茯苓硫酸酯化多糖的制备及其核磁共振波谱分析

[目的]用不同方法制备3种茯苓多糖硫酸酯（Pachyman sulfate,PS）,并对其进行核磁共振波谱对比分析,为多糖硫酸化提供基础数据。[方法]以氯磺酸为磺化试剂硫酸化茯苓多糖制备茯苓多糖硫酸酯PS—I和PSII,以氨基磺酸为磺化试剂制备PS-Ⅲ,分析磺化反应前后和不同硫酸根取代度（Degree of sulfation,DS）的多糖碳原子化学位移的变化规律。[结果]PS—I（DS=2．80）分子所有自由羟基均发生取代反应;PS—H（DS=1．55）C-6羟基被全部酯化,C-2和C-4羟基发生部分酯化反应;PS-Ⅲ（DS=0．95）C-6和C-2羟基发生部分硫酸酯化反应。[结论]发生取代反应的碳原子化学位移移动的距离与PS的DS呈正相关,核磁共振技术能够提供多糖结构解析的关键数据。     

安徽省知母药材质量研究

[目的]研究安徽产知母药材中知母皂苷BⅡ和芒果苷的含量.[方法]知母皂苷BⅡ采用Diamonsil C18柱（250 mm ×4.6 mm,5μm）、乙腈-水等度洗脱（25∶75）,流速1.0 ml/min、ELSD检测器漂移管温度60℃、载气流速3.0 ml/min、柱温为室温（25℃）;芒果苷采用Diamonsil C18柱（150 mm×4.6 mm,5μm）、乙腈-0.2％冰醋酸水溶液（15∶85）等度洗脱,流速1.0 ml/min.[结果]知母皂苷BⅡ在1.036～10.360 μg范围内（r=0.999 6）进样量的对数值与峰面积的对数值呈良好的线性关系,芒果苷在0.05 ～1.00 μg范围内（r=0.9994）进样量与峰面积呈良好的线性;知母皂苷BⅡ平均回收率为99.7％,RSD=1.19％;芒果苷平均回收率为100.3％,RSD=1.42％.[结论]安徽省知母药材中知母皂苷BⅡ与芒果苷含量均符合2010版药典含量标准.     

铅离子印迹磁性材料的制备及其对Pb(Ⅱ)的吸附去除研究

[目的]制备铅离子印迹和非印迹磁性材料,研究两种材料对Pb(Ⅱ)的吸附去除行为,考察两种材料对Pb(Ⅱ)的吸附选择性,探索其脱附和循环利用性.[方法]采用透射电镜、红外光谱、X射线衍射光谱和能量色散谱等方法对两种材料的形貌和结构进行表征;采用静态吸附法,以原子吸收为检测手段,探讨了pH值、反应时间及Pb(Ⅱ)初始浓度等因素对Pb(Ⅱ)吸附能力的影响;采用Langmuir等温吸附模型和Ho准二级动力学方程对其进行热力学和动力学模拟研究;以Cd(Ⅱ)为竞争离子,研究两种材料对Pb(Ⅱ)吸附选择性;以硝酸为脱附试剂,考察其脱附和循环利用性.[结果]1)与Fe3O4 10 nm的粒径相比,铅离子印迹磁性材料粒径增至80～90 nm;两种材料红外光谱图中557 cm-1处出现强吸收峰,证实Fe-O键存在,2 940 cm-1和1 084 cm-1处的吸收峰证实C-H和Si-O键存在;X射线衍射光谱图显示,它们都具有Fe3O4晶型及SiO2壳层;能量色散谱结果显示,它们主要构成元素为C、O、Si、S和Fe,说明Fe3O4磁核已被SiO2包覆,且巯基已成功键合至两种材料的表面.2)在低酸度时Pb(Ⅱ)基本不被两种材料吸附;当pH值从3增至7时,吸附率不断增大并达到最大,且非印迹材料对Pb(Ⅱ)的吸附率低于印迹材料对Pb(Ⅱ)的吸附率.3)铅印迹磁性材料对Pb(Ⅱ)的吸附量随时间的增加而升高,最后达到平衡吸附.4)随着溶液中Pb(Ⅱ)初始浓度的增加,铅印迹磁性材料对Pb(Ⅱ)的吸附量先是急剧上升,然后达到饱和吸附.5)铅印迹磁性材料对Pb(Ⅱ)的吸附动力学和热力学分别符合准二级吸附模型和Langmuir等温吸附模型.6)铅印迹磁性材料对Pb(Ⅱ)/Cd(Ⅱ)选择吸附系数K为29.75,对Pb(Ⅱ)/Cd(Ⅱ)的相对选择系数是非印迹磁性材料的5.86倍,说明该材料对Pb(Ⅱ)具有良好的吸附选择性.7)研究了HNO3对保留在铅印迹磁性材料上Pb(Ⅱ)的脱附影响,结果显示0.5 mol·L-1 HNO3可定量脱附Pb(Ⅱ),且材料可重复使用5次而脱附率无变化.[结论]在pH 7、反应时间为60 min及Pb(Ⅱ)初始质量浓度为10 mg·L-1时铅印迹磁性材料对Pb(Ⅱ)的最大吸附容量可达38 mg·g-1,可有效去除水中Pb(Ⅱ);该材料对Pb(Ⅱ)具有一定的选择性且具有很好的再生性.     

