Expression, purification, and in vitro refolding of soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a new member of the TNF superfamily. Here, a recombinant form of the extracellular domain of the TRAIL (sTRAIL) was expressed in Escherichia coli BL21(DE3) under the control of a T7 promoter. The resulting insoluble bodies were separated from cellular debris by centrifugation and solubilized with 8 M urea. A rapid and simple on-column refolding procedure was developed. It was applied and then the refolded sTRAIL was purified by anion-exchange chromatography. The purified final product was >98% pure by SDS-PAGE stained with Coomassie brilliant blue R-250. Mass spectroscopic analysis indicated the protein to be 19.2 kDa, which equalled the theoretically expected mass. N-terminal sequencing of refolding sTRAIL showed the sequence which corresponded to the designed protein. The renatured protein displayed its immunoreactivity with the antibodies to TRAIL protein by Western blotting. The purified sTRAIL had a strong cytotoxic activity against human cervical cancer HeLa cells with ED50 about 1.5 mg/L. Circular dichroism and fluorescence spectrum analysis showed that the refolded sTRAIL had a structure similar to that of native protein with beta-sheet secondary structure. This efficient procedure of sTRAIL renaturation may be useful for the mass production of this therapeutically important protein.     

The tumor necrosis factor-related apoptosis-inducing ligand receptors TRAIL-R1 and TRAIL-R2 have distinct cross-linking requirements for initiation of apoptosis and are non-redundant in JNK activation

Overexpression of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors, TRAIL-R1 and TRAIL-R2, induces apoptosis and activation of NF-kappaB in cultured cells. In this study, we have demonstrated differential signaling capacities by both receptors using either epitope-tagged soluble TRAIL (sTRAIL) or sTRAIL that was cross-linked with a monoclonal antibody. Interestingly, sTRAIL was sufficient for induction of apoptosis only in cell lines that were killed by agonistic TRAIL-R1- and TRAIL-R2-specific IgG preparations. Moreover, in these cell lines interleukin-6 secretion and NF-kappaB activation were induced by cross-linked or non-cross-linked anti-TRAIL, as well as by both receptor-specific IgGs. However, cross-linking of sTRAIL was required for induction of apoptosis in cell lines that only responded to the agonistic anti-TRAIL-R2-IgG. Interestingly, activation of c-Jun N-terminal kinase (JNK) was only observed in response to either cross-linked sTRAIL or anti-TRAIL-R2-IgG even in cell lines where both receptors were capable of signaling apoptosis and NF-kappaB activation. Taken together, our data suggest that TRAIL-R1 responds to either cross-linked or non-cross-linked sTRAIL which signals NF-kappaB activation and apoptosis, whereas TRAIL-R2 signals NF-kappaB activation, apoptosis, and JNK activation only in response to cross-linked TRAIL.     

The release of soluble forms of TRAIL and DR5 by neutrophils of oral cavity cancer patients

In the present study we examined the release of the soluble form of TRAIL by neutrophils (PMN) derived from patients with oral cavity cancer. Simultaneously, we estimated the ability of PMNs of these patients to release the soluble form of DR5 receptor, a natural regulatory protein of TRAIL. The obtained results were confronted with the serum levels of sTRAIL and sDR5. The cells were isolated from 21 patients with squamous cell carcinoma of oral cavity at diagnosis and three weeks after surgery treatment. For comparative purposes we performed similar examinations in autologous peripheral blood mononuclear cells (PBMC). Cytoplasmic protein fractions of the cells were analyzed for the presence of TRAIL and DR5 by western blotting. Soluble TRAIL and soluble DR5 concentrations in the culture supernatants of cells were confronted with their serum levels using ELISA kit. PMN and PBMC of the whole cancer patient group expressed decreased TRAIL protein and unchanged expression of DR5 receptor in comparison with the control group. Unchanged release of sTRAIL by PMNs of patients in Stage II was accompanying the decrease of the ability of PBMC to secrete this protein. In patients in Stage IV the secretion of sTRAIL by PMNs and PBMC was impaired. In contrast to changes in sTRAIL secretion by PMN and PBMC of oral cavity cancer patients, the secretion of sDR5 by these cells was unchanged. The serum levels of sTRAIL were increased in patients in Stage II before treatment and decreased in the same patients after treatment. The altered ability of PMN of PBMC to secrete sTRAIL may have different implications for the immune response of patients with oral cavity cancer cells at different stages of disease.     

