鞭类药材电泳及显微鉴定研究

鞭类药材即动物外肾，为中医临床常用补肾壮阳药，传统应用品种主要有鹿鞭、牛鞭、驴鞭、狗鞭等。但目前由于货源紧缺，品种混乱情况时有发生。为此我们将常见的鞭类药材进行了蛋白电泳比较分析并对粉末的显微特征进行了测量，以期寻找具有鉴别意义的特征性谱带和显微量化规律，为品种鉴定提供依据。     

布鲁氏菌强毒株16M和疫苗株M5-90诱导巨噬细胞凋亡线粒体途径相关因子的表达比较

目的比较布鲁氏菌强毒株16M和疫苗株M5-90侵染小鼠巨噬细胞RAW264.7后其凋亡相关因子的转录和表达。方法建立16M、M5-90侵染RAW264.7模型,采用CFU计数法比较16M、M5-90侵染RAW264.7 4、8、12、24h后的胞内生存情况;采用qRT-PCR检测AIF、Bcl-2、Bax、Apaf-1、Bcl-xl基因转录水平的变化;采用ELISA检测TNF-α和Cyt C在细胞内的表达;采用激光共聚焦检测Cyt C在细胞内共定位表达情况。结果布鲁氏菌16M在侵染后4h~24h胞内CFU呈增多趋势,而M5-90CFU先增多后减少。qRT-PCR显示AIF基因的转录水平在侵染后4h~24h内呈上升趋势,且M5-90侵染组与16M侵染组比较差异无统计学意义(P0.05);由M5-90侵染诱导的Apaf-1基因转录水平与16M侵染组比较有统计学意义(P0.05);Bcl-xl的转录量随着侵染时间增长而不断增加,且16M诱导组与M5-90组比较差异无统计学意义(P0.05);M5-90侵染组Bax和Bcl-2水平与16M组比较差异均有统计学意义(P均0.05);M5-90侵染组Bax/Bcl-2比值与16M侵染组比较差异无统计学意义(P0.05)。ELISA分析显示TNF-α、Cyt C分泌量随着时间增长而增加,且M5-90诱导组与16M组比较差异无统计学意义(P均0.05)。激光共聚焦显示Cyt C定位在RAW264.7内,属于胞浆表达,且M5-90诱导的表达量与16M组比较差异无统计学意义(P0.05),并随感染时间的延长不断增加。结论布鲁氏菌疫苗株M5-90侵染RAW264.7 4h~24h内,凋亡相关因子Cyt C、AIF、Bax/Bcl-2、Apaf-1的表达量高于强毒株16M侵染组,而Bcl-xl则相反,表明在此侵染阶段M5-90具有更强的促细胞凋亡作用。     

布鲁氏菌抗体胶体金免疫层析法快速检测技术的建立

目的建立一种快速检测布鲁氏菌抗体的新方法。方法利用胶体金免疫层析技术,以大肠埃希菌表达、纯化的OMP31与BP26重组蛋白作为检测抗原,以金黄葡萄球菌A蛋白(SPA)作为胶体金标记物,制备胶体金免疫层析试纸条,用于检测布鲁氏菌抗体,并分析方法的敏感性和特异性。结果以粒径为40nm胶体金制备的试纸条检测布鲁氏菌抗体的敏感性最高,胶体金最佳标记pH为6.2,SPA蛋白最适标记量为6μg/ml。交叉试验证明试纸条不与其他非相关疾病感染血清反应,特异性高。试纸条检测结果与琥红平板试验方法的符合率为92%。结论制备的布鲁氏菌抗体胶体金免疫层析试纸条具有敏感、特异、简便、快速的特点,可用于鉴别布鲁氏菌自然感染和人工免疫,并可区分牛、羊种布鲁氏菌感染,可在基层推广使用。     

