重组人L型丙酮酸激酶N末端单克隆抗体的制备与鉴定

目的 制备抗人L型丙酮酸激酶N末端(PK-N)的单克隆抗体并对其特异性进行鉴定.方法 以PK-N-GST-tag表达蛋白作为免疫原免疫BALB/c小鼠,取其脾细胞与SP2/O细胞融合,经多次筛选及克隆化,建立能稳定分泌抗PK-N单克隆抗体的杂交瘤细胞株.用ELISA、Western blot、免疫组化对此单克隆抗体特性进行鉴定.结果 筛选到两株能稳定分泌抗PK-N单克隆抗体的细胞株,亚类鉴定重链为IgGzb,轻链为kappa链.ELISA法测定腹水效价分别为1:409600和1:102400,抗体相对亲和力分别为3.54×108L/mol和2.72×108:L/mol.Western blot显示抗体能特异识别免疫原和人肝细胞株HL-7702中的天然丙酮酸激酶蛋白.免疫组化进一步显示了其在细胞及组织中的定位,均匀的分布于细胞的胞浆中.结论 成功制备了抗PK-N单克隆抗体.     

人乳腺癌及癌旁组织磷酸果糖激酶1及糖酵解水平的检测

目的:比较人乳腺癌及癌旁组织中糖酵解的水平差异,并探讨磷酸果糖激酶1的表达对糖酵解水平的影响。方法:采集3种不同临床分期共33对人乳腺癌及癌旁组织。酶法分析组织中乳酸含量、乳酸脱氢酶活力、己糖激酶活力、磷酸果糖激酶-1活力和丙酮酸激酶活力,用来反映糖酵解的水平;采用Western blot技术检测组织中磷酸果糖激酶-1的表达。结果:癌组织的糖酵解水平和磷酸果糖激酶1表达量均高于癌旁组织,而且,晚期癌组织的糖酵解和磷酸果糖激酶1表达量水平高于早期乳腺癌组织。结论:在人乳腺癌的发生发展过程中,糖酵解水平的增加可能与磷酸果糖激酶1的表达量增加有关。     

Pyruvate protects against kainate-induced epileptic brain damage in rats

Although the majority of epileptic seizures can be effectively controlled with antiepileptic drugs and/or surgery, a significant number progress to status epilepticus of sufficient duration to cause permanent brain damage. Combined treatment with antiepileptic drugs and neuroprotective agents, however, may help protect these individuals from permanent brain damage. Since toxicity induced by endogenous zinc contributes to epileptic brain injury, and since pyruvate is effective in reducing zinc-triggered neuronal death in cortical culture as well as ischemic neuronal death in vivo, we examined whether systemic pyruvate administration reduces seizure-induced brain damage. Na pyruvate (500 mg/kg) or osmolarity-matched saline (265 mg/kg NaCl, i.p.) were given to adult SD rats 30 or 150 min after 10 mg/kg kainite injection (i.p.), and there was no significant difference in the time course or severity of seizures between these groups. Zinc accumulation in neuronal cell bodies in the hippocampus, however, was much lower in the pyruvate than in the saline group. There was a close correlation between zinc accumulation and cell death, as assessed by acid-fuchsin and TUNEL staining. Pyruvate treatment markedly reduced neuronal death in the hippocampus, neocortex and thalamus. Pyruvate increased HSP-70 expression in hippocampal neurons. These results suggest that pyruvate, a natural glucose metabolite, may be useful as adjunct treatment in status epilepticus to reduce permanent brain damage.     

Glucose uptake inhibitor sensitizes cancer cells to daunorubicin and overcomes drug resistance in hypoxia

Purpose A high-rate glycolysis is a fundamental property of solid tumors and is associated with an over-expression of glucose transporters and glycolytic enzymes. We hypothesize that over-expression of glucose transporters in tumors prevents apoptosis, promotes cancer cell survival, and confers drug resistance. Inhibition of glucose transporter will preferentially sensitize the anticancer effects of chemotherapeutic drugs to overcome drug resistance in hypoxia. Methods Glucose transporter expressions were detected in cancer tissues and NCI 60 cancer cells with immunostaining and DNA microarray. Glucose uptake was measured with ³H-2-deoxy-glucose. Cytotoxicity of daunorubicin (DNR) in combination of glucose inhibitor was detected by MTS assay under hypoxic condition. Early stage apoptosis was monitored with Annexin V-FITC staining. Results Immunostaining showed that GLUT1 was significantly increased in hypoxic regions of the human colon and breast tumors. The expression profiles of all glucose transporters in NCI 60 cancer cells exhibited distinct expression patterns. Phloretin exhibited more than 60% glucose uptake inhibition. Hypoxia conferred two to fivefold higher drug resistance in SW620 and K562 to DNR. Inhibition of glucose uptake by phloretin sensitized cancer cells to DNR for its anticancer activity and apoptosis to overcome drug resistance only under hypoxia. Conclusion Cancer cells heavily rely on glucose transporters for glucose uptake to facilitate a high-rate glycolysis under hypoxia for their survival and drug resistance. Combination of glucose transporter inhibitors and chemotherapeutic drugs may provide a preferential novel therapeutic strategy to overcome drug resistance in hypoxia.     