肿瘤坏死因子相关的凋亡诱导配体(TRAIL)cDNA的克隆、表达和活性测定

肿瘤坏死因子相关的凋亡诱导配体 (TRAIL)能选择性诱导肿瘤细胞凋亡 .为利用基因工程技术获得重组TRAIL蛋白可溶性片段 (sTRAIL) ,设计 1对引物 .利用PCR技术特异性扩增出sTRAIL的cDNA ,克隆于质粒pGEM 3Zf( )的EcoRⅠ和PstⅠ位点 .经测序证明序列正确后克隆于表达质粒pBV2 2 0的EcoRⅠ和PstⅠ位点 ,转化大肠杆菌DH5α .转化菌株经温度诱导 ,SDS PAGE检测和Western印迹鉴定 ,获得重组sTRAIL的高水平非融合表达菌株 .表达量占菌体总蛋白的 2 0 % .对其表达产物进行了初步纯化 ,SDS PAGE结果显示纯度可达 90 %以上 .用L92 9细胞测定其生物学活性表明 ,重组蛋白在体外能明显诱导肿瘤细胞凋亡     

Efficacy of adenovirally expressed soluble TRAIL in human glioma organotypic slice culture and glioma xenografts

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in malignant cells, including gliomas, and is currently in anticancer clinical trials. However, the full-length and tagged forms of TRAIL, unlike the untagged ligand (soluble TRAIL (sTRAIL)), exhibits toxicity against normal cells. Here, we report the generation and testing of an adenovirus (AdsTRAIL) that expresses untagged sTRAIL in an intracranial xenograft model and a human glioma organotypic slice culture model. AdsTRAIL efficiently induced apoptosis in glioma cell lines, including those resistant to sTRAIL, but not in normal human astrocytes (NHAs). It inhibited anchorage-independent glioma growth and exerted a bystander effect in transwell assays. Intratumoral injections of AdsTRAIL in a rodent intracranial glioma model resulted in reduced tumor growth and improved survival compared with Ad-enhanced green fluorescent protein (EGFP)- or vehicle-treated controls without toxicity. Human glioma organotypic slices treated with AdsTRAIL demonstrated apoptosis induction and caspase activation.     

Generation of a novel proform of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein that can be reactivated by matrix metalloproteinases

TRAIL has been suggested to induce the cell death in various tumor cells but not in normal cells; however, several studies have provided the evidence that TRAIL can induce the cell death in some normal cells including human normal hepatocytes, suggesting that TRAIL may show hepatic toxicity in human. In this study, we designed a pro-form of TRAIL (sTRAIL:IL-18) in that soluble TRAIL (sTRAIL) is fused to IL-18, and a matrix metalloproteinases (MMPs) cleavage site is introduced at the connecting site. We showed that sTRAIL:IL-18 has significantly diminished the killing activity in HeLa cells but regains the activity by releasing the free sTRAIL through MMP-2-mediated cleavage. In addition, the killing activity of sTRAIL:IL-18 was significantly increased in HeLa cells when active MMP-2 was produced by TNF-alpha. Taken together, the data suggested that the sTRAIL:IL-18 can be reactivated at the specialized areas where MMPs are pathologically produced.     

Soluble Tumor Necrosis Factor Related Apoptosis Inducing Ligand Level as a Predictor of Severity of Sepsis and the Risk of Mortality in Septic Patients

Background

Tumor necrosis factor related apoptosis inducing ligand (TRAIL) as a member of the TNF gene superfamily induces apoptosis primarily in tumor cells. TRAIL also plays an important role in the modulation of inflammatory responses, especially in the process of immune paralysis. The aim of the present study was to examine soluble TRAIL (sTRAIL) levels in septic patients in an attempt to explore the association between sTRAIL level and the risk of mortality.

Methods

Plasma sTRAIL levels were detected by ELISA in 50 septic patients and 20 healthy volunteers. HLA-DR expression in monocytes was detected by flow cytometry. Selective biochemical parameters were recorded, and patients were monitored in a 28-day period for mortality.

