中药寒热药性表达模糊评价模式的理论与实验研究

目的:寒热药性反映了中药的作用趋向,其认知概念具有模糊性特征,符合模糊数学的应用范畴。本文旨在通过建立药性生物学表达的模糊判别模式并运用其分析实验数据,为整体、综合地评价寒热药性提供方法学指导。方法:首先,建立寒热药性表达模糊评价模式。以经典寒热复方效应作为寒热药性表达的基准方向,运用Fisher线性判别法计算寒热药性表达模糊集的隶属度函数,进一步得到寒性表达函数和热性表达函数,并以此作为评价参数。第二,对评价模式进行实践。以姜附桂方作为经典热性复方,以三黄方作为经典寒性复方,以各系统生化指标考察药性表达,建立寒、热性表达方程。在此基础上,收集热性药肉桂、仙茅,寒性药黄柏、栀子干预正常状态,以及糖皮质激素诱导的虚寒和虚热状态模型的生物学实验数据,将药物干预前后机体状态的相对变化量代入评价模式,分析其药性表达特征。结果:肉桂干预正常、虚寒和虚热3个状态的热性表达均值大于寒性表达均值,分别为0.528>0.221、0.203>-0.490、1.750>-0.479;仙茅干预正常和虚寒状态的热性表达均值大于寒性表达均值,分别为0.474>-1.601、0.288>-1.923,体现了药物的热性;黄柏干预正常状态和虚寒状态的寒性表达均值大于热性表达均值,分别为0.798>-0.870、0.194>-0.339;栀子干预虚热状态的寒性表达均值大于热性表达均值0.354>-1.802,体现了药物的寒性。结论:运用寒热药性模糊评价模式,可以有效分析出药物的寒热药性和药性表达方式。药性相同的中药,既有共性表达,也有个性表现。寒热药性表达具有多途径特点。

复杂性科学视野下的中药药性理论——药性表达的多样性(二)

中药药性是药物性质与功能的高度概括,反映了中药干预机体后的作用倾向。中药药性表达即中药药性的生物学表达,描述了将药性所蕴含的作用及功能信息表现在生物载体上的过程。由于药性表达涉及中药与机体两大复杂系统,故其具有复杂性特征。第一,药性表达的影响因素十分复杂,药材的生长环境、采收季节、炮制加工等,以及机体的体质、证候等,均可影响实际的药性表达结果;第二,鉴于药物个性特征,相同药性的不同中药会通过不同的药效作用(指标)表达药性,其药性表达方式多样。中药药性表达具有多样性特征,药性实质研究应密切关注影响药性表达的各因素。

中药辛味药性表达及在临证配伍中的应用分析

随着我国医学技术的不断发展,中药用于临床疾病治疗效果也越来越明显。中药五味中辛味药是常用药物,归经用药,分析辛味药性的表达和配伍应用情况,具有重要的应用价值。论文对辛味药临证配伍情况进行了研究,仅供参考。

中药辛味药性表达及在临证配伍中的应用价值研究

随着当前我国医学技术的发展和中药研究的不断深入,中药在医学实践的各个层面上均显示了突出的应用价值。辛味药是当前中医临床上最常见的药物之一,可归入各经,对人民群众的身体健康有积极作用。因此,本文对中药辛味药在药性表达和临证配伍上进行了应用价值研究。

中药咸味药性表达及在临证配伍中的应用

对中药五味中咸味的中医药内涵、咸味药基原的分布特征以及咸味与四气、归经等药性理论的关联关系进行归纳和总结,并对咸味味觉受体、味觉表达及物质基础研究进行阐述。最后,概述了咸味药物配伍理论在临床上的应用,为咸味药性理论更好地指导临床实践提供参考。

