硒化壳聚糖理化性质和分子结构的研究

运用高效凝胶色谱（ＨＰＧＰＣ）、乌氏粘度计、ＸＹＧ－１１０型多功能原子荧光仪、常规溶剂和ＷＺＺ－１自动指示旋光仪等方法对本实验室制备的硒化壳聚糖的理化性质进行测定,结果表明：硒化壳聚糖的分子量为３４４８;特性粘度为９９;室温干燥放置１年,硒化壳聚糖保持稳定;硒化壳聚糖在水中可溶,而且溶解度随温度升高而增加,但在有机溶剂中基本不溶;硒化壳聚糖在２５℃时的旋光度为－１４。通过紫外光谱、红外光谱、氢谱（     

硒化壳聚糖抗K562细胞作用机制的初步探讨

应用分光光度法和放免法观察不同剂量硒化壳聚糖对K562细胞株抗氧化指标及cAMP、cGMP含量的影响,结果发现硒化壳聚糖可明显升高细胞内GSH-PX活力,降低SOD活性和MDA含量,升高cAMP含量,降低cGMP含量,升高cAMP/cGMP比值.降低K562细胞内脂质过氧化水平,升高cAMP/cGMP比值可能是硒化壳聚糖诱导K562细胞凋亡机制之一.     

硒化壳聚糖和亚硒酸钠抗K562细胞的实验研究

应用MTT比色分析法比较研究有机硒化物-硒化壳聚糖和无机硒化物亚硒酸钠对白血病K562细胞的作用。结果表明：两种硒在体外实验浓度范围内均对K562细胞的增殖具有显著的抑制作用，并有剂量反应关系：硒化壳聚糖的抗白血病效应明显高于含同样硒量的亚硒酸钠，约为其3倍，有机硒比无机硒具有更高的抗白血病活性。     

硒化壳聚糖对NB_4细胞的作用及其与NF-κB信号分子的关系

目的:研究硒化壳聚糖对人急性早幼粒细胞性白血病NB4细胞抗氧化指标的影响及其与核因子κB(NF-κB)信号分子的关系。方法:取NB4细胞加入不同浓度的硒化壳聚糖(50、100、200mg·L-1)作用24h后取上清检测谷胱甘肽过氧化物酶(GSH-px)、超氧化物歧化酶(SOD)、丙二醛(MDA)、NF-κB蛋白含量或活性,并设培养液为对照组。结果:与对照组比较,硒化壳聚糖各浓度组GSH-px活性明显升高(P<0.01),SOD活性和MDA含量明显降低(P<0.01或P<0.05),NF-κB蛋白含量明显降低(P<0.01),并呈量效关系。结论:硒化壳聚糖可通过减少进入细胞核内NF-κB蛋白含量的方式下调其下游基因表达,以及增强细胞抗氧化能力的方式抑制NB4细胞增殖,诱导细胞凋亡。     

硒化壳聚糖对HL60细胞增殖分化的影响

目的 探讨硒化壳聚糖与细胞c-myc和c-fos基因表达的关系及对人早幼粒白血病HL60细胞增殖和分化的影响.方法 采用SRB法和克隆形成法检测细胞增殖;Wright-Giemsa染色法检测细胞分化;流式细胞术检测CD11b、c-myc和c-fos分子表达.结果 25、50、100 mg/L硒化壳聚糖作用HL60细胞48 h可明显促进细胞分化和抑制细胞增殖(P＜0.05,P＜0.01);各浓度硒化壳聚糖均可使细胞CD11b蛋白表达显著升高(P＜0.01),c-myc蛋白表达下降(P＜0.05或P＜0.01),c-fos蛋白表达升高(P＜0.05或P＜0.01).结论 硒化壳聚糖可通过调控c-myc和c-fos基因表达来抑制HL60细胞增殖,促进细胞分化.     

钙离子一氧化氮在硒化壳聚糖诱导的皮肤成纤维细胞增殖中的作用

目的观察硒化壳聚糖对体外培养人皮肤成纤维细胞增殖周期及细胞内一氧化氮(NO)水平及钙离子含量的影响。方法皮肤成纤维细胞培养液加入不同浓度的硒化壳聚糖(25、50、100、200、400 mg/L),应用噻唑蓝(MTT)法检测细胞增殖,改良Griess法检测NO,Huo-3AM钙荧光指标剂测定细胞内钙离子浓度,流式细胞仪检测细胞周期。结果与对照组比较,50~400 mg/L硒化壳聚糖作用48 h后,细胞对MTT的吸收明显升高(P<0.05);各浓度硒化壳聚糖组细胞NO水平(72±9)μmol/L~(44±10)μmol/L与对照组(107±9)μmol/L比较均呈不同程度下降,呈剂量依赖性(P<0.01);200、400 mg/L硒化壳聚糖组细胞内钙离子浓度(179±32)mmol/L、(179±14)mmol/L与对照组(147±10)mmol/L比较显著升高(P<0.05);流式细胞分析显示,G0~G1期细胞所占百分率显著性减少,G2~M期和S期细胞所占百分率显著性增加(P<0.01)。结论硒化壳聚糖可通过调节细胞内钙离子含量和NO水平来促进皮肤成纤维细胞增殖。     

