高效液相色谱法测定人红细胞中巯基嘌呤甲基转移酶的活性

目的:建立一种改良的高效液相色谱法测定人红细胞中巯基嘌呤甲基转移酶(thiopurinemethyltransferase,TPMT)的活性。方法:采用反相高效液相色谱法直接测定酶促反应的产物6-甲基巯基嘌呤的浓度,从而计算红细胞中TPMT的活性。以6-巯基嘌呤为底物,S-腺苷-L-甲硫氨酸作为甲基供体,红细胞裂解液在一定的条件下进行孵化,然后经乙酸乙酯萃取处理后,用HPLC分析。色谱柱为hypersilBDSC18,流动相为水-甲醇-三乙胺(磷酸调至pH3.5)(76.8∶23∶0.2),紫外检测波长为290nm。结果:6-甲基巯基嘌呤在10～200μg/L范围内呈良好的线性关系,相关系数r=0.9994,最低检测限为3μg/L(S/N=3),平均回收率为86%～94%。我们运用该法测定了273例健康中国成年人红细胞中TPMT的活性,其平均活性为(11.96±3.27)nmol/h·mlPackedRBC,男性和女性的平均TPMT活性分别为(12.08±3.38)nmol/h·mlPackedRBC和(11.73±3.07)nmol/h·mlPackedRBC,两者无显著性差异。结论:本分析方法具有灵敏准确,重复性好,安全可靠,易于推广等特点,能满足巯基嘌呤类药物药代动力学研究和临床用药监测的要求。     

葛根素注射液细菌内毒素检查方法的研究

目的:考察葛根素注射液细菌内毒素检查方法的可行性.方法:依据<中国药品检验标准操作规程>2000年版细菌内毒素检查法,确定葛根素注射液的有效稀释浓度和细菌内毒素限值.结果:葛根素注射液在最大有效稀释浓度下对鲎试剂无干扰作用,其内毒素限值符合规定.结论:可用细菌内毒素检查法取代<中国药典>2000年版(二部)规定的热原检查法.     

葡萄糖氯化钠注射液中细菌内毒素定量检测研究

①目的 探讨葡萄糖氯化钠注射液中细菌内毒素的定量检测方法。②方法 按中国(2000年版)附录中检测细菌内毒素的动态浊度法，进行葡萄糖氯化钠注射液的细菌内毒素干扰实验。③结果 葡萄糖氯化钠注射液对细菌内毒素定量检测无干扰性。④结论 应用细菌内毒素定量检测法检测葡萄糖氯化钠注射液中细菌内毒素是可行的。     

Cell-free protein synthesis inhibition assay for the cyanobacterial toxin cylindrospermopsin

The cyanobacterial toxin cylindrospermopsin (CYN) is known to be a potent inhibitor of protein synthesis. This paper describes the use of a rabbit reticulocyte lysate translation system as a protein synthesis inhibition assay for CYN. A dose response curve for protein synthesis inhibition by CYN was constructed and was modeled to a sigmoidal dose response curve with variable slope (R2 = 0.98). In this assay, CYN has an IC50 of 120 nM [95% confidence limits (Cl) = 111-130 nM] with a detection limit in the region of 50 nM in the assay solution. Application of the assay allows quantification of toxin samples within the range 0.5-3.0 microM (200-1200 micrograms/L) CYN. To assess the usefulness of this assay, a range of toxic and nontoxic Cylindrospermopsis raciborskii extracts, including both laboratory strains and environmental samples, were assayed by protein synthesis inhibition. These CYN quantifications were then compared to quantifications obtained by high performance liquid chromatography (HPLC) and HPLC-tandem mass spectrometry (HPLCMS-MS). The results demonstrate that the protein synthesis inhibition assay correlates well with both HPLCMS-MS (r2 = 0.99) and HPLC (r2 = 0.97) quantifications. We conclude that this is an accurate and rapid assay for the measurement of cylindrospermopsin in cyanobacterial extracts.     

特异性鲎试剂检测人参多糖注射液中的细菌内毒素

目的：建立快速的人参多糖注射液细菌内毒素检查法，方法：用特异性鲎试剂对不同批号的人参多糖注射液分别进行干扰试验，考察确立人参多糖注射液内毒素检查法。结果：将人参多糖注射液稀释25倍利用特异性鲎试剂检测可消除干扰作用，结果准确可靠，结论；选用特异性鲎试剂，细菌内毒素检查法代替人参多糖注射热原检查是可行的。     

Conjugated linoleic acid alters caveolae phospholipid fatty acid composition and decreases caveolin-1 expression in MCF-7 breast cancer cells

Conjugated linoleic acid (CLA) refers to a group of biologically active fatty acids that exhibit anticarcinogenic properties; however, the mechanism of action remains poorly understood. Caveolae are specialized plasma membrane structures that affect many facets of cancer cell function, including growth, cell signaling, and apoptosis. Therefore, one potential mechanism could be alteration of caveolae lipid composition and function. We hypothesized that CLA can alter the lipid microenvironment of caveolae and alter expression of the major caveolae-resident protein, caveolin-1. MCF-7 human breast cancer cells were treated with a vehicle control, linoleic acid (LA), or CLA for 3 days after which total cell lysate, plasma membrane, and caveolae membrane fractions were isolated. Our findings indicate that CLA readily incorporates into caveolae (Δ9cis,11trans-18:2 being the major isomer) and maybe preferentially enriched in specific phospholipid species. Furthermore, caveolin-1 localization to caveolae after treatment with CLA was decreased relative to either control- or LA-treated cells, without changes in total cellular levels of protein relative to vehicle-control treated cells. Taken together, our results suggest that further investigation of a potential therapeutic role for CLA in modulating caveolae function in breast cancer is merited.     