基于分子对接技术的辛热药药性表达研究

目的:基于分子对接技术,探讨辛热药药性表达与钙通道相关蛋白的关系。方法:辛热药附子、仙茅、肉桂药效成分与钙通道相关蛋白进行分子对接,通过比较、分析初步筛选出拟合适配度均较高蛋白作为潜在靶蛋白;并与苦寒药黄柏药效成分分子对接结果进行比较,从而确定相关潜在靶蛋白,最后借助生物学实验进行验证。结果:通过比较、分析,筛选出毒蕈碱型乙酰胆碱受体和钙调蛋白可能为辛热药药性表达的潜在靶蛋白,并通过Western Blot实验显示,辛热药附子、仙茅、肉桂给药组均能够纠正阳虚状态下大鼠肝中的钙调蛋白表达,而苦寒药黄柏对其作用不明显。结论:钙调蛋白与辛热药药性表达密切相关。

辛热药药性表达与钙调蛋白之间关系的实验研究

目的:在观察辛热药附子、仙茅、肉桂和苦寒药黄柏对阳虚状态大鼠下丘脑-垂体-靶腺轴功能调节的基础上,通过体内外研究其对钙调蛋白的作用,以期阐释辛热药药性表达的实质。方法:氢化可的松琥珀酸钠粉针剂肌肉注射复制阳虚大鼠模型,辛热药附子、仙茅、肉桂和苦寒药黄柏对其干预7 d后,检测下丘脑-垂体-靶腺轴的促甲状腺激素(TSH)、三碘甲腺原氨酸(T3)、甲状腺素4(T4)、17-羟皮质类固醇(17-OHCS)、皮质醇(COR)、睾酮(T)、雌二醇(E2)等相关指标及Western blot法检测各组肝脏和体外培养的L02细胞中钙调蛋白的表达。结果:辛热药附子、仙茅、肉桂对阳虚状态的下丘脑-垂体-靶腺轴指标、钙调蛋白均有明显的纠正作用,而苦寒药黄柏对其作用不明显。结论:初步确定辛热药药性表达与钙调蛋白密切相关。

基于不同状态下的辛热药附子药性表达特征指标研究

目的:通过观察附子对不同状态下客观指标的影响,初步筛选出能体现附子辛热药性的特征指标,为辛热药药性表达的实验研究提供依据。方法:SD雄性大鼠按体质量随机分为正常组、阳虚组、阴虚组、阳虚给药组、阴虚给药组;阳虚组、阴虚组分别肌肉注射氢化可的松琥珀酸钠、地塞米松磷酸钠,正常组注射等体积的0.9%氯化钠溶液;造模14d后,给药组开始灌服附子水煎液,其他组灌服等体积的蒸馏水,于灌胃7d后各组腹股静脉取血,对相关指标进行测定。结果:与正常组比较,阳虚组17-羟皮质类固醇(17-OHCS)、皮质醇(COR)、总胆固醇(TC)、尿素氮(BUN)、三碘甲腺原氨酸(T3)、促甲状腺激素(TSH)、睾酮(T)、睾酮/雌激素(T/E2)显著降低(P<0.05),葡萄糖(GLU)、甘油三酯(TG)、雌激素(E2)显著升高(P<0.05);阴虚组COR、E2显著降低(P<0.05),17-OHCS、GLU、TG、TC、T3显著升高(P<0.05);给予附子水煎液干预后,阳虚组17-OHCS、COR、GLU、TG、TC、BUN、T3、TSH、T、E2、T/E2被纠正,且与正常组比较无显著性差异;阴虚组17-OHCS、COR、GLU、TG、TC、BUN、T3、四碘甲腺原氨酸(T4)、TSH、T、T/E2指标没有被纠正或指标变化更加恶劣。结论:初步筛选出下丘脑-垂体-靶腺轴相关指标与辛热药附子药性表达密切相关。