Results

The mean plasma sTRAIL level in septic patients was significantly lower than that in healthy controls (16.9±8.3 vs. 68.3±8.6 pg/ml, P<0.01), and was significantly higher in 28-day survivors than those in non-survivors (19.4±9.8 vs. 13.9±4.7 pg/ml, P<0.05). Univariate analysis indicated that plasma sTRAIL level was positively correlated with monocyte and lymphocyte counts and HLA-DR expression level (r = 0.5, P<0.01; r = 0.3, P<0.05; r = 0.43, P<0.01, respectively). STRAIL level was negatively correlated with APACHE II score, BUN and age (r = −0.48, P<0.01; r = −0.29, P<0.05; r = −0.45, P<0.01, respectively). Multiple linear regression analysis indicated that the predictor of plasma soluble TRAIL level was HLA-DR expression (P<0.01).

Conclusion

Low plasma sTRAIL levels were associated with immune paralysis and a high risk of mortality in patients with septic shock. sTRAIL may prove to be a potential biomarker of immune function and predict the survival of septic patients.     

Simultaneous inhibition of epidermal growth factor receptor (EGFR) signaling and enhanced activation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor-mediated apoptosis induction by an scFv:sTRAIL fusion protein with specificity for human EGFR

Epidermal growth factor receptor (EGFR) signaling inhibition by monoclonal antibodies and EGFR-specific tyrosine kinase inhibitors has shown clinical efficacy in cancer by restoring susceptibility of tumor cells to therapeutic apoptosis induction. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-cancer agent with tumor-selective apoptotic activity. Here we present a novel approach that combines EGFR-signaling inhibition with target cell-restricted apoptosis induction using a TRAIL fusion protein with engineered specificity for EGFR. This fusion protein, scFv425:sTRAIL, comprises the EGFR-blocking antibody fragment scFv425 genetically fused to soluble TRAIL (sTRAIL). Treatment with scFv425:sTRAIL resulted in the specific accretion to the cell surface of EGFR-positive cells only. EGFR-specific binding rapidly induced a dephosphorylation of EGFR and down-stream mitogenic signaling, which was accompanied by cFLIP(L) down-regulation and Bad dephosphorylation. EGFR-specific binding converted soluble scFv425:sTRAIL into a membrane-bound form of TRAIL that cross-linked agonistic TRAIL receptors in a paracrine manner, resulting in potent apoptosis induction in a series of EGFR-positive tumor cell lines. Co-treatment of EGFR-positive tumor cells with the EGFR-tyrosine kinase inhibitor Iressa resulted in a potent synergistic pro-apoptotic effect, caused by the specific down-regulation of c-FLIP. Furthermore, in mixed culture experiments binding (L)of scFv425:sTRAIL to EGFR-positive target cells conveyed a potent apoptotic effect toward EGFR-negative bystander tumor cells. The favorable characteristics of scFv425:sTRAIL, alone and in combination with Iressa, as well as its potent anti-tumor bystander activity indicate its potential value for treatment of EGFR-expressing cancers.     

Association between Complement C3 and Prevalence of Fatty Liver Disease in an Adult Population: A Cross-Sectional Study from the Tianjin Chronic Low-Grade Systemic Inflammation and Health (TCLSIHealth) Cohort Study

Activation of the innate immune system plays a key role in the development of fatty liver disease (FLD). The complement system is a major humoral component of the innate immune response and complement C3 plays a central role, implying that C3 may be a powerful predictor or therapeutic target for FLD. However, few studies have assessed the association between C3 and FLD in a large population. Here we use a cross-sectional study to investigate the link between serum C3 levels and FLD. Participants were recruited from Tianjin Medical University’s General Hospital-Health Management Centre. Serum C3 was measured using immunoturbidimetry method and FLD was diagnosed by liver ultrasonography. Multiple logistic regression analysis was used to examine the association between quartiles of C3 and FLD prevalence. The overall prevalence of nonalcoholic fatty liver disease (NAFLD) and alcoholic fatty liver disease (AFLD) were 37.3% and 10.1%, respectively. After adjusting for covariates, the odds ratio of having NAFLD or AFLD (only in males) in the fourth quartile of C3 compared with the first quartile was 4.13 times greater (95% confidence interval, 2.97-5.77) (trend P values < 0.0001) and 2.09 times greater (95% confidence interval, 1.08-4.18) (trend P values = 0.02). This is the first study to demonstrate that serum C3 levels are independently associated with a higher prevalence of NAFLD and AFLD (only in males) in an adult population. Further studies are needed to establish a causal link and determine the precise role of C3 in FLD.     

