（缺氧复氧组细胞缺氧18 h，复氧6 h）；C组，PEP-1-HO-1预处理组，按PEP-1-HO-1浓度分为5亚组（C1，0．125μmol/L；C2，0．25μmol/L ；C3，0．5μmol/L；C4，1．0μmol/L；C5，2．0μmol/L，缺氧前以PEP-1-HO-1融合蛋白预处理2 h，缺氧18 h，复氧6 h）。复氧结束后，收集各组细胞及培养液上清，检测MDA、LDH、AST、ALT含量及SOD活性。结果缺氧复氧组较正常对照组相比，MDA、LDH、AST及ALT水平明显升高（P＜0．05），而 SOD 水平下降（P＜0．05）；PEP-1-HO-1预处理组与缺氧复氧组相比，预处理组能明显降低MDA、LDH、AST及ALT水平（P＜0．05），使得SOD水平上升（P＜0．05），且在一定浓度下与剂量呈正相关。结论 PEP-1-HO-1融合蛋白预处理可减轻细胞缺氧复氧损伤。

Objective To evaluate the pretreatment effects of heme oxygenase-1 mediated by cell-penetrating peptide PEP-1 on cultured human hepatocytes(L02)against hypoxia-reoxygenation injury. Methods Genetic engineering techniques were used to express and purify PEP-1-heme oxygenase-1 (PEP-1-HO-1)fusion protein.Human hepatic cell line(HL-7702)was used to establish the model of he-patic hypoxia-reoxygenation injury.According to the experimental requirement,L02 cells were fed with Dulbecco''s modified Eagle Medium supplemented with the 10%fetal bovine serum.The cultured L02 cells were divided into 7 groups randomly.Group A(CTL)received no anoxia and served as control and Group B (H/R)received 18 hours of anoxia,followed by reoxygenation for 6 hours.According to the concentration of PEP-1-HO-1 pretreatment,Group C were divided into 5 subgroups,including Group C1 (0.125 μmol/L),Group C2(0.25 μmol/L),Group C3(0.5 μmol/L),Group C4(1.0 μmol/L)and Group C5(2.0μmol/L).PEP-1-HO-1 fusion pro

目的 通过体外实验研究骨髓间充质干细胞(MSC)旁分泌肝细胞生长因子(HGF)对大鼠肾小管上皮细胞缺氧复氧损伤的修复作用及其相关机理.方法 获取和培养大鼠骨髓MSC;构建HGF siRNA质粒对MSC进行转染,将大鼠MSC进行相应的基因沉默;建立大鼠大鼠肾小管上皮细胞系NRK-52E(简称:NRK)缺氧复氧模型.在前述基础上,实验共分4组建立上下共培养体系,(1)对照组:将未进行缺氧复氧处理的NRK单独培养;(2)缺氧复氧组:将进行了缺氧复氧处理的NRK单独培养;(3)野生MSC组:上室中加入缺氧复氧后的NRK,下室中加入野生型MSC;(4)转染MSC组:上室中加入缺氧复氧后的NRK,下室中加入转染了HGF siRNA的MSC.采用MTT法检测NRK增殖情况,采用实时荧光定量聚合酶链反应试剂盒检测各组HGF、HMGB1、IL-1β及TNF-αmRNA的相对表达量,采用蛋白质印迹法检测HGF、HMGB1、IL-1β及TNF-α蛋白的表达.结果 MSC转染HGF siRNA的效果较好,转染率在70％以上;转染HGF siRNA后MSC的HGF mRNA表达水平显著降低.随培养时间的延长各组NRK均有增殖,培养32 h时野生型MSC组NRK的增殖率最高,转染MSC组次之,缺氧复氧组最低(P＜0.01).缺氧复氧组HMGB1、IL-1β及TNF-αmRNA的相对表达量均为最高,转染MSC组次之,野生MSC组最低,3组间两

Objective To preliminarily explore the effect of paracrine hepatocyte growth factor (HGF) from mesenchymal stem cells (MSCs) on the repair of hypoxia-reoxygenation injury in renal tubular epithelial cells of rats and the relevant mechanism.Method The bone marrow MSCs of rats were harvested and cultured,and transfected with HGF-siRNA plasmid.The corresponding genes of rat MSCs were silenced to build a hypoxia-reoxygenation model of the renal tubular epithelial cells (NRK-52E) in rats.On the basis of the foregoing,the experiment was divided into four groups to establish co-culture system:(1) the control group,simple culture of NRK cells not treated with hypoxia-reoxygenation; (2) hypoxia-reoxygenation group,simple culture of NRK cells treated with hypoxia-reoxygenation; (3) the wild MSCs group,coculture of hypoxia-reoxygenation-treated NRK cells (upper chamber) + wild type MSCs (lower chamber) ; (4) MSCs transfection group,coculture of hypoxia-reoxygenatiowNRK cells (upper cham

