Cloning of human brevican cDNA and expression of its mRNA in human glioma

Objective: To clone the cDNA of human brevican secreting isoform and to investigate its mRNA expression in human glioma. Methods:The full-length cDNA of human brevican secreted isoform was cloned from a human anaplastic astrocytoma by RT-PCR, and the expression of human brevican mRNA in 22 cases of human glioma and 13 cases of non-glial brain tumors were investigated by in situ hybridization. Results:The cDNA which including the whole open reading frame of human brevican secreted isoform was obtained. In situ hybridization showed that brevican positive cells were present in all of the 22 cases of gliomas (100%), whereas none were found in the 13 cases of non-glial and metastasis brain tumors examined. Conclusion: The results suggest that brevican mRNA is highly and specifically expressed in human glioma.     

The elevated expression of osteopontin and NF-κB in human aortic aneurysms and its implication

The expression and significance of osteopontin (OPN) and NF-κB in patients with thoracic aortic aneurysm (TAA) and abdominal aortic aneurysm (AAA) were investigated.Thirteen TAA specimens,20 AAA specim...     

Inhibition of NF-κB by mutant IκBα enhances TNF-α-induced apoptosis in HL-60 cells by controlling bcl-xL expression

Backgound The aim of this study was to explore whether the inhibition of nuclear factor-κB (NF-κB)activation by mutant IκBα (S32,36→A) can enhance TNF-α-induced apoptosis of leukemia cells and to investigate the possible mechanism. Methods The mutant IκBα gene was transfected into HL-60 cells by liposome-mediated techniques. G418 resistant clones stably expressing mutant IκBα were obtained by the limiting dilution method. TNF-α-induced NF-κB activation was measured by electrophoretic mobility shift assay (EMSA). The expression of bcl-xL was detected by RT-PCR and Western blot after 4 hours exposure of parental HL-60 and transfected HL-60 cells to a variety of concentrations of TNF-α. The percentage of apoptotic leukemia cells was evaluated by flow cytometry (FCM). Results Mutant IκBα protein was confirmed to exist by Western blot. The results of EMSA showed that NF-κB activation by TNF-α in HL-60 cells was induced in a dose-dependent manner, but was almost completely inhibited by mutant IκBα repressor in transfected cells. The levels of bcl-xL mRNA and protein in HL-60 cells increased after exposure to TNF-α, but changed very little in transfected HL-60 cells. The inhibition of NF-κB activation by mutant IκBα enhanced TNF-α-induced apoptosis. Thecytotoxic effects of TNF-α were amplified in a time- and dose-dependent manner. Conclusions NF-κB activation plays an important role in the resistance to TNF-α-induced apoptosis. The inhibition of NF-κB by mutant IκBα could provide a new approach that may enhance the antileukemia effects of TNF-α or even of other cytotoxic agents.     

The expression and significance of NF-κB in endothelial cells of deep venous thrombosis

目的 探讨核因子-κB(NF-κB)在孕鼠深静脉血栓形成(DVT)后其血管内皮细胞中的动态变化及意义.方法 清洁级SD孕鼠54只,其中48只采用下腔静脉结扎法建立深静脉血栓模型后,随机分为血栓模型组、吡咯烷二硫代氨基甲酸盐(PDTC)干预组,分别于术后6、12、24、72 h处死;余6只为假手术组.比较血栓模型组和PDTC干预组静脉血栓的形成情况并观察其病理学形态,采用免疫组化SP法检测各组血管内皮细胞中NF-κB的表达水平.结果 NF-κB蛋白水平于静脉血栓形成后6 h开始升高,24 h达高峰,72 h后下降.与假手术组比较,血栓模型组和PDTC干预组各时间点静脉血管内皮细胞中NF-κB的表达均显著增强(P＜0.05).NF-κB抑制剂PDTC干预后静脉血管内皮细胞中NF-κB的表达显著下降,72 h后血栓重量与长度比值显著低于血栓模型组(P＜0.05).结论 孕鼠DVT血管内皮细胞中NF-κB明显激活,并介导血管内皮的损伤,抑制NF-κB的信号通路可能对DVT有潜在的治疗价值.     