附子影响阳虚证大鼠的相关指标研究

目的初步筛选出与辛热药附子药性表达的相关指标。方法 SPF级雄性SD大鼠按体质量随机分为正常组、阳虚组、附子给药组、黄柏给药组;除正常组外其余3组均肌注氢化可的松粉针剂,正常组肌注等量的0.9%氯化钠溶液;造模14 d后,除附子组和黄柏组外均进行眼眶取血,取血后各组继续进行注射造模,给药组开始予以相应浓度的药物灌胃,正常组和阳虚组灌服蒸馏水,灌胃7 d后于腹股静脉取血,测定相关指标。结果与正常组比,阳虚组17-羟皮质类固醇(17-OHCS)、皮质醇(COR)、总胆固醇(TC)、乳酸脱氢酶(LDH)、三碘甲腺原氨酸(T3)、睾酮(T)、睾酮/雌二醇(T/E2)显著降低(P<0.05),甘油三酯(TG)、雌二醇(E2)显著升高(P<0.05);给药后,附子以及黄柏给药组LDH显著升高,TG显著减低(P<0.05);附子给药组17-OHCS、TC、T3、T、T/E2升高,E2降低,而黄柏给药组未有纠正作用或变化方向相反(P<0.05)。结论通过黄柏反证,揭示辛热药附子药性表达的相关指标主要集中在下丘脑—垂体—靶腺轴方面。

辛热示例药对正常大鼠基础代谢及内分泌系统的影响

目的以附子、肉桂、干姜为辛热示例药,观察三药合剂对正常大鼠基础代谢及内分泌系统的影响,综合评价辛热药性的表达特征。方法正常大鼠灌服附子、肉桂、干姜(1∶1∶1)配伍200%水煎液,4 mL/只,连续给药9周,2%戊巴比妥钠麻醉后,腹主静脉取血,分离血清,全自动生化仪检测血清物质能量代谢指标[糖(GLU)、三酰甘油(TG)、胆固醇(TC)、总蛋白(TP)、白蛋白(ALB)、尿酸(UA)、乳酸脱氢酶(LDH)、乳酸(LAC)],放免法检测血清内分泌系统指标[皮质醇(COR)、三碘甲腺原氨酸(T3)、甲状腺素(T4)、促甲状腺激素(TSH)、雌二醇(E2)、睾酮(T)]。结果中药组血清中TG、TC、TP、LAC、LDH明显升高,而COR、E2、T、T3含量明显降低;其他指标变化不明显。结论示例辛热药给予正常大鼠9周后,基础代谢加快,内分泌系统功能抑制,可能与辛热药性表达特征相关,具体机制有待进一步探讨。

Molecular Cloning and Characterization of the Tapetum- specific Gene RA39 from Rice

A novel tapetum-specific cDNA clone of rice, its corresponding gene designed as RA39, is isolated by RNA subtractive hybridization, differential screening and rapid amplification of cDNA ends. The RA39 cDNA is 1013 bp in length with an open reading frame encoding 298 amino acid residues. mRNA in situ hybridization reveals that RA39 is a tapetum-specific gene, and highly expressed at the meiosis stage of pollen mother cells. The deduced protein contains a signal peptide, a transmembrane region and a cytoplasmic tail, and is predicted to localize in endoplasmic reticulum by PSORT program. This cDNA sequence did not show significant homology to any known sequences in Genbank database. RA39 is the first gene identified to be expressed specifically in tapetal cells at the meiosis stage of pollen mother cells from cereals.

