银杏内酯B注射液在生理盐水中的稳定性的研究

目的:采用高效液相法对银杏内酯B注射液在生理盐水中的稳定性进行研究,又采用HPLC-MS法对银杏内酯B注射液在生理盐水中经加热后所生成的降解产物进行研究。方法:高效液相条件为:色谱柱:Venusil XBP-C18(5μm,4.6×250mm),柱温:25℃,流动相∶甲醇∶水(40∶60),流速:1.0mL/min,检测波长:220nm。结果:在上述色谱条件下,本品在0.1~2mg/mL范围呈线性关系,回归方程为:Y=313.95X+0.8054,r=0.9999;精密度良好,RSD:0.86%;主峰与其它杂质峰分离良好。结论:银杏内酯B注射液在生理盐水中至少可放置3个月。     

HPLC-ELSD法测定银杏内酯B注射剂的含量及有关物质

目的:使用HPLC-ELSD法测定银杏内酯B的含量及有关物质。方法:使用蒸发光散射检测器,采用色谱柱为ZorbaxSB-C18柱(4.6×150mm,5μm,安捷伦公司),流动相为四氢呋喃-甲醇-水(10:25:75),流速为1.0mL.min-1。ELSD漂移管温度105℃,气体流速:2.9L.min-1。结果:HPLC-ELSD法测定在1μg-100μg的范围内呈良好线性关系。r=0.9998,最低检测限为0.05μg,本方法的重复性和精密度良好(RSD<2%)。结论:采用HPLC-ELSD法测定银杏内酯B注射剂的含量及有关物质,方法简便,结果准确。     

液相色谱-串联质谱法测定人血浆中银杏内酯B的含量

建立测定人血浆中银杏内酯B的含量的LC-MS/MS方法。取正常人血浆50μL,加入2.5mol/L盐酸溶液10μL酸化,再加入内标2μg/mL银杏内酯C20μL,以1mL乙醚萃取后,取提取液500μL挥干溶剂,然后用30%的乙腈500μL溶解,取10μL进行LC-MS/MS测定。LC条件:色谱柱为ZorbaxC18柱(4.5*150mm,5μm);流动相:乙腈-水梯度洗脱;流速:0.6mL/min,柱温:35℃。质谱条件:ESI电离源;负离子模式;用于定量分析的离子对分别为m/z423.1→m/z367.2(银杏内酯B)和m/z439.1→383.2(内标:银杏内酯C)。该方法血浆中银杏内酯B的最低定量限为5ng,线性范围为5ng/mL~0.5μg/mL。本方法操作简便,灵敏度高,结果准确可靠,可用于该药物的含量测定,同时也可为临床药代动力学研究提供参考。     

用激光扫描共聚焦显微镜原位检测细胞凋亡

用激光扫描共聚焦显微镜在原位检测细胞凋亡具有多方面的优势。本文介绍用该仪器原位检测Annexin—Ⅴ试验样品、TUNEL试验样品及进行细胞凋亡形态学观察的过程和方法。     

HPLC-ELSD法测定舒血宁注射液配伍溶液中内酯含量

目的:建立高效液相色谱-蒸发光散射检测法(HPLC-ELSD)同时测定舒血宁注射液中银杏内酯A、B、C的含量。结果:舒血宁注射液中银杏内酯A进样量在2～32μL范围内线性关系良好,r=0.9998;银杏内酯B进样量在2～32μL范围内线性关系良好,r=1;银杏内酯C进样量在2～32μL范围内线性关系良好,r=0.9996。方法重复性良好。银杏内酯A、B、C平均回收率分别为97.08%、102.06%、101.6%;RSD分别为2.53%、2.91%、2.25%。结论:该法专属性强,灵敏、准确,可作为舒血宁注射液配伍溶液中内酯含量的检测方法。     

新型荧光液池的设计及其在凋亡细胞核酸检测中的应用

设计并制作了可容纳塑料离心管的新型荧光液池。该液池适用的样品体积变化宽（１０－５０μＬ）,可用于大批量样品分析,对生物和临床样品的测定尤为合适,该液池用于凋亡细胞核酸样品的检测,取得了满意的结果。     

D-柠檬烯对人胃癌MGC803细胞增殖和凋亡的影响

目的研究D-柠檬烯对人胃癌MGC803细胞生长抑制和凋亡的影响。方法噻唑蓝（MTT）法比色检测细胞生长情况;流式细胞仪检测细胞凋亡率;激光共聚焦检测细胞内活性氧（reactive oxygen species,ROS）的含量和caspase-3蛋白的表达。结果D-柠檬烯对人胃癌MGC803细胞生长抑制作用呈剂量依赖关系;流式细胞仪检测发现D-柠檬烯可引起MGC803细胞凋亡,且呈时间依赖性;激光共聚焦显微镜荧光检测发现D-柠檬烯处理的细胞内ROS明显升高（P〈0．05）,caspase-3蛋白表达明显增加（P〈0．05）。结论D-柠檬烯能够有效地抑制癌细胞的生长以及诱导凋亡,这种作用机制可能是使MGC803细胞内产生大量的活性氧,也可能与细胞内caspase-3基因的表达变化有关。     

