二烯丙基二硫诱导人白血病HL-60细胞分化差异表达cDNA文库的构建

目的　应用抑制性消减杂交技术构建二烯丙基二硫(diallyldisulfide ,DADS)诱导人白血病细胞分化的消减杂交cDNA文库 ,以期克隆DADS诱导人白血病HL 6 0细胞分化的相关基因。方法　用DADS诱导人白血病HL 6 0细胞分化 ,提取polyA+ RNA ,反转录合成cDNA ,消化成短片段后分成两组 ,分别与两种不同的接头连接 ,再与未处理的白血病细胞cDNA进行两次消减杂交及两次抑制性PCR扩增 ,将PCR产物与pGEM T线性载体连接 ,转化大肠杆菌进行文库扩增 ,随机挑取克隆进行酶切鉴定。结果　成功地构建了具有高消减效率的DADS诱导白血病细胞分化的cDNA文库 ,随机挑取消 2 0 0个克隆制备质粒并酶切分析 ,其中 84 5 %的克隆均具有 1 0 0～ 6 0 0bp左右的插入片段 ,说明每一克隆中均含有特异性的目的片段 ,从而为大批量筛选、克隆DADS诱导人白血病细胞分化的相关未知新基因奠定了基础。结论　DADS能诱导人白血病HL 6 0细胞分化 ,并引起相关的基因发生改变 ,而抑制性消减杂交技术能有效地分离差异表达的基因。     

Simultaneous qualitative and quantitative analysis of triterpenic acids, saponins and flavonoids in the leaves of two Ziziphus species by HPLC-PDA-MS/ELSD

The leaves of Ziziphus jujuba and Z. jujuba var. spinosa have been utilized as crude drugs for their health benefits in China for thousands of years. To control their quality, a reliable method based on high-performance liquid chromatography coupled with photo diode array and electrospray ionization tandem mass spectrometry detection (HPLC-PDA-ESI-MS/MS) was developed for exploration of the chemical profiles of these jujube leaves. As the results, fourteen constituents including three flavonoids, two saponins and nine triterpenic acids were identified or tentatively characterized. Then, twelve of them such as quercetin-3-O-rutinoside, zizyphus saponins I and II, ceanothic acid, alphitolic acid, maslinic acid, 2α-hydroxyursolic acid, zizyberanalic acid, epiceanothic acid, ceanothenic acid, betulinic acid, and oleanolic acid were selected as the chemical markers and were determined using an HPLC coupled with evaporative light scattering detection (ELSD) method. The separation was carried out on a Waters Sunfire C₁₈ column with 0.2% acetic acid and acetonitrile as the mobile phase under gradient elution. The operating conditions of ELSD were set as 80 °C for drift tube temperature and 2.7 l/min for nitrogen flow rate. The developed method was fully validated in terms of linearity, sensitivity, precision, repeatability as well as recovery, and subsequently applied to evaluate the quality of eight batches of Z. jujuba and Z. jujuba var. spinosa leaves from different collections.     

Four new diarylheptanoids from Rhizoma Zingiberis

Four new diarylheptanoids, (1S, 3R, 5R, 6R)-1, 5-epoxy-3, 6 dihydroxy-1-(4-hydroxy-3, 5-dimethoxyphenyl)-7-(4-hydroxy-3-methoxyphenyl) heptane (1), (1R, 3R, 5S)-1, 5-epoxy-3-acetoxy-1-(4, 5-dihydroxy-3-methoxyphenyl)-7-(3, 4- hydroxyphenyl) heptane (2), (3R, 5S, 6R, 7S)-3, 6-epoxy-7-hydroxyl-1-(4-hydroxyphenyl)-7-(3-methoxy-4-hydroxyphenyl) heptane (3), (E)-3-keto-1-(3-methoxy-4-hydroxyphenyl)-7-(4, 5-dihydroxy-3-methoxyphenyl)-4- heptene (4), were isolated from Rhizoma Zingiberis, and their structures were determined based on HR-ESI-MS and extensive spectroscopic techniques (UV, IR, 1D-NMR and 2D-NMR). Compounds 1–4 exhibited no cytotoxicity against HepG2 cell lines.