Fusion expression of Helicobacter pylori neutrophil-activating protein in E.coli

AIM: To produce a recombinant protein rMBP-NAP, which was fusionally expressed by Helicobacter pylori(H pylori)neutrophil-activating protein (NAP) and E. coli maltosebinding protein (MBP) and to evaluate its immunoreactivity and immunogenicity.METHODS: Neutrophil-activating protein gene of H pylori (HP-napA) was subcloned from the recombinant plasmid pNEB-napA, and fused to MalE gene of expressing vector pMAL-c2x. The recombinant plasmid pMAL-c2x-napA was confirmed by restriction enzyme digestion, and then transformed into E. coli TB1. Fusion protein rMBP-NAP was induced by IPTG and identified by SDS-PAGE analysis.Soluble rMBP-NAP was purified by amylose affinity chromatography. Immunoreactivity and immunogenicity of the fusion protein were evaluated by animal experiment,Western blotting with human H pylori anti-sera.RESULTS: E.coli TB1 carrying recombinant plasmid pMAL-c2x-napA was constructed and led to a high efficiency cytosol expression of fusion protein rBMP -NAP when induced by IPTG.The molecular weight of rBMP-NAP was about 57 kD,accounting for 37.55% of the total protein in the sonicated supematant of E. coli TB1 (pMAL-c2x-napA). The purity of the fusion protein after one-step affinity chromatography was 94% and the yield was 100 mg per liter of bacterial culture.The purified fusion protein could be specifically recognized by both human anti-sera from clinical patients with H pylori infection and rabbit sera immunized by rMBP-NAP itself.CONCLUSION: Recombinant protein rMBP-NAP might be a novel antigen for vaccine development against H pylori.

IMPROVING P-gp EXPRESSION IN HUMAN MONONUCLEAR CELLS IN VITRO TRANSFECTED BY MULTIDRUG RESISTANCE-1 mRNA

Objective To evaluate the expression and function activity of P-glycoprotein (P-gp) in human mononuclear cells (MNCs) in vitro transfected by multidrug resistance-1(MDR1) mRNA. Methods Two MDR1 cDNA vectors, pT7TS_MDR1 and pGEM5Zf(+)_MDR1, were constructed and transcripted in vitro. Vector pGEM5Zf(+)_MDR1 only contained the coding region of mdr1 cDNA, and pT7TS_MDR1 also included Xeponus β-globin 5’ and 3’ untranslated region. MNCs were prepared from peripheral blood of parvicellular lung cancer patient. The two human mdr1 mRNAs were then transferred into human MNCs in vitro by DOTAP. And the expression efficiency and pump function of P-gp were measured with flow cytometry. Results Expression of P-gp significantly elevated in both transferred cells compared with untransferred cells (P < 0.01). And pT7TS_MDR1 showed higher capability in elevating the expression of P-gp than pGEM5Zf(+)_MDR1 (P < 0.01). The P-gp function was elevated in both pT7TS_MDR1 and pGEM5Zf(+)_MDR1 groups. The survival ratio of MNCs in erythrocyte-lysis-solution (ELS, 86.07%) and lymphocyte-isolation-solution (LIS, 83.67%) had no significant difference. The CD34+ cells content of the MNCs used for transfection was 2.65% and 1.01% in ELS and LIS group, respectively (P < 0.01).Conclusions It is a feasible approach to improve P-gp expression in human MNCs by transfection of MDR-1 mRNA. And the ELS may be more suitable for purifing MNCs for mRNA transfection than LIS.

Effect of monocyte chemoattractant protein-1 on chemotactic gene expression by macrophage cell line U937

Objective: To study the chemotactic superfamily genes expression profiling of macrophage line U937 treated with monocyte chemoattractant protein-1 (MCP-1) using gene chip technique. Methods: Total RNA from macrophage line U937 (as control) and U937 with MCP-1 was extracted, made reverse transcript to cDNA and tested with gene expression chip HO2 human. Results: Some chemotactic-related gene expressions were changed in all analyzed genes. Regulated upon activation, normal T cell expressed and secreted (RANTES) was up-regulated over 2-fold and 7 chemotactic-related genes (CCR2, CCR5, CCL16, GROβ, GROγ, IL-8 and granulocyte chemotactic protein 2) were down-regulated over 2-fold in MCP-1 treated U937 cells at mRNA level. Conclusion: MCP-1 can influence some chemokines and receptors expression in macrophage in vitro, in which MCP-1 mainly down-regulates the chemotactic genes expression of those influencing neutrophilic granulocyte (GROβ, GROγ, IL-8 and granulocyte chemotactic protein 2). Another novel finding is that it can also down-regulate the mRNA level of CCR5, which plays a critical role in many disorders and illnesses.