目的观察蛋白质非酶糖化是否会增加心肌缺氧/复氧损伤的敏感性。方法常规培养H9C2细胞,分别给予PBS(对照组)、丙酮醛(200μmol/L)、丙酮醛(200μmol/L)+氨基胍(100μmol/L)孵育6 d后接受缺氧/复氧处理,以MTT法测定心肌细胞存活率,微量酶活性测试法检测乳酸脱氢酶(LDH)释放变化,比色法检测丙二醛(MDA)含量,并通过免疫组织化学检测心肌细胞终末糖化晚期产物(AGEs)表达。结果缺氧/复氧降低细胞存活率,增加心肌细胞MDA含量和LDH释放;而丙酮醛处理6 d的心肌细胞的AGEs含量明显增加,并对缺氧/复氧损伤的敏感性增加,与PBS孵育细胞经缺氧/复氧处理后相比,MDA含量和LDH释放显著上升,存活率明显下降(P0.01);MGO引起的上述变化可以被糖化抑制剂氨基胍部分阻断(P0.05)。结论蛋白质非酶糖化是糖代谢中间产物丙酮醛增加心肌细胞对缺氧/复氧损伤敏感性的重要机制。

Objective To investigate whether protein glycation increases the injury of myocardial cells subjected to hypoxia/reoxygenation. Methods The cultured H9C2 cells were incubated for 6 days with PBS, Methylglyoxal, and Methylglyoxal+Aminoguanidine, respectively. Then the cells were treated by hypoxia/reoxygenation, and the survival rates of cardiomyocytes in each group were detected by MTT method. LDH release and MDA content were determined in each group. AGEs contents in cells treated with PBS, MGO or MGO+AG were assayed by immunohistochemical staining. Results Preculturing cardiomyocytes with MG for 6 days caused a significant increase in AGEs production, which was dramatically reduced by cotreatment with AG, a strong AGEs formation inhibitor, and preculturing cardiomyocytes with MG for 6 days made cardiomyocytes more susceptible to H/R, as evidenced by decreased survival rate of cells and LDH release. Conclusion Protein glycation sensitized cultured cardiomyocytes to H/R injury, and the

本文研究丹参水提物对乳鼠心肌细胞缺氧-复氧损伤的保护作用。以乳鼠心室肌进行心肌细胞培养并建立缺氧-复氧损伤模型，检测不同浓度丹参水提物预处理后细胞的存活率，乳酸脱氢酶(LDH)、肌酸激酶(CK)、天冬氨酸转移酶(AST)的泄漏量及Akt的磷酸化水平。丹参水提物对缺氧-复氧损伤的心肌细胞具有明显的保护作用，这可能与它激活PI3K/Akt信号通路有关。

To study the protective effect by Salvia Miltiorrhiza aqueous extract for hypoxia-reoxygenation damage in myocardial cells of suckling mice. The hypoxia-reoxygenation damage model was made from myocardial cells culture of suckling mice. Detection was taken on cells survival rate, leakage rates of lactate dehydrogenase (LDH), creatine kinase (CK), aspartate transaminase (AST), and phosphorylation level of Akt of cells in different concentrations of Salvia Miltiorrhiza aqueous extract. Salvia Miltiorrhiza aqueous extract provides obviously protective effect for myocardial cells with hypoxia-reoxygenation damage, and that may be related with its activation of PI3K/Akt signal path.

目的 探讨P物质对新生大鼠心肌细胞缺氧复氧损伤的影响.方法 新生SD大鼠,1～3日龄,处死后取心室肌组织,分离心肌细胞,接种于6孔培养板中,培养72 h.采用随机数字表法,将心肌细胞分为4组(n=3):对照组(C组)常规培养6h;缺氧复氧组(A/R组)进行缺氧3h,复氧2 h;P物质组(SP组)给予终浓度为10-7mol/L的P物质孵育lh,行缺氧3h,复氧2h;P物质+D-SP组(SP+ D-SP组)给予终浓度为10-7mol/L的P物质和终浓度为10-mol/L的速激肽1型受体拮抗剂D-SP孵育lh,行缺氧3h,复氧2h.于复氧结束时,测定心肌细胞凋亡率和乳酸脱氢酶(LDH)活性.结果 与C组比较,A/R组、SP组和SP+ D-SP组细胞凋亡率和LDH活性升高(P＜0.01);与A/R组比较,SP组和SP+ D-SP组细胞凋亡率和LDH活性降低(P＜0.01);与SP组比较,SP+ D-SP组细胞凋亡率和LDH活性升高(P＜0.01).结论 P物质可减轻新生大鼠心肌细胞缺氧复氧损伤,其机制与激活速激肽1型受体有关.

Objective To investigate the effects of substance P on anoxia/reoxygenation (A/R) injury to cardiomyocytes of neonatal rats.Methods Cardiomyocytes of neonatal rats were isolated from Sprague-Dawley rats,aged 1-3 days,and were cultured in 6-well plates for 72 h.The cardiomyocytes were then assigned into 4 groups (n =3 each) using a random number table:control group (group C),A/R group,substance P group (group SP) and substance P + D-SP group (group SP + D-SP).The cells were cultured routinely for 6 h in group C and the cells were exposed to 99.9 ％ N2 in an incubator at 37 ℃ for 3 h followed by reoxygenation for 2 h in the other groups.The cells were incubated with substance P with the final concentration of 10-7 mol/L for 1 h before anoxia in group SP.The cells were incubated for 1 h with substance P with the final concentration of 10-7 mol/L and D-SP (a specific antagonist of neurokinin-1 receptor) with the final concentration of 10-6 mol/L before anoxia in group SP + D-SP.