Purification and characterization of recombinant sTRAIL expressed in Escherichia coli

As a potential anti-tumor protein, tumor necrosis factor-related apoptosis-inducing ligand(TRAIL) has drawn considerable attention. This report presented the purification and characterization ofsoluble TRAIL, expressed as inclusion bodies in E. coli. sTRAIL inclusion bodies were solubilized andrefolded at a high concentration up to 0.9 g/L by a simple dilution method. Refolded protein was purifiedto electrophoretic homogeneity by a single-step immobilized metal affinity chromatography. The purifiedsTRAIL had a strong cytotoxic activity against human pancreatic tumor cell line 1990, with EDs0 about 1.5mg/L. Circular dichroism and fluorescence spectrum analysis showed that the refolded sTRAIL had astructure similar to that of native protein with 13-sheet secondary structure. This efficient procedure ofsTRAIL renaturation may be useful for the mass production of this therapeutically important protein.     

Selective induction of apoptosis in leukemic B-lymphoid cells by a CD19-specific TRAIL fusion protein

Although the treatment outcome of lymphoid malignancies has improved in recent years by the introduction of transplantation and antibody-based therapeutics, relapse remains a major problem. Therefore, new therapeutic options are urgently needed. One promising approach is the selective activation of apoptosis in tumor cells by the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). This study investigated the pro-apoptotic potential of a novel TRAIL fusion protein designated scFvCD19:sTRAIL, consisting of a CD19-specific single-chain Fv antibody fragment (scFv) fused to the soluble extracellular domain of TRAIL (sTRAIL). Potent apoptosis was induced by scFvCD19:sTRAIL in several CD19-positive tumor cell lines, whereas normal blood cells remained unaffected. In mixed culture experiments, selective binding of scFvCD19:sTRAIL to CD19-positive cells resulted in strong induction of apoptosis in CD19-negative bystander tumor cells. Simultaneous treatment of CD19-positive cell lines with scFvCD19:sTRAIL and valproic acid (VPA) or Cyclosporin A induced strongly synergistic apoptosis. Treatment of patient-derived acute B-lymphoblastic leukemia (B-ALL) and chronic B-lymphocytic leukemia (B-CLL) cells resulted in strong tumoricidal activity that was further enhanced by combination with VPA. In addition, scFvCD19:sTRAIL prevented engraftment of human Nalm-6 cells in xenotransplanted NOD/Scid mice. The pre-clinical data presented here warrant further investigation of scFvCD19:sTRAIL as a potential new therapeutic agent for CD19-positive B-lineage malignancies. This work was supported by Schickedanz KinderKrebs Stiftung (JS) and grants from the Association “Kaminkehrer helfen krebskranken Kindern” (CK, GHF), the Association of supporters of the University of Erlangen Childrens’s Hospital (GHF) and the Dutch Cancer Society (RUG 2002-2668 and 2005-3358) (EB, BC, WH). Julia Stieglmaier and Edwin Bremer contributed equally.     

Delivery of sTRAIL variants by MSCs in combination with cytotoxic drug treatment leads to p53-independent enhanced antitumor effects

Mesenchymal stem cells (MSCs) are able to infiltrate tumor tissues and thereby effectively deliver gene therapeutic payloads. Here, we engineered murine MSCs (mMSCs) to express a secreted form of the TNF-related apoptosis-inducing ligand (TRAIL), which is a potent inducer of apoptosis in tumor cells, and tested these MSCs, termed MSC.sTRAIL, in combination with conventional chemotherapeutic drug treatment in colon cancer models. When we pretreated human colorectal cancer HCT116 cells with low doses of 5-fluorouracil (5-FU) and added MSC.sTRAIL, we found significantly increased apoptosis as compared with single-agent treatment. Moreover, HCT116 xenografts, which were cotreated with 5-FU and systemically delivered MSC.sTRAIL, went into remission. Noteworthy, this effect was protein 53 (p53) independent and was mediated by TRAIL-receptor 2 (TRAIL-R2) upregulation, demonstrating the applicability of this approach in p53-defective tumors. Consequently, when we generated MSCs that secreted TRAIL-R2-specific variants of soluble TRAIL (sTRAIL), we found that such engineered MSCs, labeled MSC.sTRAIL^DR5, had enhanced antitumor activity in combination with 5-FU when compared with MSC.sTRAIL. In contrast, TRAIL-resistant pancreatic carcinoma PancTu1 cells responded better to MSC.sTRAIL^DR4 when the antiapoptotic protein XIAP (X-linked inhibitor of apoptosis protein) was silenced concomitantly. Taken together, our results demonstrate that TRAIL-receptor selective variants can potentially enhance the therapeutic efficacy of MSC-delivered TRAIL as part of individualized and tumor-specific combination treatments.     