Expression of MMP-9 and TIMP-1 in Lesions of Systemic Sclerosis and Its Implications

In order to investigate the role of MMP-9 and TIMP-1 in the pathogenesis of systemic sclerosis, the expression of MMP-9 and TIMP-1 was immunohistochemically detected in skin lesions of the patients with diffuse cutaneous systemic sclerosis, skin lesions of the patients with limited cutaneous systemic sclerosis, and skin tissues of normal subjects. The results showed that the expression of MMP-9 in lesions of diffuse cutaneous systemic sclerosis was significantly lower than that of normal skins （P〈0.05）. However, no significant difference in the level of MMP-9 in the limited cutaneous systemic sclerosis and normal skin was found. Meanwhile, the expression of TIMP-1 in lesions of diffuse cutaneous systemic sclerosis and limited cutaneous systemic sclerosis were significantly higher than that of normal skins （both P〈0.05）. It was suggested that the expression of MMP-9 and TIMP-1 might play an important role in the development of systemic sclerosis.     

The Role of IκBα in TNF-α-induced Apoptosis in Hepatic Stellate Cell Line HSC-T6

To investigate the role of NF-κB in TNF-α induced apoptosis in HSC-T6, a mutant IκBα was transfected into HSC-T6 cells by lipofectin transfection technique and its transient effect was examined 48 h after the transfection. The activation of NF-κB was detected by immune fluorescence cytochemistry and Western blotting with anti-p65 antibody. The apoptosis and the rate of inhibition by TNF-α in both transfected and untransfected HSC-T6 cells were measured respectively by FAC-Scan side scatter analysis and MTF methods. Our results showed that TNF-α could activate NF-κB in untransfected cells but not in transfected HSC-T6 cells. The percentage of apoptosis in transfected cells were significantly higher than that in the untransfected ones （P〈0.01） and it was also true of the inhibition rate （P〈0.01）. It is concluded that the resistance of HSC-T6 towards apoptosis induced by TNF-α can be mediated by NF-κB activation. The inhibition of NF-κB activation by mutant IκBα can attenuate the resistance of HSC-T6 cells and increase its sensitivity to TNF-α.     

Effect of Atorvastatin on Serum MMP-2,MMP-9 and TIMP-1 in Rabbits with Chronic Heart Failure

Objective: To observe the effects of atorvastatin on serum matrix metalloproteinase-2(MMP-2), matrix metalloproteinase-9(MMP-9), and the tissue inhibitor of metalloproteinase-1(TIMP-1) in the development of chronic heart failure. To investigate the role of atorvastatin in the therapy of chronic heart failure and determine its possible mechanism of action. Methods: Thirty Japanese Big Ear rabbits were randomly selected and divided into 3 groups: sham-operated group(SO group), heart failure control group(HC group) and heart failure atorvastatin therapy group(HA group), with 6, 12 and 12 animals in the respective groups. Volume overloading was produced in the HC group and HA group animals by creating an aortic insufficiency, induced by damaging the aortic valve with a catheter introduced through the carotid artery. After 14 days, abdominal aorta constriction was performed in order to obtain a pressure overload. Six weeks later rabbits in the HA group were administered atorvastatin 3mg. Kg-1.d-1 for 4 weeks, at which time the experiment was terminated. Arterial blood was drawn and serum levels of MMP-2, MMP-9 and TIMP-1 were measured in all groups at the same time using an ELISA method. Results: Structural and functional indicators of chronic heart failure(CHF) were seen in both the HC and HA groups, but atorvastatin significantly reduced the observed effects. The serum concentrations of MMP-2, MMP-9 and TIMP-1 were at low levels in all three groups at the start of the study, with no difference between them(P < 0.05). At the end of 6th week concentrations were significantly increased in the HC and HA groups compared with the SO group(P < 0.05), but there were no differences between the HC group and HA group(P > 0.05). The increased concentrations in HC group continued to the end of the experiment, but values in the HA group were all lower than those in the HC group by the end of the experiment(P < 0.05). Conclusion: Serum concentrations of MMP-2, MMP-9 and TIMP-1 increase significantly during the course of CHF, paralleling the pathological progress of CHF. Atorvastatin benefits CHF, and the decreased serum levels of MMP-2, MMP- 9 and TIMP-1 may be one of the drug's mechanism of action.     