硒化壳聚糖促进体外培养皮肤成纤维细胞增殖

目的研究硒化壳聚糖对体外培养的皮肤成纤维细胞增殖的影响。方法用不同剂量（25、50、100、200、400mg/L）硒化壳聚糖作用于成纤维细胞,光镜下观察药物对细胞形态的影响;MTT法、3H-TdR掺入法、细胞生长动力学研究用于检测药物对细胞增殖影响,LDH漏出率检测药物对细胞增殖及损伤的影响,并以等体积细胞培养液处理为对照组。结果各药物浓度组处理细胞后,细胞形态未见明显改变,均可使细胞吸光度值增加,细胞倍增时间缩短,促进表皮细胞对3H-TdR的掺入（P〈0.05）,降低LDH漏出率（P〈0.05）。结论硒化壳聚糖对体外培养皮肤成纤维细胞增殖具有促进作用。     

硒化壳聚糖对HL60细胞的诱导分化作用

目的:研究硒化壳聚糖对人早幼粒细胞性白血病HL60细胞分化的影响,并探讨该影响与细胞c-myc和c-fos蛋白表达的关系。方法:取HL60细胞分为5组,分别为25、50、100mg·L-1硒化壳聚糖组、空白对照组(培养液)和阳性对照组(二甲基亚砜)。各组经相应试药作用后,采用硝基蓝四氮唑(NBT)还原法检测HL60细胞分化情况,观察NBT阳性细胞数;流式细胞术检测c-myc和c-fos蛋白表达。结果:与空白对照组比较,硒化壳聚糖各组作用与阳性对照组相似,NBT还原阳性细胞明显增多,c-myc蛋白表达显著下降(P<0.01或P<0.05),c-fos蛋白表达显著升高(P<0.01或P<0.05)。结论:硒化壳聚糖可能通过增强HL60细胞c-fos表达及下调c-myc表达来诱导细胞分化。     

低分子量半乳糖化脂肪酰壳聚糖的制备及其形成胶束的性质研究

目的制备具有主动肝靶向作用的低分子量半乳糖化脂肪酰壳聚糖聚合物胶束材料并进行结构表征,对其形成的胶束特性进行研究。方法以甲磺酸为溶剂及反应介质,利用脂肪酰氯(棕榈酰氯及月桂酰氯)与壳聚糖的OH进行酰化反应制备具有不同碳链长度及取代度的脂肪酰壳聚糖,再利用EDC.HCl活化乳糖酸,与所得的脂肪酰壳聚糖的2位氨基反应制备半乳糖化脂肪酰壳聚糖。用红外光谱、1H核磁共振谱对其结构进行表征及取代度的计算,并考察其溶解性能;制备其聚合物胶束,测定临界胶束浓度(CMC)和粒径。结果壳聚糖脂肪酰化的反应条件为20℃、1 h,半乳糖化反应的条件为30℃、24 h;壳聚糖棕榈酰化及月桂酰化反应中酰氯/壳聚糖的合适摩尔比分别为2∶1～8∶1、2∶1～10∶1。此条件下制得的脂肪酰壳聚糖及半乳糖化脂肪酰壳聚糖在二甲基亚砜(DMSO)中有较好的溶解性能;红外光谱、1H核磁共振光谱结果表明,成功合成了半乳糖化脂肪酰壳聚糖;脂肪酰基的取代度范围为0.28～1.13 mol,胶束的CMC约为0.39×10 2～2.82×10 2mg·mL-1,粒径大小119.8～546.0 nm。结论成功制备低分子量半乳糖化脂肪酰壳聚糖聚合物胶束材料。相同碳链长度条件下,脂肪酰氯与壳聚糖摩尔比越大,脂肪酰基的取代度越大,所得胶束的CMC及粒径越小;相同脂肪酰氯与壳聚糖摩尔比时,制得的半乳糖化脂肪酰壳聚糖的粒径小于脂肪酰壳聚糖的粒径。     

