从绞股蓝转录组挖掘可能与绞股蓝皂苷生物合成相关修饰酶基因细胞色素P450 (cytochrome P450,CYP)及UDP-糖基转移酶(UDP-dependent glycosyltransferase,UGT)基因的cDNA进行克隆并开展了以下研究.根据课题组前期绞股蓝转录组数据,筛选CYP及UGT Unigenes,利用RT-PCR克隆ORF全长,并结合生物信息学对其编码蛋白进行功能分析,最后通过荧光定量PCR验证其在根、茎、叶的差异性表达.结果 克隆得到两个ORF序列,分别命名为CYP94A1和UGT91A1;CYP94A1序列包含1 509 bp的开放阅读框架,编码一条含503个氨基酸残基的肽链,具有CYP保守结构域;UGT91A1序列包含1 383 bp的开放阅读框架,编码一条含461个氨基酸残基的肽链,具有UGT保守结构域.这两个基因的转录表达均具有组织特异性,在叶或茎中的表达均高于根.CYP94A1和UGT91A1的克隆、转录表达及生物信息学分析为进一步挖掘绞股蓝中CYPs、UGTs及功能验证提供了帮助,增加对CYP和UGT两个超基因家族的认识.

目的 探讨单核细胞趋化蛋白-1(MCP-1)A-2518G基因多态性与葡萄糖耐量异常(IGT)、新发2型糖尿病(T2DM)的关系.方法 对111例健康人(NC组)、82例IGT患者(IGT组)及76例新发T2DM患者(T2DM组)采用聚合酶链反应-限制性片段长度多态性技术,观察MCP-1 A-2518G基因多态性,分析其与IGT和T2DM的关系.结果 IGT、T2DM组与NC组的MCP-1等位基因和基因频率比较差异无统计学意义(P＞0.05),logistic回归分析结果显示,腰臀比、胰岛素抵抗指数、MCP-1 AA是IGT及新发T2DM发病的独立危险因素(P＜0.05).结论 MCP-1 A-2518G基因多态性可能参与IGT及新发T2DM的发病机制,AA基因携带增加人群IGT及新发T2DM的患病风险.

Objective To investigate the effects of Rosa Laevigata Michx Flavoid( RLMF) and Rosa laevigata Michx Polysaccharose (RLMP) on expression of TRPV5 in IgA Nephropathy (IgAN) rat renal tissue. Methods Forty Sprague-Dawley rats were randomly assigned to four groups.The rat model of IgA nephropathy was induced by intragastric administration of bovine serumalbumin and injections of LPS and CC14.Eight weeks later,the rats with IgAN were treated with RLMF or RLMP (4 weeks), or normal saline.Rats was sacrificed at thirteenth weeks, and RNA was extracted from the kidney.Expression of TRPV5 in tubulointerstitial tissues were analyzed by fluorescent quantitative RT-PCR. Results After RFLP intervention,the expression levels of TRPV5 were markedly increased (P<0.01) than model control group,while decreased (P<0.05) than normal control group but had no significance with model control group after RFLF intervention. Conclusion TRPV5 expression is decreased in IgAN,and RLMP can adjust TRPV5 expression and improve renal function of IgAN.

[目的]探讨金樱子总黄酮抗氧化及清除自由基的作用.[方法]分别采用水杨酸法和邻苯三酚自氧化法观察金樱子总黄酮对羟自由基(·OH)和超氧阴离子(O2-·)的清除作用,同时对金樱子总黄酮总还原能力进行测定.[结果]金樱子总黄酮具有良好的抗氧化能力,并对·OH和O2-·等自由基具有较强的清除能力,但对不同的自由基敏感程度不同.[结论]金樱子总黄酮具有良好的抗氧化活性,且对·OH和O2-·的清除能力与其浓度呈正相关关系,提示其具有一定抗衰老作用.

目的:探讨金樱子多糖体外抗氧化能力.方法:分别采用Smirnoff水杨酸法和邻苯三酚自氧化法观察金樱子多糖对羟自由基(·OH)和超氧阴离子(02-·)的清除作用,同时观察了金樱子多糖对1,1-二苯基-2-三硝基苯肼(DPPH自由基)的清除作用和总还原能力.结果:金樱子多糖具有良好的抗氧化能力,并对·OH、O2-·及DPPH自由基具有较强的清除能力,但对不同的自由基敏感程度不同,对·OH和O2-·的清除能力在实验浓度范围内随浓度的增高而增强.结论:金樱子多糖具有良好的抗氧化活性,提示其具有良好的抗衰老作用.