斜纹夜蛾核型多角体病毒Ⅱ型(SpltMNPV Ⅱ)ORF117基因的结构和功能研究

为了研究斜纹夜蛾核型多角体病毒Ⅱ型(Splt MNPVⅡ)中ORF117基因(编码GP117蛋白)的结构和功能,根据其核苷酸序列和氨基酸序列进行了生物信息学分析,以此为基础对该基因的启动子活性和转录时相进行分析,表达纯化了GP117蛋白的部分序列以制备豚鼠来源多克隆抗体,并利用该抗体对ORF117基因的表达时相和GP117蛋白在细胞中的定位进行了分析。生物信息学分析表明该基因全长1 203 bp,编码400个氨基酸的蛋白质,预测分子量为45.5 k Da,等电点为5.06;启动子活性和转录时相分析结果表明ORF117是一个晚期表达的基因;原核表达抗原免疫制备的多克隆抗体效价可以达到1∶3 200,利用该抗体对其表达时相的分析进一步验证了ORF117为晚期表达基因,免疫荧光检测表明编码的蛋白多存在于细胞质中。     

自然感染鲤疱疹病毒Ⅱ的病毒载量研究

通过建立靶向于鲤疱疹病毒Ⅱ(Cyprinid herpesvirus 2,Cy HV-2)胸苷激酶基因(TK基因)的荧光定量PCR法,分别检测了自然感染Cy HV-2的金鱼、鲫样品,并结合人工感染试验,分析了脾和肾组织内Cy HV-2的载量及其引发的组织病理变化。结果表明,自然感染Cy HV-2的金鱼和鲫的病毒拷贝数范围为102~105,人工感染Cy HV-2的异育银鲫在感染后第五天脾和肾组织内病毒的拷贝数高达1010。人工感染条件下鱼体呈现系统性病理损伤,自然感染状态下鱼体脾和肾的病理变化分别表现为细胞嗜性的转变和炎症。     

Inhibition of proliferation and induction of apoptosis by tanshinone ⅡA in C6 cells

AIM: This study was designed to investigate the inhibition of tanshinone ⅡA on C6 glioma cell line and its mechanism. METHODS: MTT was used to measure the levels of the proliferation of C6 cultured with tanshinone ⅡA at different concentrations. The effects of tanshinone ⅡA on cell cycle of C6 were observed by FCM. The change of DNA was observed by Sepharose electrophoresis. The expression of proto-oncogenes c-myc was measured by RT-PCR. RESULTS: The proliferation of C6 was obviously inhibited by tanshinone ⅡA in a dose-dependent manner. The outcome of FCM showed that the apoptotic cell rate was 7.7%, when cultured with tanshinone ⅡA at 1.0 mg/L for 3 days. The apoptotic cell rate was 21.6%, when cultured with tanshinone ⅡA at 2.0 mg/L in 3 days. CONCLUSION: Tanshinone ⅡA inhibits the proliferation of C6 cells, induces apoptosis and inhibits the expression of proto-oncogene c-myc.     

