Down-regulation of TSPO expression doesn't affect the productions of TNF-α,IL-1β and IL-6 in LPS-stimulated BV-2 microglia

目的研究相对分子质量(Mr)18 000转运蛋白(TSPO)基因表达下调对脂多糖(lPS)诱导bV-2小胶质细胞分泌Tnf-α,Il-1β和Il-6的影响。方法以rnA干扰技术建立TSPO基因表达下调的细胞模型,实时荧光定量PCr(QrT-PCr)和WESTErn blOT法检测转染TSPO SIrnA bV-2细胞TSPO MrnA及蛋白水平表达的效果;用QrT-PCr法检测TSPO基因下调后小胶质细胞bV-2在lPS作用下分泌Tnf-α、Il-1β和Il-6的MrnA水平表达的情况;ElISA检测TSPO基因下调小胶质细胞bV-2在lPS作用下分泌Tnf-α、Il-1β和Il-6的蛋白水平表达的变化。结果成功建立了TSPO基因下调的细胞模型,稳定表达TSPO SIrnA细胞的TSPO MrnA和蛋白水平表达均明显下降,TSPO基因下调后bV-2细胞在lPS作用下分泌Tnf-α、Il-1β和Il-6的量无变化。结论下调TSPO的表达对lPS刺激引起的小胶质细胞Tnf-α、Il-1β和Il-6的分泌无明显影响。Objective To evaluate the effect of translocator protein( TSPO,Mr18 000) on the productions of TNF-α,IL-1βand IL-6 in lipopolysaccharide( LPS)-stimulated BV-2 cells.Methods RNA interference technique was used to decrease TSPO expression in BV-2 cells.Western blotting was performed to assess the level of TSPO protein in BV-2 cells transfected with TSPO siRNA.Then the mRNA and protein levels of TNF-α,IL-1β,and IL-6 were measured in LPS-stimulated BV-2 cells by real-time quantitative PCR( qRT-PCR) and ELISA,respectively.Results The level of TSPO protein obviously decreased in BV-2 cells transfected with TSPO siRNA.However,knockdown of TSPO had no effect on the productions of TNF-α,IL-1βand IL-6 in LPS-stimulated BV-2 cells.Conclusion Down-regulated TSPO is not directly involved in regulating TNF-α,IL-1βand IL-6 productions induced by LPS in microglia.国家自然科学基金(81071182); 福建省医学创新项目(2009-CXB-46); 厦门大学生命科学学院细胞生物和肿瘤工程教育部重点实验室开发基金(2009101); 福建医科大学非直属附属医院科研发展专项基金(2008031

重组人骨形成蛋白-5 cDNA的克隆及其在大肠杆菌中的表达和活性

学位：理学硕士院系专业：生命科学学院生物学系_细胞生物学学号：1993510

The role of FGFR4 in the regulation of bile acid metabolism

成纤维细胞生长因子受体4(fIbrOblAST grOWTH fACTOr rECEPTOr4,fgfr4)是一种能够介导成纤维细胞生长因子(fgf)信号传导的细胞表面跨膜酪氨酸激酶受体。在成熟肝实质细胞中,fgfr4在其配体fgf15或fgf19的刺激下通过激活Jnk信号通路来抑制胆汁酸合成限速酶胆固醇7α-羟化酶的表达,进而抑制胆汁酸的合成。本文介绍fgfr4及其配体在胆汁酸代谢过程中的作用及其相关机制方面的研究进展。Fibroblast growth factor receptor 4(FGFR4) is a transmembrane tyrosine kinase receptor which mediates the transduction of FGF signals to the cytoplasm.The expression of CYP7A1,a rate-limiting enzyme in bile acid synthesis,is repressed by FGFR4 and its ligands,FGF15 or FGF19,through activation of JNK signaling pathway in mature hepatocytes.This review presents the research progress of the role of FGFR4 in the regulation of bile acid metabolism.福建省自然科学基金计划项目(2008J0110)资

IdentiFication of BMP-3 & BMP-5 in Various Human Tissues and Clouing & Expression of Them in E. coli.