马尔尼菲青霉是温度依赖双相性条件性致病真菌,可感染免疫缺陷人群,区域流行于东南亚和我国南方.马尔尼菲青霉己成为研究真菌双相形态转换和病原-宿主相互作用分子机制的模式系统.功能基因组学研究为深入探讨马尔尼菲青霉双相转换和致病分子机制提供了线索,大量的候选基因功能需要高通量的基因修饰方法验证.在许多真菌中已建立基于阻断DNA非同源重组修复途径提高基因打靶效率的实验研究模型.本文报道在马尔尼菲青霉中通过缺失马尔尼菲青霉非同源重组修复途径的关键组分PKUB同源基因pkuB构建了一个高效基因打靶系统.结果表明,以pkuB基因缺失菌株(△pkuB)为出发菌株能降低外源DNA片段的非同源末端连接重组的概率而显著提高基因打靶效率,pkuB基因缺失不影响菌落形态和双相转换能力.高效基因打靶分子遗传实验模型的建立为大规模进行马尔尼菲青霉基因功能研究提供了有力的工具.

Paclitaxel is one of the best anticancer agents that has been isolated from plants, but its major disadvantage is its dose-limiting toxicity. In this study, we obtained evidence that the active mutant IPP5 (8-60hIPP5m), the latest member of the inhibitory molecules for protein phosphatase 1, sensitizes human cervix carcinoma cells HeLa more efficiently to the therapeutic effects of paclitaxel. The combination of 8-60hIPP5m with paclitaxel augmented anticancer effects as compared to paclitaxel alone as evidenced by reduced DNA synthesis and increased cytotoxicity in HeLa cells. Furthermore, our results revealed that 8-60hIPP5m enhances paclitaxel- induced G2/M arrest and apoptosis, and augments paclitaxel-induced activation of caspases and release of cytochrome C. Evaluation of signaling pathways indicated that this synergism was in part related to down- regulation of NF-?B activation and serine/threonine kinase Akt pathways. We noted that 8-60hIPP5m down- regulated the paclitaxel-induced NF-?B activation, I?Bα degradation, PI3-K activity and phosphorylation of the serine/threonine kinase Akt, a survival signal which in many instances is regulated by NF-?B. Together, our observations indicate that paclitaxel in combination with 8-60hIPP5m may provide a therapeutic advantage for the treatment of human cervical carcinoma.

Protein phosphatase 1 (PP1) is a major serine/threonine phosphatase that controls gene expression and cell cycle progression. The active mutant IPP5 (8-60hIPP5 m ), the latest member of the inhibitory molecules for PP1, has been shown to inhibit the growth of human cervix carcinoma cells (HeLa). In order to elucidate the underlying mechanisms, the present study assessed overexpression of 8-60hIPP5 m in HeLa cells. Flow cytometric and biochemical analyses showed that overexpression of 8-60hIPP5 m induced G2/M-phase arrest, which was accompanied by the upregulation of cyclin B1 and phosphorylation of G2/M-phase proteins ATM, p53, p21 cip1/waf1 and Cdc2, suggesting that 8-60hIPP5 m induces G2/M arrest through activation of the ATM/p53/p21 cip1/waf1 /Cdc2/ cyclin B1 pathways. We further showed that overexpression of 8-60hIPP5m led to delayed nuclear translocation of cyclin B1. 8-60hIPP5 m also could translocate to the nucleus in G2/M phase and interact with pp1α and Cdc2 as demonstrated by co-precipitation assay. Taken together, our data demonstrate a novel role for 8-60hIPP5m in regulation of cell cycle in HeLa cells, possibly contributing to the development of new therapeutic strategies for cervix carcinoma.

RT-qPCR is a commonly used method for evaluating gene expression; however, its accuracy and reliability are dependent upon the choice of appropriate reference gene(s), and there is limited information available on suitable reference gene(s) that can be used in mouse testis at different stages. In this study, using the RT-qPCR method, we investigated the expression variations of six reference genes representing different functional classes (Actb, Gapdh, Ppia, Tbp, Rps29, Hprt1) in mice testis during embryonic and postnatal development. The expression stabilities of putative reference genes were evaluated using five algorithms: geNorm, NormFinder, Bestkeeper, the comparative delta C t method and integrated tool RefFinder. Analysis of the results showed that Ppia, Gapdh and Actb were identified as the most stable genes and the geometric mean of Ppia, Gapdh and Actb constitutes an appropriate normalization factor for gene expression studies. The mRNA expression of AT1 as a test gene of interest varied depending upon which of the reference gene(s) was used as an internal control(s). This study suggested that Ppia, Gapdh and Actb are suitable reference genes among the six genes used for RT-qPCR normalization and provide crucial information for transcriptional analyses in future studies of gene expression in the developing mouse testis.