中药药性理论复杂性特征分析

中药药性是在中医理论指导下总结的反映药物及其作用性质的概念。中药药性理论是研究中药药性的形成机制及运用规律的理论,包括四气、五味、归经、升降沉浮、有毒无毒等。从发生学上看,中药药性理论与药物本体及药物作用密切相关,并且深受阴阳五行、取象比类等哲学思想影响,具有复杂性特征。主要表现在第一,药性的形成具有多源性特征,其早期的确定途径不唯一,其对应的功效作用不唯一;第二,药性的表达具有多样性特征,其表征指标多样,其实际表达的影响因素多样。中药药性理论的现代研究应关注这些复杂性特征,注意药性与功效的辩证统一,在尊重个性的基础上寻找共性。

Epigenetic regulation mechanism of ABCG2 induced drug-resistant phenotype

The aim of this study is to investigate epigenetic mechanism of ABCG2 induced drug-resistance. It is not only expatiate for drug-resistance regulation mechanism in all-round, but also to provide scientific experimental basis for selecting target to reverse its drug-resistance. Apply methylation-specific PCR （MSP） to have tested methylation of ABCG2 promoter region -359 to -353 specific positions in breast cancer tissues and paired adjacent tissue of 22 cases and test their methylation positions with MSP products for sequencing; and adopt fluorescent quantitation RT-PCR to test expression level DNMT1, DNMT3A, DNMT3B and ABCG2; to make analysis on relationship between them with statistical spearman correlation. Specific positions of ABCG2 gene promoter region of 18 cases among the 22 cases with breast cancer （18/22, 82%） existed high methylation （P〈0.05）, MSP products sequencing proved methylation of the specific position, and mRNA expression level was relative higher in remarkable positive correlation （P〈0.05） ABCG2, DNMT1, DNMT3A, DNMT3B mRNA expression levels in breast cancer tissues were obviously higher than adjacent tissues （P〈0.01）, and DNMT3B expression level was obviously higher than DNMT1 and DNMT3A （P〈0.01） in negative correlation with ABCG2 gene expression （P=0.001）. -359 to -353 positions of promoter regions of ABCG2gene existed high methylation capable to push expression of this gene in beast cancer tissue. DNMT3B is involved in expression regulation in ABCG2 gene, and provides new scientific basis for drug-resistance target as reverse ABCG2 induction

UP REGULATION OF UBIQUITIN C TERMINAL HYDROLASE IN THE RESPONSE OF THE MOSQUITO ANOPHELES STEPHENSI TO PLASMODIUM YOELII INFECTION

The isolation and study of genes that are differentially expressed in malaria infected mosquitoes is important for the elucidation of basic molecular mechanisms underlying vector parasite interactions. When screening against a previously established cDNAs pool representing specifically expressed genes in the mosquito Anopheles stephensi infected by Plasmodium yoelii, it was found that one of these encodes a protein with extensive sequence similarity to the Drosophila melanogaster ubiquitin C terminal hydrolase(UCTH). Similarity alignment showed that the fragment is 89% identical at amino acid level to the corresponding region of the known An. gambiae EST sequence, as well as 63% identical to that of both the fruitfly and human sequence. Virtual Northern blot expression dynamics of the gene indicated that it was up regulated significantly in the mosquito at least 1 7 days post infection, consistent with the critical transition stages of midgut invasion and relocation of sporozoites from the oocysts to the salivary glands during parasite development. Rather little is known about the role of the ubiquitin pathway in the activation of the mosquito innate immune system. The results indicate that the gene is related to malaria infection in mosquito. The cloning and expression profile analysis of As UCTH enables us to make predictions as to the roles it may play during malaria infection.