刺五加、葡萄籽提取物方剂对衰老模型小鼠肝脏细胞凋亡及氧化应激的影响

目的研究刺五加、葡萄籽提取物方剂对衰老模型小鼠肝脏细胞凋亡及氧化应激的影响。方法SPF级昆明种雄性小鼠50只,随机分1组阴性对照组(10只)和4组D-半乳糖模型组(每组各10只),模型组以1ml/(kg.bw)的剂量连续腹腔注射150mg/(kg.bw)的D-半乳糖2个月后,分为模型对照组和刺五加、葡萄籽提取物方剂低、中、高3个剂量组。各剂量组灌胃分别给予0.16、0.33、1.00g/(kg.bw)的刺五加、葡萄籽提取物方剂,模型对照组灌胃给予相应体积的纯净水,同时各组腹腔注射150mg/(kg.bw)的D-半乳糖,连续60d后取肝脏。通过AnnexinV/PI联合标记染色肝细胞,以流式细胞仪在Ex=488nm条件下,CellQuest软件分析肝细胞凋亡率;同时以分光光度法检测肝脏氧化应激状态。结果在Contourplot图上,根据肝细胞标记染料AnnexinV/PI的强度可见,中、高剂量组肝细胞凋亡百分率分别为(30.94±7.59)%、(29.35±5.58)%,均低于模型对照组[(47.99±11.95)%](P<0.01)。中、高剂量组MDA含量[(1.64±1.30)、(1.57±1.72)nmol/mg]均低于模型对照组[(3.92±0.73)nmol/mg](P<0.01);中、高剂量组SOD活性[(162.92±18.04)、(175.53±24.65)NU/mg]均高于模型对照组[(135.77±29.16)NU/mg](P<0.05);而各剂量组GSH-Px活性与模型对照组比较差异均无统计学意义(均P>0.05)。结论刺五加、葡萄籽提取物方剂可减少衰老模型小鼠肝细胞凋亡的发生,通过降低MDA含量和升高SOD活性增加机体对氧化应激反应的能力。     

In vitro evaluation method of bioartificial liver function: constant infusion test

 Various types of bioartificial livers (BALs), which are extracorporeal medical devices incorporating living hepatocytes in cartridges, have been developed. However, it is difficult to compare metabolic functions among BAL types or to know what proportion of the normal liver functions could be replaced by a BAL, because there is not a well-established method for the quantitative evaluation of BAL functions. In our series of studies, we have proposed methods for performing drug-loading tests and procedures to analyze drug concentration changes for the quantitative evaluation and expression of BAL metabolic functions. In this study, constant infusion tests of lidocaine were performed on a BAL device developed in our laboratory, and lidocaine concentration changes in the perfusion medium were analyzed by using pharmacokinetic equations. The lidocaine clearance value of the BAL was precisely determined by a constant infusion test, demonstrating the usefulness of the constant infusion test for quantitative evaluation of BAL functions. Received: February 12, 2002 / Accepted: September 10, 2002 Acknowledgments This work was supported by grant JSPS – RFTF 96I 00204 from the Japan Society for the Promotion of Science and by the New Energy and Industrial Technology Development Organization. Correspondence to:H. Iwata     

N-亚甲基磷酸盐壳聚糖衍生物的设计、合成和表征

目的：合成和表征N-亚甲基磷酸盐壳聚糖衍生物作为潜在的肝靶向基因载体：方法：以天然聚合物壳聚糖为原料，与甲醛和磷酸反应，制得双取代的N-亚甲基磷酸壳聚糖，然后在其剩余的2-NH2和乳糖酸反应，制得N-亚甲基磷酸-N-乳糖酰化壳聚糖；与乳糖反应，用KBH4还原，制得N-亚甲基磷酸-N-乳糖胺化壳聚糖。结果与结论：分别用FTIR、1HNMR、13CNMR和元素分析对其进行了表征。用粉末X-衍射、DSC、TG对其物理性质进行了分析。制得的N-亚甲基磷酸壳聚糖、N-亚甲基磷酸-N-乳糖酰化壳聚糖和N-亚甲基磷酸-N-乳糖胺化壳聚糖的取代度分别为1．22，0．23和0．21，所制得的栽体有望作为潜在的肝靶向基因载体：     

Galactose removal kinetics during hypoxia in perfused pig liver: reduction of Vmax, but not of intrinsic clearance Vmax/Km

The galactose elimination kinetics was examined in five perfused pig livers of 1.2 kg during hypoxia induced by administration of 2, 4 or 7% oxygen in the oxygenator instead of 20% as used in nine control experiments, previously published. Galactose was given as four to five successive constant infusion rates so that successive steady-state period with galactose concentrations from 0.04 to 5 mmol l-1 were obtained in each experiment. From the relationship between the calculated elimination rate and the perfusate galactose concentration, values of the maximal elimination rate Vmax and the half saturation concentration Km were calculated. Both Vmax and Km were reduced by hypoxia: the lower the oxygen supply, the greater the reduction. Vmax was about 0.08 mmol min-1 kg-1 liver at 2% oxygen and about 0.18 mmol min-1 kg-1 liver at 4-7% oxygen; both being significantly lower than the value of 0.43 mmol min-1 kg-1 liver at 20% oxygen. Km was about 0.07 mmol l-1 at 2% oxygen and 0.13 mmol l-1 at 7% oxygen; both significantly lower than the value of 0.23 mmol l-1 at 20% oxygen. A nearly parallel reduction of liver ATP concentration and galactose Vmax indicates that the galactose Vmax may reflect the phosphorylation capacity of the liver cells. The Vmax/Km ratio (intrinsic hepatic clearance) was unchanged during hypoxia.     