Multifactor Regulation on Expressions of MMP-9 and TIMP-1 in Endometrial Stromal Cells

Recent evidence suggests that preparation of the endometrium for implantation is notmerely a question of adequate hormonal stimulation but that implantation also depends on theinteraction between the blastocyst and the endometrium and is mediated by cytokines,growthfactors,and adhesion molecules,which are produced and secreted by the endometrium andthe blastocyst[1].Implantation is a complex process that involves embryo apposition andattachment to the maternal endometrial epithelium,the extrace…     

The role of human leukocyte antigen in susceptibility and clinical manifestations of sarcoidosis.

ＴＨＥＲＯＬＥＯＦＨＵＭＡＮＬＥＵＫＯＣＹＴＥＡＮＴＩＧＥＮＩＮＳＵＳＣＥＰＴＩＢＩＬＩＴＹＡＮＤＣＬＩＮＩＣＡＬＭＡＮＩＦＥＳＴＡＴＩＯＮＳＯＦＳＡＲＣＯＩＤＯＳＩＳＸｕＺｕｏｊｕｎ徐作军，ＺｈａｏＹａｎ赵岩，ＺｈｕＸｉｌｉｎ朱席琳，ＱｉｕＣｈａｎ...     

Effects of Fucoidan on the expression of MMP2 and TIMP-2 in human mesangial cells cultured with growth factor β1

目的 观察褐藻多糖硫酸酯(Fucoidan FPS)对TGF-β1下刺激人肾小球系膜细胞MMP-2、TIMP-2分泌的影响,探讨其在慢性肾衰竭(Chronic Renal Failure CRF)防治中的意义.方法 用TGF-β1作为刺激因子,孵育体外培养的人肾小球系膜细胞(Human Mesangial cell HMC),并分别用不同浓度的FPS干预,具体分组如下:模型组(TGF-β14 ng/ml)、FPS高剂量组(TGF-β14 ng/ml +FPS 100 ug/ml)、FPS中剂量组(TGF-β14 ng/ml + FPS 50 ug/ml)、FPS低剂量组(TGF-β14 ng/ml + FPS 25 ug/ml)、FPS对照组(FPS 100ug/ml)、正常对照组.ELISA技术检测HMC培养上清液中MMP-2、TIMP-2蛋白表达水平,Realtime PCR技术检测HMC 的MMP-2、TIMP-2 mRNA表达水平.结果 与正常对照组相比,TGF-β1能减少HMC的MMP-2蛋白及mRNA表达 (P＜0.01),增加HMC的TIMP-2蛋白及mRNA表达 (P＜0.01);FPS能阻断TGF-β1刺激下HMC的MMP-2蛋白及mRNA表达减少、TIMP-2蛋白及mRNA表达增加(P＜0.01,P＜0.05);FPS对照组与正常对照相比MMP-2、TIMP-2蛋白及mRNA表达差异无统计学意义(P＞0.05).结论 褐藻多糖硫酸酯能阻断TGF-β1刺激下人肾小球系膜细胞MMP-2、TIMP-2表达的比例失衡,阻断了由MMP-2、TIMP-2比例失衡导致的ECM积聚,从而延缓了慢性肾衰竭的进展.     