银杏达莫联合奥扎格雷对缺血性脑卒中血液流变影响及疗效观察

目的:探讨银杏达莫联合奥扎格雷对缺血性脑卒中血液流变影响及临床疗效。方法:选取本院2011年9月²013年4月诊治的缺血性脑卒中患者172例,根据治疗方案分为两组,各86例,观察组采用银杏达莫联合奥扎格雷注射液治疗,对照组采用银杏达莫注射液治疗,疗程2周。比较两组患者治疗前后神经功能缺损评分、血流动力学指标变化及不良反应。结果:治疗后,两组患者的神经功能缺损评分、红细胞积压、血浆黏度、纤维蛋白原、血小板凝集指数均显著降低。观察组上述指标显著低于对照组,差异均有统计学意义(P<0.05)。观察组患者治疗总有效率高于对照组,差异均无统计学意义(P>0.05)。结论:银杏达莫联合奥扎格雷注射液可明显改善患者的神经功能缺损状况和血流动力学指标,疗效肯定。     

应用多参数技术方法对脑缺血皮质区神经细胞凋亡的检测

随着生物科学研究技术的不断提高和对细胞凋亡认识的不断深入,在细胞凋亡确认过程中,若单独使用一种方法检测会出现一定概率的假阳性,往往需要采用多参数技术方法相互印证。本实验结合透射电镜、流式细胞仪和共聚焦显微镜的检测技术及原理,通过多技术相结合观察判断了急性脑缺血皮质神经元细胞凋亡,并进行了定性、定量分析。     

高温对体外培养海马神经元细胞的影响及细胞凋亡机制研究

目的 :研究不同温度对体外培养海马神经元细胞的影响及其机制。方法 :取出生1~3天的SD幼鼠海马神经细胞体外培养,分为37℃和42℃2组,分别孵育12h和24h。利用CCK8、TUNEL分析、Western blot等技术,观察12h和24h时海马神经细胞存活率及凋亡细胞数,以及Bcl-2、Bax和cleaved-caspase3蛋白表达。结果 :与37℃组相比、42℃组海马神经元细胞存活率下降,凋亡阳性细胞数明显增加,并随着时间延长凋亡阳性细胞数增多。42℃组中,神经元细胞Bcl-2表达减弱,Bax、cleaved-caspase3表达增强,上述指标与37℃组同时点比较,差异均有统计学意义(P<0.05)。结论 :高温可以诱导体外培养海马神经元细胞凋亡,可能通过PI3K/Akt信号传导通路诱导神经细胞凋亡。     

Dihydroartemisinin enhances cell apoptosis in diffuse large B cell lymphoma by inhibiting the STAT3 activity

Background: Dihydroartemisinin (DHA) is reported to be a potential anticancer agent, and the mechanisms underlying the effects of DHA on diffuse large B cell lymphoma however are still obscure. This study aimed to assess the antitumor effect of DHA on diffuse large B cell lymphoma cells and to determine the potential underlying mechanisms of DHA-induced cell apoptosis. Methods: Here, the Cell Counting Kit 8 assay was conducted to study cell proliferation. We performed Annexin V-FITC/propidium iodide staining, real-time polymerase chain reaction, and western blot analysis to analyze cell apoptosis and potential molecular mechanisms. Results: The results showed that DHA substantially suppressed cell proliferation and induced cell apoptosis in vitro in a time- and concentration-dependent fashion. Moreover, STAT3 activity could be inhibited after stimulation with DHA. Conclusion: These results imply that the underlying anti-tumoral effect of DHA may increase apoptosis in diffuse large B cell lymphoma cells via the STAT3 signaling pathway. In addition, DHA might be an effective drug for diffuse large B cell lymphoma therapy.     