Adeno-associated virus-mediated gene transfer of a secreted form of TRAIL inhibits tumor growth and occurrence in an experimental tumor model

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces cell death in various tumor cells, but relatively spares normal cells. Recombinant adeno-associated virus (rAAV) vectors have a number of advantages including in vivo long-term gene expression. Here, we assessed the biological activity of a novel, secreted form of TRAIL (sTRAIL) for cancer gene therapy using a rAAV2 vector. METHODS: A plasmid and rAAV2 vectors were constructed encoding sTRAIL composed of a leader sequence, the isoleucine zipper, and the active domain of TRAIL (aa 95-281). The functionality of sTRAIL was validated by cell viability, FACS analysis, caspase-3 activity, and TUNEL staining. rAAV-sTRAIL was injected intratumorally to nude mice bearing human A549 lung tumor cells. Nude mice received A549 tumor cells after intravenous delivery of rAAV-sTRAIL. The antitumor effect was then evaluated by measuring tumor regression and occurrence in the experimental animal. RESULTS: sTRAIL was released from cells transfected with the sTRAIL expression construct or transduced with rAAV-sTRAIL, and induced apoptosis in cancer cells, but spared normal fibroblast cells. Secreted sTRAIL formed oligomers including trimers with intersubunit disulfide. Purified sTRAIL exerted much lower cytotoxicity on primary human hepatocytes compared to recombinant TRAIL. Intratumoral delivery of rAAV-sTRAIL significantly inhibited growth of A549 tumors established in nude mice. A number of apoptotic tumor cells were detected by TUNEL staining in mice treated with rAAV-sTRAIL. Systemic pretreatment with rAAV-sTRAIL significantly inhibited tumor formation in nude mice. CONCLUSION: The results suggest that rAAV-sTRAIL may be useful for local or systemic cancer gene therapy for treating TRAIL-sensitive tumors.     

Construction and expression of sTRAIL–melittin combining enhanced anticancer activity with antibacterial activity in Escherichia coli

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), as an anticancer protein with tumor-selective apoptotic activity, has been examined for use in clinical application. Melittin, an antibacterial peptide isolated from the bee Apis mellifera, has shown strong cytotoxicity to both tumor and normal cells. To ameliorate the cytotoxicity of melittin on cells and enhance the activity of TRAIL on cancer cells, we constructed a novel fusion protein, sTRAIL–melittin, containing a small ubiquitin-related modifier (SUMO) tag and expressed this fusion protein in Escherichia coli. Data showed that expression of the soluble fusion protein with the SUMO tag was approximately 85 % of total target protein which was much higher than that without the SUMO tag (approximately 10 %); sTRAIL–melittin was easily purified using Ni-NTA affinity chromatography and the tag was removed easily using SUMO-specific protease. To assay anticancer activity and side effects, methyl thiazolyl tetrazolium, hemolytic, and apoptosis assays were employed. Results demonstrated that sTRAIL–melittin had cytotoxic and apoptotic activity in K562 leukemia cells and HepG2 liver carcinoma cells, while it had only a minimal effect on erythrocytes and normal HEK293 cells. This indicates that the cytotoxicity of sTRAIL–melittin in normal cells was low and the anticancer activity of the fusion protein in tumor cells was significantly enhanced compared with sTRAIL (P?<?0.01). Furthermore, we found that sTRAIL–melittin also showed antibacterial activity to Staphylococcus aureus due to the presence of the melittin domain. Therefore, TRAIL fused with an antibacterial peptide may be a promising novel TRAIL-based anticancer treatment strategy.     