Cloning of human melanoma antigen MAGE-A9 and its expression in hepatocellular carcinomas

Objective：To express the melanoma associated gene MAGE-A9 recombinant protein, obtain the anti-MAGE-A9 monoclonal antibody and to examine the expression of MAGE-A9 in hapatocellular carcinoma specimens. Methods：MAGE-A9 cDNA was cloned from human hepatocellular carcinoma tissue by using RT-PCR, and then subcloned into the plasmid pMD18-T. After sequencing, the MAGE-A9 was cloned into the prokaryotic expression vector pBAD/gⅢ to construct the recombinant expression vector pBAD/gⅢ - MAGE-A9, and was transformed into E. coli TOP10. The recombinant MAGE-A9 protein was expressed under induction of L-Arabinose, and was purified through Hitrap column. The anti-MAGE-A9 monoclonal antibody was generated. The expression of MAGE-A9 in hepatocellular carcinoma specimens was examined through ABC assay. Results：The cDNA sequence of the cloned MAGE-A9 gene was consistent with the reported sequence. By affinity column and SDS-PAGE, the purified MAGE-A9 fusion protein displayed a band of Mr 35,000, and subsequently the anti-MAGE-A9 monoclonal antibody was obtained. We found that MAGE-A9 expressed in the cytoplast of positive cells and MAGE-A9 antigen was detected in 8 cases out of 39 （21%） hepatocellular carcinoma specimens. Conclusion：MAGE-A9 antigen was expressed in a fair proportion of hepatocellular carcinoma specimens, these patients might be suitable candidates for immune involving antigen, encoded by the MAGE-A9 gene.     

LMP1 activates NF- κB via degradation of IκBα in nasopharyngeal carcinoma cells

Objective To elucidate the mechanisms by which Epstein- Barr virus- encoded latent membran e protein 1 activates NF- κB in nasopharyngeal carcinoma cells. Methods A tetracycline- regulated LMP1- expressing nasopharyngeal carcinoma cell line, T et- on- LMP1- HNE2, was used as the cell model. The kinetics of the expression of proteins, including LMP1, IκBα and IκBβ, was analyzed by Western blotting . The subcellular localization of NF- κB (p65) was detected by indirect immuno fluorescence assay. The NF- κB transactivity was studied by transient transfec tion and reporter gene assay. Results IκBα was phosphorylated and degraded after the inducible expression of LMP1, a lthough the total protein levels remained stable. The steady- state level of to tal IκBβ protein may have resulted from the initiation of an autoregulation lo op after the activation of NF- κB. No change in the IκBβ level was detected . NF- κB (p65) was translocated from the cytoplasm to the nucleus following de gradation of IκBα. After the introduction of the dominant- negative mutant of IκBα (Del 71) into Tet- on- LMP1- HNE2 cells, both nuclear translocation and transactivation of NF- κB induced by LMP1 was significantly inhibited. Conclusions The results indicated that in nasopharyngeal carcinoma cells, LMP1 activated NF - κB via phosphorylation and degradation of IκBα, but not IκBβ. The do minant- negative mutant of IκBα (Del 71) could completely inhibit both the nuc lear translocation and transactivation of NF- κB induced by LMP1.     

The expression of Fas, FasL and their biological behavior in human cervical carcinoma

Objective： To investigate the relationship between the expression of Fas and Fas ligand （FasL） and its biological behavior in human cervix carcinoma. Methods： Immunohistochemisty technique was used to detect the expression of Fas and FasL in 47 cases of cervical carcinoma, 16 cases of cervical interaepithelial neoplasia, 10 cases of chronic cervicitis and 10 cases of normal cervix. TUNEL technique was used to observe the apoptic cells in 47 cases of cervical carcinoma. Retrospective study was carried out to find the relationship between the expression of Fas and FasL and cell apoptosis, clinical stage, pathological classification, lymph node metastasis, prognosis and age. Results： The expression of Fas and FasL was significantly different in different cervix （P 〈 0.01）, and also related to the degree of differentiation, lymph node metastasis and prognosis （P 〈 0.05）. But had no relation with clinical stage or age （P 〉 0.05） ; Cervix carcinoma cells apoptosis in different pathological classification appeared negative relation （Rs=-0.35, P 〈 0.05 ）. Cervix carcinoma cell apoptosis was significantly higher in Fas-positive and FasL- positive than that in Fas-negative and FasL-negative （P 〈 0.05）. By retrospective investigation, Fas-negative and FasL-positive were related to poor prognoses of the patients with cervical carcinoma （P 〈 0.05 ）. Conclusion： The development of apoptosis in cervix carcinoma has a promoting regulation function in Fas and FasL expression. Gene treatment can alter apoptosis abnormality, thus induce apoptosis in cancerous cell expressing Fas and FasL. Fas or FasL may be taken as a marker in the prognostic character- ization.     