The role of baicalin on carbon tetrachloride induced liver fibrosis

The effect of the baicalin, a bio-active flavonoid extracted from Scutellaria baicalensis Georgi, on the carbon tetrachloride (CCl₄) induced liver fibrosis was investigated. To compare the effect of baicalin on the liver fibrosis, five different groups of rats treated by 100, 200, and 400 mg/kg baicalin were studied. Upon CCl₄ treatment, the levels of procollagen type III, aspartate aminotransferase, aminotransferase, hyaluronic acid, and hydroxyproline were significantly increased, whereas the superoxide dismutase and glutathione peroxidase content were decreased. These changes in the biochemical parameters, which are associated with liver function, were significantly attenuated by the baicalin treatment, suggesting that baicalin can suppress the liver fibrosis induced by CCl₄. Moreover, the histological staining analysis demonstrated that baicalin could effectively inhibit the degree of liver cell injury. The protein expression of AKT/JAK2/ERK in the serum were markedly increased by CCl₄ but suppressed by the treatment of baicalin in a dose-dependent manner, implying that baicalin can attenuated cell apoptosis induced by CCl₄. Overall, these results suggest that baicalin effectively protects hepatocytes from the CCl₄ oxidative damage, likely due to the inhibition of free radical generation and cell apoptosis during the liver injury.     

Vitamin B3 inhibits apoptosis and promotes autophagy of islet β cells under high glucose stress

Background: Hyperglycemia is a typical symptom of diabetes. High glucose induces apoptosis of islet β cells. While autophagy functions in cytoprotection and autophagic cell death. The interaction between autophagy and apoptosis is important in the modulation of the function of islet β cells. Vitamin B3 can induce autophagy and inhibit islet β apoptosis.Method: The mechanism of vitamin B3-mediated protective effect on the function of islet β cells was explored by the method of western blot, immunofluorescence and flow cytometry.Results: In the present study, high glucose stress increased the apoptosis rate, while vitamin B3 reduced the apoptosis rate. The effect of vitamin B3 on autophagy flux under normal and high glucose stress was also investigated. Vitamin B3 increased the number of autophagosomes and increased the light chain (LC)3-II/LC3-I ratio. In contrast, vitamin B3 decreased sequestosome 1 (SQSTM1)/p62 protein expression and inhibited the phosphorylation of mammalian ribosomal protein S6 kinase β-1 (p70S6K/S6K1), which was a substrate of mammalian target of rapamycin (mTOR) under normal and high glucose stress. To further verify the protective effect of vitamin B3 on apoptosis, we treated islet β cell RIN-m5F with autophagy inhibitor 3-methyladenine (3-MA). Vitamin B3 decreased the apoptosis rate under high glucose stress, while the inhibition of apoptosis by vitamin B3 was blocked after adding 3-MA.Conclusion: Our data suggested that vitamin B3 reduced the apoptosis rate of β cells, possibly through inducing autophagy under high glucose stress.     

Effects of substrate texture and curvature on the morphology of cultured cells

The ultrastructural characteristics of several growth matrices were examined using two cell types chosen for their distinct growth habits. Chinese hamster ovary cells and Balb-c 3T3 mouse fibroblasts were grown on flat substrates (glass, tissue culture plastic, Millipore filters) as well as spherical (glass, tissue culture plastic, cross-linked dextran) substrates. Cells were plated maintaining equal densities and growth surface area. Once the majority of the cells reached confluency, the cell's morphology on each matrix was examined using scanning electron microscopy. Digital analysis was performed on cell attachment area to compare the effect of each matrix on cell spreading. Variation in cell shape was dramatic between matrices, being most noticeable between a textured surface (filter, dextran bead) and that of a smooth (glass) surface. Even within smooth surfaces, some variation was observed. There was also an effect of matrix curvature on cell attachment area, the greatest being in the 3T3-c Balb cells, causing an overall decrease in the area of attachment between cell and matrix. The changes seen could also be related to the particular cell type used. Hamster ovary cells tended to be cylindrical and showed little effect between matrices, whereas the mouse fibroblasts, which are more flattened, showed the matrix effect to a greater degree. This study demonstrates the necessity of being aware of substrate-induced cell changes in tissue culture, where some variation in cell shape may be due to the surface on which the cells are grown as opposed to the experimental procedure.     

An in vitro study to explore the role of prolylcarboxypeptidase in non-small cell lung cancer

Prolylcarboxypeptidase (PRCP) belongs to the S28 family of proteases, which is also a dipeptidyl peptidase.In this study, we demonstrate the expression pattern of PRCP in Non-small cell lung cancer (NSCLC). We found thatthe repression of PRCP expression by small interfering RNA successfully inhibited cell proliferation, migration, andinvasion. Further, we explored the involvement of PRCP in the regulation of epithelial-mesenchymal transition (EMT).The epithelial marker E-cadherin was significantly increased, meanwhile mesenchymal markers MUC1, vimentin, andSNAIL were markedly decreased in PRCP knockdown cells. Moreover, the downregulation of PRCP in the NSCLCcells induced the expression of apoptosis-related proteins in vitro. We performed RT-PCR in 30 pairs of clinical NSCLCtissues and adjacent non-cancerous tissues, which revealed significantly higher PRCP expression levels in cancer tissuesthan in adjacent non-cancerous tissues. Collectively the results from our study suggest a possible cancer promotion roleof PRCP in NSCLC.