硒化壳聚糖对体外培养人皮肤成纤维细胞分泌细胞因子与一氧化氮的影响

目的观察硒化壳聚糖对体外培养人皮肤成纤维细胞分泌细胞因子及一氧化氮（NO）的影响。方法测定经25,50,100,200,400 mg&#8226;L 1硒化壳聚糖作用后,人皮肤成纤维细胞培养上清液中转化生长因子 α（TGF α）、TGF β1、白细胞介素 1β（IL 1β）、IL 6、IL 8、肿瘤坏死因子（TNF）及NO的水平;对照组用等体积细胞培养液处理。结果与对照组比较,硒化壳聚糖作用后皮肤成纤维细胞培养液中TGF α、TGF β1、IL 1β、IL 6、IL 8和TNF水平不同程度升高,差异有显著性或极显著性（P＜0.05或P＜0.01）;而NO水平与对照组比较显著下降（均P＜0.01）。结论硒化壳聚糖通过促进细胞分泌TGF α、TGF β1、IL 1β、IL 6、IL 8及TNF,抑制NO释放来促进皮肤成纤维细胞增殖和胶原合成。     

低频超声辐照对顺铂抑制人肝癌细胞体内外生长增效作用的实验研究

目的观察低频超声辐照对顺铂抑制人肝癌细胞的体内外生长增效作用,探讨低频超声在肿瘤化学治疗增效中的应用价值。方法①体外实验：将人肝癌细胞SMMC7721分为A组（实验空白对照）;B组（SMMC7721细胞常规培养）;C组（单纯使用顺铂治疗,药物终浓度为2．5和5mg／L;D组（低频超声治疗,频率为30kHz,强度分别为1．4和2．8W／cm2,每个强度分别辐照2min、5min）;E组（顺铂＋低频超声,用法同c组和D组）。计算联合治疗指数值,用噻唑蓝比色法检测对人肝癌细胞SMMC7721生长抑制率。②体内实验：16只裸鼠体内移植人肝癌肿瘤SMMC7721模型分为对照组、顺铂组、低频超声组和观察组（顺铂＋低频超声）,各4只。采用裸鼠体内移植人肝癌细胞SMMC7721,用低频超声波（频率30000Hz,超声强度2．8W／cm2）对腹腔内注射顺铂（5m/kg）的裸鼠进行肿瘤局部辐射,测量肿瘤体积,绘制肿瘤生长曲线,实验结束称瘤重和体重,计算肿瘤抑制率。结果D组1．4W／cm。和2．8w／cm2辐照2min的生长抑制率分别为5．3％和8．0％,辐照5min的生长抑制率分别为12．2％和23．8％;E1组（顺铂终浓度2．5mg／L＋低频超声强度1．4W／cm2）辐照2和5min的生长抑制率分别为55．4％和69．5％,E2组（顺铂终浓度2．5mg／L＋低频超声强度2．8w／cm。）分别为70．9％和77．9％,E3（顺铂终浓度5．0mg／L＋低频超声强度1．4W／cm2）分别为70．8％和75．5％,E4组（顺铂终浓度5．0mg／L＋低频超声强度2．8W／cm。）分别为80．1％和84．9％。低频超声波联合顺铂对SMMC7721细胞生长抑制率明显高于单独低频超声。体内实验中,不同治疗方法干预后,对照组、顺铂组、低频超声组和观察组裸鼠平均瘤重分别为（0．65±0．31）、（0．42±0．19）、（0．50±0．17）、（0．29±0．18）g,观察组与对照组间差异有统计学意义（P〈0．05）。顺铂组、低频超声组和观察组的肿瘤抑制率分别为36．0％、23．8％、55．2％,低频超声组和顺铂组的差异无统计学意义（P〉0．05）,观察组与低频超声组的差异有统计学意义（P〈0．05）。结论低频超声在体内、外均明显增强抗癌药顺铂对人肝癌细胞SMMC7721的抑制作用。低频超声波的作用时间是增加化学治疗药物细胞毒作用的关键因素,低频超声波局部透射可以增加全身给药化学治疗的疗效,有望成为临床治疗的新方法。     

Low Ca2+ plus tetraethylammonium medium: its usefulness in studying the effect of Ca antagonist on adrenergic neurotransmission.

The low Ca2+ + tetraethylammonium (TEA) medium enhanced electrically evoked norepinephrine release by 3.5-fold in canine saphenous veins. omega-Conotoxin GVIA (omega-CTX) inhibited the neurotransmission more markedly in low Ca2+ + TEA medium than in normal Krebs medium. This is the case for the inhibition by tetrodotoxin, though to a lesser extent. The inhibition by omega-CTX was competitively antagonized by elevating external Ca2+. The results indicate that low Ca2+ + TEA medium is useful for studying the effect of Ca antagonists on adrenergic neurotransmission, because omega-CTX acts more effectively in this medium.     