糖酵解关键酶PFKFB3对婴幼儿血管瘤内皮细胞增殖、迁移及凋亡的影响

【摘要】 目的 研究糖酵解关键酶6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶3（PFKFB3）对婴幼儿血管瘤（IH）内皮细胞（HemEC）生物活性的影响。方法 收集4例增殖期及4例消退期IH组织,从增殖期IH组织中提取原代HemEC,以脐静脉内皮细胞（HUVEC）为对照。通过免疫组化与Western印迹法分别检测PFKFB3在IH组织与HemEC中的表达水平。采用CCK8实验检测0 ~ 10 μmol/L PFKFB3特异性抑制剂PFK15对HemEC细胞增殖的影响。体外培养HemEC,采用5 μmol/L PFK15干预细胞,采用Transwell法观察HemEC迁移能力,流式细胞仪检测HemEC凋亡水平。组间比较采用t检验或方差分析。结果 免疫组化显示,增殖期IH组织中PFKFB3表达丰度（74.34% ± 5.26%）高于消退期（41.46% ± 2.99%,t = 9.40,P < 0.001）。Western印迹显示,HemEC中PFKFB3相对表达水平（0.73 ± 0.05）高于HUVEC（0.45 ± 0.04,t = 8.50,P < 0.001）。CCK8实验结果显示,0.625、1.25、2.5、5、10 μmol/L PFK15组HemEC增殖活性均低于对照组（均P < 0.01）。Transwell实验显示,PFK15干预组HemEC迁移数（297 ± 15）低于对照组（422 ± 8,t = 12.59,P < 0.001）。流式细胞仪检测显示,PFK15干预组HemEC凋亡率（6.69% ± 0.64%）高于对照组（0.34% ± 0.07%,t = 17.07,P < 0.001）。结论 糖酵解关键酶PFKFB3在IH增殖期组织与HemEC中高表达,PFKFB3抑制剂PFK15可抑制HemEC增殖、迁移并促进其凋亡。     

ESET通过lncRNA GMDS-AS1调控肝癌细胞增殖、凋亡、糖酵解的研究

目的 探究组蛋白甲基转移酶集合域分叉1(ESET)、长链非编码RNA甘露糖4,6-脱水酶反义RNA1(lncRNA GMDSAS1)对肝癌细胞增殖、凋亡、糖酵解的调控及二者之间的潜在关系。方法 收集右江民族医学院附属医院和百色市人民医院2019年1月至2020年1月期间手术切除的肝癌组织及癌旁组织49例。脂质体法将空白组（不做任何处理）、空载体组（转染pcDNA或si-con）、敲减ESET组（转染si-ESET）、过表达lncRNA GMDS-AS1组（转染pcDNA-GMDS-AS1）转染至HepG2细胞;实时荧光定量逆转录聚合酶链反应（qRT-PCR）实验、蛋白质印迹法实验检测ESET、lncRNA GMDS-AS1、细胞增殖抗原标志物Ki-67、葡萄糖转运蛋白1(GLUT-1)、乳酸脱氢酶A(LDHA)、活化胱天蛋白酶-3(C-caspase-3)的表达;细胞计数试剂盒（CCK8）法、流式细胞术检测细胞增殖、凋亡;RNA结合蛋白免疫沉淀（RIP）实验检测ESET与lncRNA GMDS-AS1的关系。结果 肝癌组织中ESETmRNA和蛋白表达量为5.18±0.94、0.69±0...     

O2 consumption,aerobic glycolysis and tissue phosphagen content during activation of the NA+/K+ pump in rat portal vein