利用rT-PCr技术，检测了bMP-3及bMP-5在不同组织中的表达，并将PCr扩增出的bMP-3和bMP-5CdnA克隆入PgEX表达载体.在大肠杆菌中得到高效表达。The expressions of bmp-3 and bmp-5 gelles were identiFied in the various human tissues and cell lines by RT-PCR.The ampliFications of PCR were,cloned into PGEX expression vectors and transFormed into E.colt..The E.coli.transFormed by the above plasmids yield high level of GST-BMP-3 and GST-BMP-5 Fusion proteins.福建省自然科学基

Cloning and Expression of rhBMP 3 in Escherichia coli

重组人骨形成蛋白3的克隆及其在大肠杆菌中的表达陈亚兵俞春东温龙平曾定（厦门大学肿瘤细胞工程国家专业实验室厦门361005）骨形成蛋白（bOnEMOrPHOgEnETICPrOTEIn，bMP）是一类能诱导异位骨及软骨形成，并在动物的发育和分化中起作...Here is shown the cloning and expression of BMP 3 cDNA in E.coli .Utilizing speciFic primers designed according to published sequences,a 0 3kb band was assessed in various human tissues,namely brain,liver,thymus,spleen,placenta and testis,as well as human neuroblastoma SK cells by RT PCR.The 0 3kb Fragment was inserted into pCRII vector and the sequencing result identiFied it the expected 288 bp BMP 3 cDNA.The expression of this BMP gene Fragment was acquaired by ligating it with pGEX 2T and transForming E.coli .The transFormed E.coli yielded high level of BMP 3 GST Fusion proteins.Furthermore,the activities of the Fusion proteins was domestrated by Western blotting

Establishment of a cell-based assay for examining the expression of tumor necrosis factor alpha (TNF-alpha) gene

Tumor necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine produced by activated macrophages and lymphocytes and involved in many inflammatory diseases. Preventing the production or action of TNF-alpha is a potent therapeutic strategy for these inflammatory diseases. Since there is a lack of rapid and effective assay for examining the expression TNF-alpha in macrophages, we attempt to establish a reporter system to assess TNF-alpha gene expression through measuring luciferase activity. In this study, mouse macrophage cell line RAW 264.7 was stably transfected with a luciferase reporter pGL3-TNFPro-UTR, which contains TNF-alpha promoter and 3'-untranslated region (3'-UTR). The TNF-alpha-luciferase reporter cell line is used for assessing the expression of TNF-alpha gene induced by LPS in the presence or absence of chemicals that inhibit the biosynthesis of TNF-alpha such as dexamethasone and emodin, and also for measuring change of expression of TNF-alpha gene under downregulation of the expression of steroid receptor coactivator-3, a modulator for TNF-alpha. The luciferase activity correlated well with the ELISA results for TNF-alpha production, therefore, the TNF-alpha-luciferase reporter cell line is a sensitive, effective tool for studying the expression of TNF-alpha gene

EFFect of EnForced Bcl 2 Over expression on OA induced Cell Death in Human Neuroblastoma SK Cell Line

OA能诱发人神经母细胞瘤Sk细胞进行编程死亡.死亡细胞缩小变圆，细胞质凝聚，dnA有控降解成约200bP左右的片段，且这一过程可由蛋白质合成抑制剂亚胺环己酮（CHX）抑制.将编码bC1-2全长蛋白质的CdnA植入PXJ41nEO载体中，使其表达由HCMV病毒启动子控制.形成的顺义（PbCl-2-S）及反义（PbCl-2-AS）表达质粒经转染导入Sk细胞中获得稳定转染子，WEST-Ern印迹表明顺义转染子表达较大量的26kdbC1-2蛋白，而反义转染子则不表达.增强表达的bC1-2基因产物对OA引Sk细胞编程死亡无抑制效应.OA induce cell death in the human neuroblastoma SK cell line with the characteristic Feather of apoptosis.Cells are rounded and shrink;cytoplasm condenses;DNA is Fragmented into a 200 bp ladder.Moreover,the above phenomenon can be partially inhibited by protein synthesis inhibitor cycloheximide.A 1.6 kb NDA encoding the Full length human Bcl 2 protein was inserted into mammalian expression vector pXJ41neo and placed under the control of HMCV promoter in either the sense (pBcl 2 S) or antisense (pBcl 2 As) orientation.The above two plasmids were transFected into SK cell and stable transFectants were obtained.pBcl 2 s transFected Sk cells expressed high level of 26 kd Bcl 2 protein,as evidenced by Western blotting,while pBcl1 2 As transFected cells showed no detectable Bcl 2 expression.Over expressed Bcl 2 protein cannot inhibit OA induced cell death.国家自然科学基