Low dosage of levomepromazine did not increase plasma concentrations of fluvoxamine

The cytochrome enzyme P450 2D6 (CYP2D6) is thought to play a role in the human metabolism of fluvoxamine. Levomepromazine is a potent inhibitor of CYP2D6. We coadministered a low dosage of levomepromazine and fluvoxamine in 15 patients and found that the low dosage of levomepromazine was effective in counteracting the fluvoxamine-induced insomnia and did not increase plasma fluvoxamine levels. These results suggest that the inhibition of CYP2D6 by levomepromazine has little effect on fluvoxamine metabolism. Therefore, a low dosage of levomepromazine, used as a hypnotic agent, appears to be effective and safe when coadministered with fluvoxamine. Since this was a pilot study without a placebo control, a double-blind placebo-controlled study is needed to confirm our preliminary findings.     

Polymorphism in CYP2C9 as a non-critical factor of warfarin dosage adjustment in Korean patients

Cytochrome P4502C9(CYP2C9) is largely responsible for terminating anticoagulant effect by hydroxylation of S-warfarin to inactive metabolites. Mutations in the CYP2C9 gene result in the expression of allelic variants, CYP2C9*2 and CYP2C9*3 with reduced enzyme activity compared to wild type CYP2C9*1. The aim of this study was to assess relationship between requirement of warfarin dose and polymorphism in CYP2C9 in Korean population. Patients on warfarin therapy for longer than 1 year were included from July 1999 to December 2000 and categorized as one of four groups; regular dose non-bleeding, regular dose bleeding, low dose non-bleeding and low dose bleeding. Low dose was defined as less than 10 mg/week for 3 consecutive monthly follow-ups. Bleeding complications included minor and major bleedings. Blood samples were processed for DNA extraction, genotyping and sequencing to detect polymorphism in CYP2C9. Demographic data, warfarin dose per week, prothrombin time (INR), indications and co-morbid diseases were assessed for each group. Total 90 patients on warfarin were evaluated; The low dose group has taken warfarin 7.6 +/- 1.7 mg/week, which was significantly lower than 31.4 +/- 0.9 mg/week in the regular dose group (p<0.0001). The measured INR in the low dose group was similar to that of the regular dose group (2.3 +/- 0.7 vs. 2.3 +/- 0.6, p=0.9). Even though there was a higher possibility of CYP2C9 variation in the low dose group, no polymorphism in CYP2C9 was detected. All patients were homozygous C416 in exon 3 for CYP2C9*2 and A1061 in exon 7 for CYP2C9*3. The DNA sequencing data confirmed the homozygous C416 and A1061 alleles. In conclusion, polymorphism in CYP2C9 is not a critical factor for assessing warfarin dose requirement and risk of bleeding complications in a Korean population.     

Comparison of ascorbic acid and ascorbic acid 2-O-alpha-glucoside on the cytotoxicity and bioavailability to low density cultures of fibroblasts.

Ascorbic acid 2-O-alpha-glucoside (AA-2G) is a stable ascorbate derivative which has vitamin C activity in vivo and in vitro. We studied whether AA-2G exerts a prooxidant action in cultured fibroblasts from chick embryo and human skin, as does ascorbic acid. At concentrations of 0.1-1.0 mM, ascorbic acid markedly reduced the viable cell number of low density cultures within 24 hr, whereas AA-2G had no such effect. The ascorbate cytotoxicity was dependent on the cell density at the time of its addition and it was characteristic of low density cultures. This cytotoxicity was completely prevented by catalase and partially by an Fe3+ ion chelator, desferrioxamine. In the early culture stage at which a morphological change in the fibroblasts began to occur, intracellular ascorbate concentrations in low density cultures after addition of ascorbic acid were much higher than in high density cultures. However, at the same concentrations, AA-2G did not cause an elevation even in low density cultures and it was also effective on collagen synthesis at high and medium densities. These results suggest that the abnormally accumulated ascorbic acid in the cells cultured at low density possibly amplifies the generation of oxygen radicals through the reduction of Fe3+ ions and subsequent oxidative reactions, leading to cell death. Therefore, it is concluded that AA-2G which supplies an adequate amount of ascorbic acid during culture period is a bioavailable ascorbate source without cytotoxicity.     