Expression of MHC Ⅱ on rat corneal keratocytes is inhibited by special siRNA targeting CⅡTA

AIM: To investigate the feasibility to inhibit the expression of MHCⅡ by special siRNA targeting class Ⅱ major histocompatibility complex (MHC Ⅱ) transactivator (CⅡTA), which might regulate MHC Ⅱexpression for suppressing immune rejection. METHODS: Five different siRNA were designed, synthesized and transfected into freshly isolated rat corneal keratocytes. At 24 hours posttransfection, the changes of MHC Ⅱexpression were detected by flowcytometry, and the mRNA abundance of CⅡTA and MHC Ⅱ was measured by FQ-PCR after inducing with recombinant rat interferon-gamma (IFN-γ). RESULTS: Different siRNA showed different reduction in MHC Ⅱ and CⅡTA expression compared with the control (P<0.01). Among the five groups, the siRNA-4 was the most efficient. The mRNA content of CⅡTA and MHC Ⅱ were reduced by 95.10%±1.25% and 82.70%±1.95% respectively and the expression of MHC Ⅱ was inhibited by over 80% in siRNA-4 group at 24 hours posttransfection. CONCLUSIONS: The special siRNA targeting to CⅡTA inhibits CⅡTA mRNA and further inhibits its regulation of MHC Ⅱ molecular expression. The blockade of MHC Ⅱ by siRNA may be useful for further studying allogeneic corneal limbal transplantation.     

Changes of IFN-γ, IL-6 and TNF-α levels in rats with severe pneumonia

AIM:To study the variety of cytokines in severe bacterial pneumonia of Sprague Dawleg (SD) rats. METHODS:A total of 60 SD rats were randomly divided into three groups: model Ⅰ group (n=24), model Ⅱ group (n=24), and control group (n=12). Rats in the model Ⅰ group and the model Ⅱ group were intratracheally instilled with suspension of klebsiella pneumoniae at different doses. Rats in the control group were intratracheally injected with 1 mL saline. On the 2nd, 4th and 6th day after intratracheal instillation, 1/3 rats in each group were killed to determine the concentration of IFN-γ, IL-6 and TNF-α in blood. RESULTS:The levels of IL-6 and TNF-α in model groups were higher than those in control group, while the level of IFN-γ was lower. The change of cytokines was more significant in the model Ⅱ group (severe pneumonia) than those in the model Ⅰ group. CONCLUSION:The cytokines we studied may play an important role in the pathogenesis of severe pneumonia. The change of cytokines is more significant in severe pneumonia than those in common pneumonia.     

Role of PARP in endothelial cell apoptosis induced by angiotensin Ⅱ

AIM: To explore the role of poly-(ADP-ribose)polymerase (PARP) in the cultured endothelial cell apoptosis induced by angiotensin Ⅱ.METHODS: The cultured endothelial cells were treated with angiotensin Ⅱ at concentration of 1 μmol/L.The apoptosis of endothelial cells was assessed by TUNEL.Meanwhile,the activity of PARP and the content of nitric oxide (NO) were also measured.RESULTS: Angiotensin Ⅱ induced apoptosis in endothelial cells in a time-dependent manner.The content of NO begun to increase at 6 h (P<0.05),and peaked at 24 h.The activity of PARP also increased at 6 h (P<0.05),peaked at 12 h,and was lower than that in the control at 48 h (P<0.05).CONCLUSION: The cytotoxicity of NO has a relevant role in apoptosis of endothelial cells induced by angiotensin Ⅱ,and can increases the activity of PARP.     

Effects of atorvastatin on cardiac myocyte hypertrophy of neonatal rat induced by angiotensinⅡ in vitro and TLR4 expression

AIM: To assess effects of atorvastatin (Ator) on cardiac myocytes (CM) hypertrophy of neonatal rat induced by angiotensinⅡ (AngⅡ) in vitro and Toll like receptor 4 (TLR4) expression for theoretical bases of preventing and treating myocardial hypertrophy.METHODS: CM of neonatal Sprague-Dawley (SD) rats were isolated with trypsin digestion method and those growth-arrested cells were stimulated with 10^-7 mol/L AngⅡ in the presence of various concentrations of Ator.The method of coomassie brilliant blue was adopted to evaluate the protein contents of CM.The changes in β-MHC,AT₁ receptor and TLR4 mRNA expression were observed by RT-PCR.RESULTS: ① AngⅡ increased the protein content of CM and β-MHC mRNA expression significantly,upregulated AT₁ receptor and TLR4 gene expression.② In a dose-dependent manner,Ator inhibited the increases in the protein contents of CM and β-MHC mRNA expression induced by AngⅡ.③ In a dose-dependent manner,Ator downregulated the AT₁ receptor and TLR4 mRNA expression of CM hypertrophy of neonatal rat induced by AngⅡ.CONCLUSION: Ator inhibits CM hypertrophy,downregulates the AT₁ receptor and TLR4 gene expression.