加巴喷丁胶囊联合小计量泼尼松对带状疱疹神经痛患者免疫功能及疼痛物质的影响

目的 探讨加巴喷丁胶囊联合小剂量泼尼松治疗对带状疱疹患者清神经肽Y（NPY）、P物质（SP）及T细胞亚群的影响及近期临床疗效。方法 选择海南省人民医院2015年1月-2016年12月期间收治的带状疱疹患者128例,随机数表法分为对照组与观察组,每组各64例。其中对照组患者给予小剂量泼尼松治疗,观察组患者给予加巴喷丁胶囊联合小剂量泼尼松治疗。比较两组患者近期疗效,治疗前后患者视觉模拟尺评分（VAS）、T淋巴细胞亚群变化情况（CD3⁺和CD4⁺、CD8⁺、CD4⁺/CD8⁺）及NPY和SP水平。结果 经不同方案治疗后,观察组的总有效率为95.31%,对照组总有效率为85.94%,两组相比差异显著且具有统计意义（χ²=8.67,P < 0.05）。与治疗前相比,两组患者治疗后的VAS评分显著降低,同组治疗前后比较差异有统计学意义（P < 0.05）;治疗后,观察组VAS评分显著低于对照组,差异有统计学意义（P < 0.05）。与治疗前相比,两组患者治疗后T淋巴细胞亚群CD3⁺和CD4⁺、CD4⁺/CD8⁺明显增高,同组治疗前后比较差异有统计学意义（P < 0.05）;观察组治疗后T淋巴细胞亚群CD3⁺和CD4⁺、CD4⁺/CD8⁺较对照组增加更显著,差异有统计学意义（P < 0.05）。两组NPY和SP水平较治疗前均显著下降,同组治疗前后比较差异有统计学意义（P < 0.05）;治疗后,观察组NPY和SP水平均显著低于对照组,差异有统计学意义（P < 0.05）。结论 加巴喷丁胶囊联合小剂量泼尼松治疗对带状疱疹患者能够有效改善患者临床症状,缓轻患者的神经疼痛,改善患者机体免疫功能功能,降低外周血NPY、SP水平,值得临床推广和应用。     

灯笼草的生药鉴别研究

对灯笼草药材进行生药学研究，分别从基源鉴别、性状鉴别、组织构造、粉末特征等方面进行研究，为更好的开发应用、药材鉴定奠定了基础。     

HPLC测定长形肉豆蔻及其不同炮制品挥发油中甲基丁香酚、异甲基丁香酚及黄樟醚含量

长形肉豆蔻为肉豆蔻科植物长形肉豆蔻Myristica argent-ea.Warb.的干燥种仁.同肉豆蔻具有相似的功效,温中行气,涩肠止泻.用于脾胃虚寒,久泻不止,脘腹胀痛,食少呕吐.在印度尼西亚市场上圆形的肉豆蔻(Myristica fragrans Houtt.)常作香料,而长形则作为药用,并且长形肉豆蔻在性状上与肉豆蔻较为相近,其主要成分为挥发油和脂肪油等,据文献报道止泻成分为甲基丁香酚和异甲基丁香酚,毒性成分为黄樟醚 [1,2]故本试验采用高效液相色谱法以甲基丁香酚,异甲基丁香酚、黄樟醚为指标,测定长形肉豆蔻及其炮制品中这3种成分的含量变化,方法简便、灵敏、准确,为建立科学的长形肉豆蔻及其炮制品的质量标准及炮制原理解析提供了依据.     

基于慢病毒的SHARP-2特异的RNA干扰延长肾移植受者大鼠存活时间

Objective To investigate if rat enhancer of split- and hairy-related protein-2 (SHARP-2) short hairpin RNA interference (shRNAi) prolongs the survival time of rat kidney transplant recipients. Methods Gene recombinant procedures, transfection and co-transfection were carried out to introduce short hairpin RNA interference sequences target for SHARP-2 into 3rd generation self-inactivated lentiviral-ViraPower packaging mix. Limiting dilution method was used for viral titration. Real-time PCR was employed for quantification of gene expression. Rat kidney transplantation was utilized to investigate the effect of SHARP-2 gene silence on the recipient survival. Results A lentiviral-based shRNAi construct LV-SHARP-2iC showed 84% SHARP-2 gene silence efficiency in normal rat kidney cells. At multiplicity of infection 20, 57% T cells could be transfected by lentivirus with spinoculation method. In activated T cells, SHARP-2 g ene silence resulted in 61.3% and 68.7% reduction of intedeukin 2 (IL-2) and interferon γ (IFN-γ) gene expression. When donor kidney was perfused with 5×107 TU LV-SHARP-2iC, the median survival time prolonged for 4-5 days as compared to blank and scramble control groups. Conclusions A recombinant lentivirus LV-SHARP-2iC that effectively silence SHARP-2 gene expression is constructed successfully, leading to the inhibition of IL-2 and IFN-γ. LV-SHARP-2iC treatment can prolong the survival time of rat kidney transplant recipients.     