     

重组 GST-β-GT 融合蛋白的原核表达、纯化及活性鉴定

目的：构建噬菌体β‐GT蛋白的原核重组体系,诱导表达、纯化GST‐β‐GT 融合蛋白并对其进行酶活性测定。方法利用PCR技术从T4噬菌体中扩增β‐GT基因;经T‐A克隆,获得β‐GT基因片段,经酶切连接,构建原核表达载体pGEX‐6P‐1‐β‐GT ;经测序鉴定序列正确后,将所构建的载体转化进入大肠埃希菌BL21（DE3）中进行表达。利用IPTG诱导,经10％ SDS‐PAGE鉴定目的蛋白表达后,采用GST柱纯化目的蛋白;通过酶切和qPCR鉴定其活性。结果成功扩增了噬菌体β‐GT基因;重组质粒在大肠埃希菌BL21（DE3）中诱导表达出GST‐β‐GT融合蛋白;通过GST柱纯化后获得了GST‐β‐GT融合蛋白,纯度达95％;通过酶切和qPCR验证,此GST‐β‐GT融合蛋白具有T4‐β‐GT酶的活性。结论。原核表达GST‐β‐GT融合蛋白具有糖基转移酶活性。     

脂蛋白脂酶活化剂NO-1886抑制糖尿病兔动脉粥样硬化的形成

目的　合成化合物NO 1886 ,一种脂蛋白脂酶活化观察脂蛋白脂酶活化剂是否降低高脂 /高蔗糖饲料诱发的糖尿病新西兰兔的血浆葡萄糖并减轻其动脉粥样硬化。方法　给予高脂 /高蔗糖饲料升高新西兰兔血浆总胆固醇 ,甘油三酯和葡萄糖及降低高密度脂蛋白胆固醇而导致动脉粥样硬化。饲料中加入 1 0 %NO 1886进行治疗观察。分别在 0、4、8、12、16、2 0及 2 4wk从禁食过夜的兔耳静脉抽取血样测定葡萄糖和脂质水平。第 2 4周末 ,处死动物 ,分离主动脉 ,经苏丹Ⅳ染色固定脂质后 ,计算脂纹病变面积。结果　应用NO 1886后 ,实验动物血浆葡萄糖 ,总胆固醇和甘油三酯降低 ,高密度脂蛋白胆固醇增加。对动脉粥样硬化的发展具有抑制作用。结论　NO 1886不仅可改善脂质紊乱 ,而且可降低血浆葡萄糖 ,减轻糖尿病兔动脉粥样硬化     

葛根黄酮滴丸对离体豚鼠心脏冠脉血流量的影响

目的探讨葛根黄酮滴丸对离体豚鼠心脏冠脉血流量的影响。方法采用Langendorff氏法，给药后记录冠脉流量和心率变化。结果乐氏液1组与葛根黄酮滴丸低剂量（150mg／L）组冠脉流量分别为（5.80±0.75）mL／min和（7．82±0．66）mL／min，两组之间比较有显著性差异（P〈0．05）；乐氏液2组与葛根黄酮滴丸高剂量（250mg／L）组冠脉流量分别为（5．78±0．69）mL／min和（7．95±0．85）mL／min，两组之间比较有显著性差异（P〈0．05）：结论葛根黄酮滴丸使冠脉流量明显增加。     

牡荆素鼠李糖苷的大鼠在体肠吸收动力学

目的 研究牡荆素鼠李糖苷(RHV)的大鼠在体肠吸收动力学特征.方法 采用HPLC法测定RHV在肠循环液中的药物浓度;采用UV法测定肠循环液中酚红浓度;以大鼠原位灌注模型考查RHV的肠吸收动力学情况.结果 RHV 浓度为20、10、5μg·ml-1的吸收速率常数(Ka)分别为0.0416、0.0478、0.0312 h-1;肠循环液pH4、6、8时RHV的Ka分别为0.0253、0.0478、0.0588 h-1;RHV在十二指肠、空肠、回肠和结肠时的Ka分别为0.0479、0.0308、0.0322、0.0305 h-1.结论 RHV 浓度对RHV的Ka无显著性影响;在pH4～8时,随肠循环液pH的增大,RHV的Ka增加;RHV在大鼠十二指肠、空肠、回肠和结肠的吸收无显著性差异(P＞0.05);RHV在大鼠肠道的吸收呈一级动力学过程,吸收机制为被动扩散.     