Protein phosphatase 1 (PP1) is a major serine/threonine phosphatase that controls gene expression and cell cycle progression. The active mutant IPP5 (8-60hIPP5(m)), the latest member of the inhibitory molecules for PP1, has been shown to inhibit the growth of human cervix carcinoma cells (HeLa). In order to elucidate the underlying mechanisms, the present study assessed overexpression of 8-60hIPP5(m) in HeLa cells. Flow cytometric and biochemical analyses showed that overexpression of 8-60hIPP5(m) induced G2/M-phase arrest, which was accompanied by the upregulation of cyclin B1 and phosphorylation of G2/M-phase proteins ATM, p53, p21(cip1/waf1) and Cdc2, suggesting that 8-60hIPP5(m) induces G2/M arrest through activation of the ATM/p53/p21(cip1/waf1)/Cdc2/ cyclin B1 pathways. We further showed that overexpression of 8-60hIPP5(m) led to delayed nuclear translocation of cyclin B1. 8-60hIPP5(m) also could translocate to the nucleus in G2/M phase and interact with pp1α and Cdc2 as demonstrated by co-precipitation assay. Taken together, our data demonstrate a novel role for 8-60hIPP5(m) in regulation of cell cycle in HeLa cells, possibly contributing to the development of new therapeutic strategies for cervix carcinoma.

To investigate the anti-proliferative mechanism of mangiferin in a human nasopharyngeal carcinoma cell line, CNE2 cells were incubated with different concentrations of mangiferin (12.5, 25, 50, 100, 150 and 200 μM) or with PBS as a control for 72 hours. Analyses were made of the cell cycle and apoptosis with measurement of mRNA and protein levels of two apoptosis-related genes, Bcl-2 and Bax. Flow cytometry assays showed mangiferin could inhibit CNE2 cell proliferation via G2/M arrest and induction of early apoptosis. Real time PCR and Western blotting showed the mRNA and protein level of Bcl-2 to be down-regulated, while those of Bax were up-regulated, when CNE2 cells were treated with mangiferin. This investigation indicated anti-proliferation effects of mangiferin through induction of cell apoptosis regulated by Bcl-2 and Bax expression.

马尔尼菲青霉菌是一种区域性流行于东南亚及中国南部地区的条件致病性温度依赖性双相型病原真菌,它能对免疫功能低下者造成致命的全身性感染,25℃时呈现菌丝状生长,而在37℃或在宿主体内为酵母相生长.为深入研究马尔尼菲青霉菌致病性和双相转化的分子机制,原有的遗传转化筛选标记基因已不能满足研究需要.为此,我们通过用马尔尼菲青霉菌微管蛋白β亚基基因启动子替换粗脉孢菌苯菌灵抗性基因的启动子,构建了一个苯菌灵抗性基因盒,并成功地将其运用于马尔尼菲青霉菌的遗传转化研究中.

在医学教育国际标准"本土化"的背景下,进行课程整合,将传统的生物化学课程中与人体代谢有关的部分内容,结合临床问题,设计人体代谢与疾病整合课程,为这门整合课程设计相应的教学案例,从而为开展PBL、CBL、TBL等模式的教学提供相应的素材。

目的 探讨血清单核细胞趋化蛋白-1 (MCP-1)水平与T2DM、IGT及其危险因素的相关性.方法 检测新诊断T2DM患者32例、IGT患者46例和健康(NC)者111名血清MCP-1水平及其他生化指标,分析血清MCP-1与T2DM、IGT及其危险因素的相关性.结果 T2DM、IGT组血清MCP-1水平高于NC组(P＜0.01).MCP-1与WHR、TC、胰岛素抵抗指数(HOMA-IR)呈正相关,与年龄、BMI、血压、FPG、2hPG和HbA1c无相关性.结论 血浆MCP-1水平与T2DM、IGT均有相关性,与WHR、TC和HOMA-IR均独立相关.MCP-1可能对T2DM和IGT发病有影响.

目的 探讨洗洁精中的主要成分十二烷基苯磺酸钠(SDBS)对小鼠肝脏的毒性作用.方法 不同组别的小鼠分别自由饮用不同浓度的SDBS溶液1周,之后检测各组小鼠血清中的GPT活性和计算肝脏体重比值,并与对照组对照.结果 随着研究范围内SDBS溶液浓度的增高,小鼠血清中GPT活性逐渐增高、肝脏体重比值逐渐增大.结论 SDBS对小鼠肝脏具有一定的毒性作用,当SDBS浓度超过0.279g/L时可造成肝脏的不可逆损害.