Expression of survivin, a novel apoptosis inhibitor and cell cycle regulatory protein, in human gliomas

Recently, a novel anti-apoptosis gene, named survivin,was identified as a structurally unique member of the inhibitor of apoptosis protein (IAP) family. The gene is located on chromosome 17q25. Survivin is a 16. 5 kDa protein that is expressed in vivo in common human cancers, but not in normal adjacent tissue,^1 during the     

Adenovirus-mediated overexpression of novel mutated IκBα inhibits nuclear factor κB activation in endothelial cells

Nuclear factor κB （NF-κB） overactivation, requiring phosphorylation and degradation of its inhibitor IκBα, is the basis for chronicity of airway inflammation in asthma. Based on our previous plasmid pShuttle-IκBα, carrying an IκBα gene from human placenta, we optimized a novel IκBα mutant （IκBα） gene, constructed and characterized its replication-deficient recombinant adenovirus （AdIκBαM）, and tested whether AdIκBαM-mediated overexpression of IκBαM could inhibit the NF-κB activation in endothelial cells.     

Inhibitory effects of polymyxin B on NF-κB activation and expression of procollagen I, III in pre-eclamptic umbilical artery smooth muscle cells

Background It has been identified that in patients with pre-eclampsia, several factors were released into the serum, which can regulate the proliferation of vascular smooth muscle cells and stimulate the synthesis of extracellular matrix （ECM）. However, the signal transduction pathway of the growth factors and cytokines synthesized by endothelial cells leading to the proliferation and ECM synthesis of human umbilical arterial smooth muscle cell （HUASMC） is unknown. The aim of this study was to research the effects of protein kinase C （PKC） inhibitor, polymyxin B （PMB）, on the proliferation, nuclear factor-kappa B （NF-KB） activation, and expression of procollagen Ⅰ and Ⅲ mRNA in HUASMC cultured with pre-eclamptic umbilical sera. Methods Normal HUASMCs were treated with pre-eclamptic umbilical serum （pre-eclamptic group）, preeclamptic umbilical serum plus PMB （PMB group）, or normal umbilical serum （normal group）. The expression of Ⅰ-kB, NF-kB was detected by Western blotting after the HUASMC was incubated for 2 hours. The proliferation of HUASMC was evaluated by MTT, the cell cycle was analyzed by flow cytomerty, and the expression of procollagen Ⅰ,Ⅲ mRNA was measured by RT-PCR assay after HUASMC was incubated for 48 hours. ANOVA was used for statistical analysis. Results Umbilical sera in pre-eclampsia could stimulate the proliferation, the DNA synthesis, the transition of G0＋G1 phase or G2/M phase to S and phase, the activation of NF-kB, and the expression of procollagen Ⅰ mRNA of HUASMC as compared with normal umbilical sera （P〈0.01）. PMB could inhibit the proliferation, the DNA synthesis, the transition of G0＋G1 or G2/M phase phase to S phase, the activation of NF-KB, and the expressions of procollagen Ⅰ mRNA of HUASMC stimulated by umbilical sera in pre-eclampsia （P〈0.01）. Conclusion PKC-NF-KB signal transduction pathway may play a key role in SMC phenotype modulation, which is more important in the pathogenesis of placental blood vessel in pre-eclampsia.