Stimulation of steroid receptor coactivator-3 (SRC-3) gene overexpression by a positive regulatory loop of E2F1 and SRC-3

Steroid receptor coactivator 3 (SRC-3, amplified in breast cancer 1, or ACTR) is a transcriptional coactivator for nuclear receptors and certain other transcription factors such as E2F1. SRC-3 is overexpressed in breast cancers, and its overexpression is sufficient to cause mammary carcinomas in vivo. However, the mechanisms controlling endogenous SRC-3 overexpression are unknown. In this study, we identified the first exon and analyzed the 5' regulatory sequence of the SRC-3 gene. We found three evolutionarily conserved regions (ECRs) in the 5' SRC-3 regulatory sequence, and ECR2 makes a major contribution to the SRC-3 promoter activity. The ECR2 region (bp -250/+350) contains several specificity protein 1 (Sp1) binding sites and two E2F1 binding sites. We show that E2F1 can significantly activate the ECR2 promoter activity in a dose-dependent manner. Furthermore, overexpression of E2F1 significantly increases the promoter activity of the endogenous SRC-3 gene and boosts SRC-3 expression in vivo. Conversely, knockdown of E2F1 reduces SRC-3 expression. We demonstrate that the mechanism of E2F1 activity on SRC-3 promoter is independent of the E2F binding sites but relies on the Sp1 element located at bp +150/+160. Sp1, E2F1, and SRC-3 are specifically recruited to this Sp1 site and the interaction between E2F1 and Sp1 is essential to modulate SRC-3 expression. Moreover, SRC-3 coactivates E2F1 activity and thereby additively stimulates a further increase in SRC-3 expression in vivo. These results suggest that in cells with hyperactive E2F1, such as the case encountered in breast cancer cells, there is a positive feedback regulatory loop consisting of E2F1 and SRC-3 to maintain high levels of SRC-3 and E2F1 activity, which may partially interpret the oncogenic role of SRC-3 overexpression

bax基因促进细胞编程死亡的机理研究

bAX基因促进细胞编程死亡的机理研究曾桂生，陈亚兵，温龙平，俞春东，蔡毓（厦门大学肿瘤细胞工程国家专业实验室厦门361005）1980年WyllIEA.H.等通过糖皮激素诱导胸腺细胞死亡的实验，提出了细胞编程死亡（PrOgAMMEdCElldEATH..

Mycoepoxydiene suppresses RANKL-induced osteoclast differentiation and reduces ovariectomy-induced bone loss in mice

Mycoepoxydiene (MED) is a compound isolated from the marine fungal Diaporthe sp. HLY-1 associated with mangroves. MED has various biological effects such as anti-microbial, anti-cancer, and anti-inflammatory activities. However, the effect of MED on the differentiation of osteoclasts, the multinucleated bone-resorbing cells which play a crucial role in bone remodeling, is still unknown. In this study, we showed that MED could inhibit receptor activator of NF-kappa B ligand (RANKL)-induced osteoclast differentiation and the expression of three well-known osteoclast markers such as tartrate-resistant acid phosphatase, calcitonin receptor, and cathepsin K in bone marrow-derived macrophages. Furthermore, we found that MED inhibited the expression of nuclear factor of activated T cells c1, a key transcriptional factor in osteoclast differentiation, via inhibiting the phosphorylation of TAK1 and then blocking the activation of NF-kappa B and ERK1/2 pathways. Moreover, MED could prevent bone loss in ovariectomized mice. Taken together, we demonstrate for the first time that MED can suppress RANKL-induced osteoclast differentiation in vitro and ovariectomy-induced osteoporosis in vivo, suggesting that MED is a potential lead compound for the development of novel drugs for osteoporosis treatment