Protein phosphatase 1 (PP1) is a major serine/threonine phosphatase that controls gene expression and cell cycle progression. Here, we report the characterization of a novel human bone marrow stromal cell (BMSC)-derived protein called protein phosphatase 1 inhibitor 5 (IPP5), which was obtained by large-scale random sequencing of a human BMSC cDNA library. The human IPP5 cDNA encodes a protein of 116 amino acid residues, which shares high homology with human protein phosphatase 1 inhibitor-1 (PPI-1). The effect of IPP5 on tumor growth and the underlying molecular mechanisms were investigated by overexpression of IPP5 in HeLa cells, a human cervical carcinoma cell line. Our results demonstrated that overexpression of the active mutant IPP5 inhibited the growth of HeLa cells both in vitro and in vivo. Biochemical analysis demonstrated that active mutant IPP5-mediated G2/M arrest of HeLa cells involved regulation of cyclin A1, cyclin B1, CDK1, p21, and p53, as well as increased inhibition of ERK activation. Furthermore, overexpression of the active mutant IPP5 leads to the formation of dikaryons following the failure of cytokinesis. Therefore, IPP5 might be a potential growth inhibitor for human tumor cells, especially for cervical carcinoma cells, and it could contribute to the development of new therapeutic strategies for human cervical cancer treatment.

Objective To explore the impact of Agkihpin on the expression of cyclooxygenase-2 in nasopharyngeal carcinoma CNE-2 cell line and to describe the mechanisms of Agkihpin inhibition against CNE-2.Methods The cultured cells were treated with different concentrations of Agkihpin for 72 h.The expression levels of COX-2 in CNE-2 cell line were assayed by immunocytochemistry,Western blotting,and RT-PCR.Results The average COX-2 absorbance among different Agkihpin concentrations showed significant differences after 72 hours of treatment(P0.05);more specificly,the measurement was lower in group A,B,C than in group D(P0.05) and lower in group A than in group B,C,D(P0.05).Western Blotting showed significant differences among different Agkihpin concentrations after 72 hours of treatment(P0.05);COX-2 expression increased as Agkihpin concentration decreased(P0.05).Expression levels of COX-2 mRNA showed significant differences among different Agkihpin concentrations after 72 hours of treatment(P0.05);COX-2 expression increased as Agkihpin concentration decreased(P0.05).Conclusion Agkihpin inhibits the expression of COX-2 in CNE-2 cells in a dose-dependent manner,which is likely one of the reasons for Agkihpin inhibition of the cellular vitality of nasopharyngeal carcinoma cells;Agkihpin might be applied in the treatment of nasopharyngeal carcinoma.

Objective To explore the effects of Agkihpin on the expression of E-Cadherin in nasopharyngeal carcinoma CNE-2 cell line,and to explore the mechanism of Agkihpin inhibiting CNE-2.Methods The cultured cells were treated with different concentrations of Agkihpin for 72 h.The expression level of E-Cadherin in CNE-2 cell line was assayed by immunocytochemistry and RT-PCR.Results Compared with Agkihpin absent group,the E-Cadherin expression levels in CNE-2 cells were reduced significantly after the treatment of different concentrations of Agkihpin(P0.05),in a concentration-dependent manner,which showed that Agkihpin could down-regulate the expression of E-Cadherin in CNE-2 cells significantly.Conclusion Agkihpin can inhibit the expression of E-Cadherin in CNE-2 cells,which may be one of reasons for Agkihpin inhibiting the cellular vitality of nasopharyngeal carcinoma cell.

Objective To explore the effect of Agkihpin on the expression of multidrug resistance associated protein 1(MRP1) in nasopharyngeal carcinoma CNE-2 cell line,and try to describe the mechanism of Agkihpin inhibiting CNE-2.Methods The cultured cells were treated with different concentrations of Agkihpin for 72 h.The expression level of MRP1 in CNE-2 cell line was assayed by immunocytochemistry,Western blot and RT-PCR.Results Comparing with Agkihpin absent group,the MRP1 expression levels in CNE-2 cells were reduced significantly after the treatment of different concentrations of Agkihpin(P0.05),in a concentration-dependent manner,which shows that Agkihpin could significantly down-regulate the expression of MRP1 in CNE-2 cells.Conclusion Agkihpin can inhibit the expression of MRP1 in CNE-2 cells,which is likely to be one of reasons for Agkihpin inhibiting the cellular vitality of nasopharyngeal carcinoma cell,and be likely to enhance the sensitivity of CNE-2 cells to chemotherapeutic drugs to some extent.