The influence of chronic lobular hepatitis on pharmacokinetics of cefoperazone—a novel galactose single-point method as a measure of residual liver function

Cefoperazone is a semisynthetic cephalosporin antibiotic containing a piperazine side chain, which results in antipseudomonal activity. Unlike the other cephalosporins, it is mainly cleared by the liver (60–80%) and it may be more sensitive to changes in the liver function and/or plasma protein binding than other cephalosporins, which are not primarily cleared by the liver. In order to study the influence of chronic lobular hepatitis on the pharmacokinetics of cefoperazone, a dose of 1 g of cefoperazone was administered to 11 normal, healthy volunteers and 16 subjects with chronic lobular hepatitis. In each volunteer or patient, a novel galactose single-point (GSP) method, the galactose elimination capacity (GEC) test, and the modified galactose elimination capacity (MGEC) test were also performed as a measure of residual liver function. Cefoperazone was administered intravenously over a period of 3–5 min. Blood and urine samples were collected at appropriate intervals after drug administration and stored at ?30°C until high-pressure liquid chromatographic (HPLC) analysis. The cefoperazone hepatic clearance, mean residence time, and renal clearance in hepatitis patients were significantly different from those of normal healthy volunteers, whereas the plasma protein binding was unaltered between the two groups. Urinary excretion of cefoperazone showed a highly significant increase in patients, 23.95 ± 5.06% and 37.54 ± 13.61% for normal men and hepatitis patients respectively. Hepatic clearance and fraction excreted in urine significantly correlated with values of GSP and MGEC respectively (p <0.05). These results suggest (i) cefoperazone kinetics was significantly altered in patients with chronic lobular hepatitis; (ii) GSP, a novel simple, clinically useful quantitative liver function test, can predict the cefoperazone hepatic clearance in patients with liver dysfunction.     

葛根素对D-半乳糖诱导大鼠抑制蛋白非酶糖基化及增强胰岛素敏感性的作用

目的观察葛根素对D-半乳糖(D-gal)诱导大鼠体内蛋白非酶糖基化反应、胰岛素抵抗、糖耐量和醛糖还原酶(AR)活性的作用。方法除空白对照组外,其余大鼠均采用腹腔注射(ip)D-gal150mg/kg,1次/d×56d致病,D-gal处理大鼠2周后,将大鼠按体重均衡随机分成:模型对照、葛根素和氨基胍3组,然后继续ipD-gal处理同时灌胃给予葛根素和氨基胍(每10ml300mg/kg)或蒸馏水,1次/d×42d;实验结束时,观察葛根素和氨基胍对D-半乳糖诱导大鼠体内晚期糖基化终末产物(AGEs)、糖化血红蛋白(HbA1c)、果糖胺(FRA)、糖耐量、血糖和胰岛素含量,并计算胰岛素敏感指数(ISI),以及醛糖还原酶(AR)活性的作用。结果与空白对照组比较,D-gal处理大鼠出现血糖升高、糖耐量减退(IGT)、糖基化产物(包括HbA1c,FRA和AGEs)和胰岛素含量增高,ISI降低,以及红细胞内AR活性增强(P<0.01);葛根素和氨基胍均能改善D-gal诱导大鼠IGT和降低空腹血糖、糖基化产物和胰岛素含量(P<0.01～0.05),提高ISI(P<0.01),并明显抑制AR活性(P<0.01～0.05)。结论D-gal能诱导大鼠体内糖基化反应、高胰岛素血症、胰岛素抵抗和醛糖还原酶活性增强;而葛根素和氨基胍能抑制蛋白非酶糖基化反应和醛糖还原酶活性,提高胰岛素敏感性,改善高胰岛素血症,提示葛根素可用于治疗糖尿病某些慢性并发症。     

Membrane protein antigens in multiple sclerosis

Rabbits were immunized with white matter (WM) membrane fractions isolated from autopsy brain specimens of three patients with multiple sclerosis (MS), and three controls. All the rabbits developed high serum antibody titers to the MS and control WM fractions, as tested by enzyme immunoassay. Antibodies against WM membrane components were analyzed further by immunoprecipitation of radiolabeled WM proteins and subsequent polyacrylamide gradient gel electrophoresis. Antigenic membrane components with molecular weights of 138 000, 111 000, 86 500, 79 600, 69 000, 63 000, 58 000, 53 400, 45 700, 24 500 and 22 300 were found in both MS and control WM. Although there may have been some quantitative differences in these immunogenic proteins of MS and normal WM, no multiple sclerosis-specific membrane antigen could be demonstrated.The hyperimmune anti-WM sera did not precipitate ³⁵S-labeled polypeptides from cells infected with herpes simplex type 1, adeno type 5, measles, mumps, rubella, respiratory syncytial, parainfluenza type 2 or cytomegaloviruses, which suggests that the MS brain WM membrane proteins do not share common antigenic determinants with the viral polypeptides.     