Oxygen consumption, lactate production and tissue contents of ATP, phosphocreatine (PCr) and lactate were measured following readdition of K⁺ to K⁺-depleted rat portal veins, in order to study the energy turnover associated with Na⁺/K⁺ pumping. During incubation in K⁺-free medium at 37° C spontaneous contractions disappeared in 10–20 min. Readdition of K⁺ (5.9 mM) after 40 min K⁺-free incubation caused hyperpolarization of the cell membrane for the first 5–10 min and then gradual depolarization with return of spontaneous action potentials and contractions by 10–20 min. During the first 4–6 min after K⁺ readdition aerobic lactate production was about doubled and then gradually returned to the original level (0.17 mol/min g) at about 20 min. The increase in glycolytic rate was prevented by 1 mM ouabain. In contrast, O₂ consumption (in K⁺-free medium, 0.38 mol/min g) rose by about 10% when K⁺ was added and this increase lasted about 5 min. By 8 min after K⁺ addition the increased glycolysis and oxidative phosphorylation had accounted for each about the same amount of extra ATP generation over that extrapolated from the steady rate before K⁺ addition. The average total increase in ATP turnover in the first 8 min was 15%. During this period there was no change in the cellular content of ATP, PCr, or extractable ADP. The results indicate that Na⁺/K⁺ pumping utilizes a relatively small share of the total energy turnover in the vascular smooth muscle but is to a large extent dependent on aerobic glycolysis and therefore a major site of carbohydrate usage.     

基因沉默缺氧诱导因子-1α对BxPC-3细胞株糖酵解基因表达的影响

目的 观察沉默缺氧诱导因子(HIF)-1α基因表达对于缺氧微环境下人胰腺癌BxPC-3细胞株中糖酵解相关基因表达的影响.方法 通过合成小于扰RNA((siRNA))转染沉默BxPC-3细胞株中HIF-1α基因表达.将细胞分为空白对照组(BxPC-3)、空白质粒转染对照组(GFP)和HIF-1α基因沉默组(sh-HIF-1α),分别在正常环境(21.0％O2)和缺氧环境(0.5％～1.0％ O2)中培养48 h后,检测各组细胞中糖酵解相关基因[如丙酮酸激酶1(PDK-1)、乳酸脱氢酶A(LDH-A)和柠檬酸合成酶(CS)]的表达以及细胞糖酵解产物乳酸含量的改变.结果 与正常环境比较,缺氧培养后sh-HIF-1α组细胞内HIF-1αmRNA增加了12.4％,蛋白增加了1.6倍,显著低于BxPC-3组和GFP组(P＜0.05),PDK-1和LDH-A的表达也明显低于两个对照组(P＜0.05).相应的sh-HIF-1α组的乳酸含量为(4.8 ±0.3)nmol/106个细胞,明显低于BxPC-3组(9.1±0.5)nmol/106个细胞和GFP组(9.2±0.5) nmol/106个细胞(P＜0.05).相反,缺氧时BxPC-3组和GFP组细胞中与线粒体氧化磷酸化相关的CS基因表达明显下降,低于其在sh-HIF-1α组细胞中的表达(P＜0.05).结论 缺氧可以诱导BxPC-3细胞株中HIF-1α基因高表达并促进糖酵解相关的PDK-1和LDH-A等基因表达.而通过沉默HIF-1α基因,可以抑制上述基因的表达,并促进CS基因表达,从而抑制肿瘤细胞的糖酵解代谢.     

磷酸甘油酸脱氢酶基因与肿瘤

磷酸甘油酸脱氢酶(phosphoglycerate dehydrogenase,PHGDH)基因编码3-磷酸甘油酸脱氢酶,是糖酵解-丝氨酸生物合成途径中的第一个分支酶.PHGDH氧化糖酵解中间产物3-磷酸甘油酸为磷酸羟基丙酮酸,后通过一系列酶的作用最终合成丝氨酸.丝氨酸在蛋白和细胞增殖所需其他生物分子(如核苷酸、磷脂丝氨酸、鞘氨醇)的合成中起着重要的作用.最新研究发现PHGDH高表达于一系列肿瘤中,且与肿瘤细胞生长、凋亡相关.该文就PHGDH基因结构、功能及与肿瘤的关系作一综述.     

Lactate balance in perfused rat liver: Effects of glucose concentration,flow and low pH on glucose to lactate flux

The effects of medium glucose concentration (0-20 mmol 1^?1), pH (7.4 and 6.8) and flow (100 to 33% normal) on lactate uptake and glycolytic flux from 6-³H glucose were studied in perfused livers from 48-h starved rats. At both pH values, the glycolytic flux increased proportionally with the medium glucose concentration. Maximum glycolytic flux at 20 mmol L¹ glucose in the medium was 0.5 umol mirT¹ g^?1 liver (C₆-units) at pH 7.4. At pH 7.4 and 20 mmol 1^?1 glucose the glycolytic flux decreased approximately proportional with flow. At pH 6.8 the glycolytic flux was extremely low and independent of flow. At flow 33% normal and pH 7.4 a net lactate production was accounted for by glycolysis from medium glucose concentration, indicating virtually no simultaneous lactate uptake. In contrast, at pH 6.8 net lactate production accounted for only half the glycolytic rate, indicating that lactate uptake occurred simultaneously with glycolysis. Thus, glucose-to-lactate flux in liver (as in muscle and brain) is subject to inhibition by low pH, and lactate uptake is enhanced by low pH.