注射用泮托拉唑钠细菌内毒素检查法的研究

目的研究注射用泮托拉唑钠的临床用药安全，保证其质量，建立注射用泮托拉唑钠细菌内毒素检查（BET）方法。方法按《中国药典》2005年版附录细菌内毒素检查法要求进行试验。结果注射用泮托拉唑钠稀释为0.4mg·ml^-1的浓度，采用灵敏度0.25EU·ml^-1的鲎试剂经干扰实验无干扰作用。结论本试验具有准确性、可靠性，细菌内毒素检查法适用于检测注射用泮托拉唑钠中的内毒素。     

Are adipose‐derived stem cells cultivated in human platelet lysate suitable for heart valve tissue engineering?

Tissue‐engineered heart valves represent a promising strategy for the growing need for valve replacements in cardiovascular medicine. Recent studies have shown that adipose‐derived stem cells (ADSC) are a viable cell source, as they are readily available in both the young and the elderly, show diverse differentiation potential and adapt their extracellular matrix (ECM) to a varying mechanical load. In vitro culture medium is usually enriched with fetal calf serum (FCS). However, a promising substitute has recently been found in human platelet lysate (HPL), which is superior in terms of proliferation speed and allogenicity. This study sought to elucidate the suitability of ADSC and HPL for heart valve tissue engineering (TE). ADSC harvested from five healthy individuals were cultured in both FCS and HPL. The cells were observed for differentiation potential, proliferation speed and immunophenotype, using immunohistochemistry and FACS analysis. Neotissue was assessed for ECM composition, human collagen I (hColl1) formation, histomorphology and mechanical stiffness under uniaxial tensile stress. Neotissue cultured in HPL was found to be significantly inferior in mechanical rigidity; it showed a three‐fold higher proliferation rate and a more dense ECM, but also a more heterogeneous hColl1 distribution. ECM analysis showed significantly higher amounts of DNA and glycosaminoglycans (GAG) in HPL‐cultured tissue. No significant differences were observed for differentiation potential and immunophenotype, apart from a lower CD166 expression in HPL. The mechanical inferiority of neotissue cultured in HPL represents a limitation to the use of HPL‐enriched media for heart valve TE with ADSC. This result concurs with data published about HPL and myofibroblasts derived from the venous wall. Similarly, the mechanical inferiority is not rooted in a difference in ECM composition, but rather in hColl1 architecture. Stem cell properties, as documented in the literature, are retained with HPL. A possible connection between the mechanical inferiority and the observed decrease in CD166 needs further investigation. Copyright © 2016 John Wiley & Sons, Ltd.     

动态浊度法检测5种中药注射液的内毒素含量

目的:通过特异性及非特异性鲎试剂(TAL)动态浊度法对5种中药注射液的内毒素含量进行检测,探讨该方法对中药注射液检测的适用性。方法:采用特异性及非特异性动态浊度试剂盒,对不同批次的鱼腥草注射液、双黄连注射液、清开灵注射液、葛根素注射液、香丹注射液进行检测。结果:无色的中药注射剂如鱼腥草注射剂、葛根素注射剂对检测干扰较少,非特异性及特异性动态比浊法对其无显著影响;对非单一成分,颜色较深的中药注射液,如清开灵、香丹、双黄连注射液,采用动态比浊法进行检测,干扰大,检测结果不稳定;清开灵注射剂采用特异性试剂盒进行检测能有效的消除干扰,但香丹、双黄连注射液仍不能消除干扰。结论:用特异性或非特异性动态浊度法可以检测鱼腥草和葛根素注射液中的内毒素含量,采用特异性动态浊度法可以检测清开灵注射液中内毒素含量,双黄连和香丹注射液的检测结果不稳定。     

乳酸依沙吖啶溶液(冲洗剂)细菌内毒素的检测

目的:建立乳酸依沙吖啶溶液（冲洗剂）细菌内毒素的检测方法。方法:按照《中国药典》2010年版（二部）附录ⅪE和附录ⅠS对5批乳酸依沙吖啶溶液进行干扰试验和细菌内毒素检查。结果:质量浓度为1 mg·ml^-1的乳酸依沙吖啶溶液对鲎试剂与细菌内毒素之间的凝集反应无干扰作用,5个批号的样品细菌内毒素检查均符合规定。结论:建立的鲎试剂方法可用于乳酸依沙吖啶溶液细菌内毒素的检查,方法具有简便、快捷、可靠等优点。