A microelectrode for continuous recording of volume fluxes in isolated perfused tubule segments

Manufacture, properties and use of a micro enzyme electrode for continuous monitoring of volume fluxes in the isolated tubule preparation is described. The specific electrode is a galactose-oxidase enzyme electrode, which can be used to detect changes in raffinose concentrations. The electrode's response to raffinose is almost linear over concentrations from 0–12 mmol/l. The electrode equally responds to galactose as to raffinose but is insensitive to other sugars, to pH changes (from 6.0–8.0), CO₂ (from 1–10%) and electrolytes tested. Reducing O₂ from 100 to 10% and to 1%, leads to a reduction of the reading by 10% and 30%, respectively. The reading is almost doubled when the temperature is increased from 20–40° C. Furthermore, reducing agents such as uric acid and ascorbic acid interfere with the reading. If these substances and raffinose are omitted from the perfusate for isolated perfused proximal mouse tubules, the reading is identical in perfusate and collected fluid, indicating that the tubular epithelium does not produce substances in sufficient amounts to interfere with the electrode reading. After addition of 6 mmol/l raffinose to the perfusate the raffinose concentration in the collected fluid of 0.76±0.05 mm segments of straight proximal mouse tubules (perfusion rate = 3.4±0.45 nl/min) is 10.2±0.3 mmol/l, indicating a volume reabsorption of 1.5±0.3 nl/min. Peritubular application of acetazolamide reduces the volume reabsorption by 42±4%.Supported by Österr. Forschungsrat, Proj. No. 4366     

Galactose Metabolism in a Patient with Hereditary Galactokinase Deficiency

Abstract. The ability of a galactokinase deficient patient to metabolize galactose, galactitol and galactonate was quantitated. In galactokinase deficiency, conversion of galactose to CO₂ is minimal. Apparently the defect is extensive, involving all tissues. Galactitol and galactonate, injected intravenously, were not metabolized. The administration of C-1 and C-2 labelled galactose resulted in ¹⁴CO₂ excretory patterns similar to that observed in uridyltransferase deficient mutants. The different fates of C-1 and C-2 observed in this galactokinase deficient patient give support to the existence of a direct oxidative pathway for galactose. Galactonate, although present in urine during the period of observation following injection of radioactive galactose failed to become labelled.     

透明质酸-半乳糖化壳聚糖复合支架与牛磺酸联合治疗脑创伤大鼠的初步研究

目的 探索透明质酸-半乳糖化壳聚糖复合支架（HGC）联合牛磺酸（Tau）对脑创伤（TBI）大鼠的治疗作用.方法 选择成年SD大鼠40只,随机分为假手术组（Sham组）、脑创伤组（TBI组）、透明质酸-半乳糖化壳聚糖复合支架组（HGC组）、透明质酸-半乳糖化壳聚糖复合支架与牛磺酸联合治疗组（HGC-Tau组）,每组10只.TBI、HGC及HGC-Tau组采用液压打击法在大鼠左侧大脑半球制作TBI模型,Sham组仅在相同位置开骨窗,不予打击.HGC组于造模后7d将30μl HGC植入大鼠脑皮质损伤区,HGC-Tau组以同样方法植入相同体积HGC与牛磺酸的混合物.TBI后1、3、7、10、14、21 d进行改良型神经功能评分（mNSS）,其中17～21 d进行Morris水迷宫（MWM）实验.TBI后28 d采用实时定量PCR及免疫荧光染色检测胶质纤维酸性蛋白质（GFAP）的基因及蛋白表达水平.结果 HGC-Tau组mNSS评分在TBI后14d及21d显著低于TBI组（P＜0.05）,HGC组各时间点评分与TBI组相比,差异均无统计学意义（P＞0.05）,HGC-Tau组与HGC组相比,评分在TBI后14、21 d显著降低（P＜0.05）;HGC-Tau组MWM检测目标象限百分比从创伤后18d开始明显大于TBI组（P＜0.05）,HGC组创伤后21 d目标象限百分比显著大于TBI组（P＜0.05）,HGC-Tau组仅在18、19 d目标象限百分比明显大于HGC组（P＜0.05）;HGC组及HGC-Tau组TBI后28 dGFAP mRNA及蛋白表达量与TBI组相比显著下降（P＜0.05）,且较HGC组,HGC-Tau组下降更加明显（P＜0.05）.结论 单纯使用HGC仅能抑制创伤性脑损伤后胶质细胞增生,对神经功能改善无明显作用.HGC联合Tau治疗可抑制重型脑损伤后胶质细胞增生,改善神经功能,表明生物材料与药物联合治疗脑外伤具有一定的应用前景.