基于慢病毒的SHARP-2特异的RNA干扰延长肾移植受者大鼠存活时间

Objective To investigate if rat enhancer of split- and hairy-related protein-2 (SHARP-2) short hairpin RNA interference (shRNAi) prolongs the survival time of rat kidney transplant recipients. Methods Gene recombinant procedures, transfection and co-transfection were carried out to introduce short hairpin RNA interference sequences target for SHARP-2 into 3rd generation self-inactivated lentiviral-ViraPower packaging mix. Limiting dilution method was used for viral titration. Real-time PCR was employed for quantification of gene expression. Rat kidney transplantation was utilized to investigate the effect of SHARP-2 gene silence on the recipient survival. Results A lentiviral-based shRNAi construct LV-SHARP-2iC showed 84% SHARP-2 gene silence efficiency in normal rat kidney cells. At multiplicity of infection 20, 57% T cells could be transfected by lentivirus with spinoculation method. In activated T cells, SHARP-2 g ene silence resulted in 61.3% and 68.7% reduction of intedeukin 2 (IL-2) and interferon γ (IFN-γ) gene expression. When donor kidney was perfused with 5×107 TU LV-SHARP-2iC, the median survival time prolonged for 4-5 days as compared to blank and scramble control groups. Conclusions A recombinant lentivirus LV-SHARP-2iC that effectively silence SHARP-2 gene expression is constructed successfully, leading to the inhibition of IL-2 and IFN-γ. LV-SHARP-2iC treatment can prolong the survival time of rat kidney transplant recipients.     

基于慢病毒的SHARP-2特异的RNA干扰延长肾移植受者大鼠存活时间

Objective To investigate if rat enhancer of split- and hairy-related protein-2 (SHARP-2) short hairpin RNA interference (shRNAi) prolongs the survival time of rat kidney transplant recipients. Methods Gene recombinant procedures, transfection and co-transfection were carried out to introduce short hairpin RNA interference sequences target for SHARP-2 into 3rd generation self-inactivated lentiviral-ViraPower packaging mix. Limiting dilution method was used for viral titration. Real-time PCR was employed for quantification of gene expression. Rat kidney transplantation was utilized to investigate the effect of SHARP-2 gene silence on the recipient survival. Results A lentiviral-based shRNAi construct LV-SHARP-2iC showed 84% SHARP-2 gene silence efficiency in normal rat kidney cells. At multiplicity of infection 20, 57% T cells could be transfected by lentivirus with spinoculation method. In activated T cells, SHARP-2 g ene silence resulted in 61.3% and 68.7% reduction of intedeukin 2 (IL-2) and interferon γ (IFN-γ) gene expression. When donor kidney was perfused with 5×107 TU LV-SHARP-2iC, the median survival time prolonged for 4-5 days as compared to blank and scramble control groups. Conclusions A recombinant lentivirus LV-SHARP-2iC that effectively silence SHARP-2 gene expression is constructed successfully, leading to the inhibition of IL-2 and IFN-γ. LV-SHARP-2iC treatment can prolong the survival time of rat kidney transplant recipients.     

同步或序贯腹腔镜内镜联合手术治疗胆囊合并胆总管结石的对比研究

目的探讨同步或序贯腹腔镜胆囊切除术（1aparoscopic cholecystectomy,LC）联合内镜下乳头括约肌切开术（endoscopic sphlncterotomy,EST）治疗胆囊合并胆总管结石的疗效。方法2009年6月-2012年2月,85例胆囊结石合并胆总管结石分为同步手术组42例（Lc联合术中EST）和序贯手术组43例（LC联合术前EST）,比较2组手术时间、并发症发生率、手术成功率、术后住院时间、术后随访情况。结果2组手术时间无明显差异[（115．3±64．8）minvs．（107．8±57．9）min,r=0．563,P=0．575]。与序贯手术组比较,同步手术组高淀粉酶血症发生率显著降低[2．4％（1／42）vs．18．6％（8／43）,χ2=5．907,P=0．015],住院时间明显缩短[（5．9±1．7）dvs．（7．8±2．4）d,t=-4．203,P=0．000]。结论同步LC联合EST治疗胆囊结石合并胆总管结石术中选择性应用腹腔镜内镜会合或逆行胆管插管方案可降低并发症发生率,缩短住院时间。