妇科盆腔手术后下肢深静脉血栓形成的临床研究

目的探讨妇科盆腔手术后下肢深静脉血栓(LEDVT)的发生率、高危因素及早期诊断方法。方法前瞻性比较141例妇科盆腔手术患者LEDVT的发生率、发病特点、高危因素、诊断方法及凝血功能变化,分别于术前1周、术后48h及术后5～7d检测患者血小板计数、凝血酶原时间、活化的部分凝血活酶时间、纤维蛋白原、凝血酶时间、D二聚体、抗凝血酶Ⅲ、组织型纤溶酶原活化物、纤溶酶原活化物抑制剂水平,并行双下肢静脉超声检查。结果(1)术后发生LEDVT22例(血栓组),发生率为15.6%,发生时间为术后2～7d,平均(4.1±1.2)d;术后未发生LEDVT119例(非血栓组)。(2)发生LEDVT的高危因素是术后开始床上活动时间晚、术后常规应用止血药、合并心血管疾病、淋巴清扫术、年龄、全身麻醉、恶性肿瘤等,但多因素分析显示,年龄及术后应用止血药是LEDVT发生的独立影响因素。(3)血栓组术后D二聚体平均为(0.9±0.6)mg/L,非血栓组为(0.5±0.4)mg/L,两组比较,差异有统计学意义(P<0.01);D二聚体用于预测LEDVT的阳性预测值为31.0%,阴性预测值为98.6%;手术后较手术前抗凝血酶Ⅲ有所升高。(4)22例行静脉超声诊断的血栓患者中,有4例行双下肢静脉造影,结果均与超声诊断相符。(5)两组手术前后组织型纤溶酶原活化物、纤溶酶原活化物抑制剂等水平比较,差异无统计学意义(P>0.05)。结论妇科盆腔手术后LEDVT的发生率较高,D二聚体、抗凝血酶Ⅲ水平检测结合静脉超声检查,可作为LEDVT的早期筛查手段。建议对有LEDVT高危因素的患者,手术后2～7d常规筛查静脉血栓,争取早发现、早治疗。     

Computed tomography and magnetic resonance imaging characteristics of giant pituitary adenomas.

PURPOSE: The purpose of this study was to characterize the computed tomography (CT) and magnetic resonance (MR) imaging features of giant pituitary adenoma (GPA) and to demonstrate the pathways of the spread of GPA. METHODS: CT and/or MR imaging scans of 356 patients evaluated preoperatively for single pituitary tumor were reviewed. Fourteen tumors (4%) fulfilled the radiologic criteria for GPA. There were 10 male and four female patients, ranging in age from 22 to 71 years (mean, 52 yr). We retrospectively reviewed the CT and MR imaging scans of these patients to characterize tumor appearances and identify the pathways of tumor extension. RESULTS: Thirteen tumors (93%) extended upward to the suprasellar cistern, and/or hypothalamus and third ventricle. Infrasellar extension through the sellar floor and sphenoid sinus to the skull base, or to the ethmoid sinus or the nasopharynx, was identified in seven patients (50%). Eight patients (57%) had lateral invasion to the cavernous sinus. Temporal and frontal extension was apparent in seven patients (50%) and six patients (43%), respectively. Five patients (36%) had posterior subtentorial extension to the posterior fossa. Histologically, only two GPAs showed invasive features. There was no correlation among histologic features, pituitary hormone concentrations, and evidence of tumor aggressiveness on CT and MRI scans. CONCLUSIONS: GPA has the potential for widespread, multi-directional extension. Our results indicate that any type of pituitary adenoma, regardless of its endocrinologic activity, may invade surrounding structures. Suprasellar invasion is the most common pathway of tumor spread, followed by infrasellar, lateral, anterior, and posterior routes.