Presynaptic effects of snake venom toxins which have phospholipase A2 activity (beta-bungarotoxin, taipoxin, crotoxin)

The presynaptic effects of beta-bungarotoxin, crotoxin and taipoxin were studied in the mouse phrenic nerve-diaphragm preparation (27 degrees C). The phospholipase A2 activity, assayed by pH-stat titration, was reduced to 4-10% at 27 degrees C compared with that at 37 degrees C. The late neuromuscular blocking activity was also reduced by more than three fold for all toxins. In contrast, the early biphasic response to the toxins, i.e. immediate depression followed by facilitation, was not delayed. The evoked quantal release of acetylcholine was enhanced by all toxins at low Ca2+-concentrations during the phase of facilitation, without an increase of the maximal release. At the late phase of treatment with beta-bungarotoxin and taipoxin, the curve relating the quantal contents of endplate potentials with Ca2+-concentration was shifted parallel to the right at low Ca2+, but marked depression of the maximal release occurred at high Ca2+. When diaminopyridine was added at the time of the late phase block by beta-bungarotoxin, the quantal release could still be enhanced at low Ca2+-concentrations, even beyond control; however, the maximal release was not simultaneously restored. It is concluded that the late phase block, but not the early biphasic response, is due to an enzymatic action and the release mechanism is abolished when the hydrolysis of membrane phospholipids proceeds to a certain critical level.     

不同浓度吸氧对新生大鼠视网膜影响的研究

目的 观察不同浓度吸氧对新生大鼠视网膜的影响.方法 新生SD大鼠56只,分娩完成后随机分为高氧组24只、低氧组24只、对照组8只,高氧组给予75％浓度的氧气吸入,低氧组给予35％浓度的氧气吸入,对照组于空气中饲养.高氧组与低氧组又随机分为高氧1～3组和低氧1～3组.高氧1、低氧1组于持续吸氧7d后,第8天行视网膜组织病...     

卡培他滨维持性口服治疗宫颈癌的临床研究

目的探讨卡培他滨维持性口服治疗宫颈癌的近期疗效及不良反应。方法将96例宫颈癌患者随机分为高剂量维持组、低剂量维持组及对照组各32例。对照组单纯予以紫杉醇联合顺铂化疗方案治疗。在对照组治疗基础上,高剂量维持组应用卡培他滨1000mg/m2进行维持性治疗,低剂量维持组应用卡培他滨500mg/m2进行维持性治疗。比较3组的临床近期疗效及不良反应。结果高剂量维持组、低剂量维持组及对照组有效率分别为50.0%、46.9%、46.9%,3组间比较差异无统计学意义(P>0.05)。在1年生存率、中位进展时间和中位生存时间方面,均为低剂量维持组>高剂量维持组>对照组(P<0.05)。且低剂量维持组不良反应发生情况优于高剂量维持组,差异有统计学意义(P<0.05)。结论卡培他滨维持性口服治疗晚期宫颈癌疗效佳,且低剂量药物引起的不良反应明显较高剂量轻,临床应用方便,利于长期使用。     

The role of extracellular Ca2+ and Na+ in paf-acether-induced human platelet activation

The dependence of paf-acether (paf)-induced human platelet activation on extracellular Ca2+ and Na+ was examined by quantitating aggregation, secretion and thromboxane (Tx) formation in the presence of physiological and/or low concentrations of Ca2+ and/or Na+. In the presence of 2 mM Ca2+ and 140 mM Na+, paf induced a dose-dependent reversible aggregation and less than 25% of [14C]serotonin release. These responses were insensitive to aspirin or Tx antagonist SQ 29548 treatment and negligible amounts of Tx were formed. In low Ca2+ buffer, paf induced irreversible aggregation and the 14C-serotonin release could exceed 60%. These increases in platelet response were associated with the formation of Tx and were suppressed by aspirin and SQ 29548 treatments, or by substituting NaCl with N-methylglucamine hydrochloride. Thus, in low Ca2+ medium, Tx synthesis is favored during platelet activation and is dependent on Na+ concentrations. A decrease in extracellular Na+ inhibited the paf-acether-induced Tx synthesis observed in low Ca2+ medium but not that induced by the Tx direct precursor, arachidonic acid (AA). Therefore, the increase observed in low Ca2+ medium, no longer seen when the Na+ level is decreased is not related to an impairment of the cyclooxygenase activity but rather implicates an effect on the activity of phospholipase A2. A decrease in extracellular Na+ (2 mM Ca2+ present), inhibited [14C]serotonin release induced by paf from platelets which had, or had not been, treated with aspirin. In this medium, the AA-induced release reaction was also affected whereas Tx formation was not altered, thus suggesting that other mechanisms involved in platelet response apart from Tx synthesis are dependent on extracellular Na+.