Impact of soluble tumor necrosis factor-related apoptosis-inducing ligand released by engineered adipose mesenchymal stromal cells on white blood cells

Background aimsThe proapoptotic protein tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is physiologically expressed by immune cells and performs regulatory functions in infections, autoimmune diseases and cancer, where it acts as a tumor suppressor. Adipose-derived mesenchymal stromal cells (AD-MSCs) also may play immunomodulatory roles in both primary and acquired immune responses. We have previously demonstrated the efficacy of an anticancer gene therapy based on AD-MSC engineered to secrete a soluble TRAIL variant (sTRAIL) against pancreatic cancer. However, the impact of AD-MSC sTRAIL on leukocyte subsets has been not yet considered also to predict a possible immunotoxicity profile in the clinical translation of this cell-based anticancer strategy.MethodsMonocytes, polymorphonuclear cells and T lymphocytes were freshly isolated from the peripheral blood of healthy donors. Immunophenotype and functional (DR4 and DR5) and decoy (DcR1 and DcR2) TRAIL receptors were tested by flow cytometry. The viability of white blood cells treated with sTRAIL released by gene-modified AD-MSC or co-cultured with AD-MSC sTRAIL was then evaluated by both metabolic assays and flow cytometry. In addition, cytokine profile in co-cultures was analyzed by multiplex enzyme-linked immunosorbent assay.ResultsMonocytes and polymorphonuclear cells showed high positivity for DR5 and DcR2, respectively, whereas T cells revealed negligible expression of all TRAIL receptors. Irrespective of TRAIL receptors’ presence on the cell membrane, white blood cells were refractory to the proapoptotic effect displayed by sTRAIL secreted by gene-modified AD-MSC, and direct cell-to-cell contact with AD-MSC sTRAIL had negligible impact on T-cell and monocyte viability. Cytokine crosstalk involving interleukin 10, tumor necrosis factor alpha, and interferon gamma secreted by T lymphocytes and vascular endothelial growth factor A and interleukin 6 released by AD-MSC was highlighted in T-cell and AD-MSC sTRAIL co-cultures.ConclusionsIn summary, this study demonstrates the immunological safety and thus the clinical feasibility of an anticancer approach based on AD-MSC expressing the proapoptotic molecule sTRAIL.     

Determination of paralytic shellfish poisoning (PSP) toxins in UK shellfish

A study was conducted to aid the interpretation of data generated by parallel testing of the qualitative Jellett Rapid Test (JRT) and the mouse bioassay (MBA) for detection of paralytic shellfish poisoning (PSP) toxins within the UK statutory shellfish biotoxin monitoring programme. A selection of stored sample extracts subjected to testing by MBA and/or JRT were further analysed by liquid chromatography with fluorescence detection (LC–FLD) to provide additional information on the concentrations of PSP toxins and toxin profiles.Results, from this study, demonstrate the potential of the JRT to effectively screen out PSP toxin negative shellfish samples and samples containing low concentrations of toxins from UK monitoring programmes. Additionally, data generated using LC–FLD highlights the potential of introducing alternative analytical techniques to completely replace the requirement for the MBA.     

代谢正常肥胖个体发生非酒精性脂肪性肝病的相关性研究

目的:探讨代谢正常肥胖(Metabolically healthy obese,MHO)个体与非酒精性脂肪性肝病(Nonalcoholic fatty liver disease,NAFLD)发生的相关性。方法:选择2006年4月～2010年1月来湖南省人民医院体检中心体检人群共4076例,排除过量饮酒者、乙肝标志物阳性者及相关资料不全者共2830例纳入本研究。其中1367例在1～3年后再次体检。记录受检者身高、体重、血压、血脂、空腹血糖、腹部B超结果。NAFLD采用2010年中华医学会肝病学分会诊断标准中影像学诊断定义,行腹部B超检查进行诊断。结果:1.我院体检人群中MHO合并NAFLD者占51.34%,明显高于正常对照组(P=0.000),MHO组发生NAFLD的OR值为19.967(95%CI,12.646-31.533;P=0.000)。2.随访1～3年后,MHO中NAFLD发病率高于正常对照组(44.44%vs 7.02%,OR=10.600,95%CI,4.873-23.058;P=0.000)。结论:MHO个体合并NAFLD比例较正常对照者升高,MHO个体增加NAFLD患病风险。     

可溶性人TRAIL分子的制备及其抗肿瘤活性

TRAIL(TNF relatedapoptosis inducingligand)是1 995年发现的一个新的TNF超家族成员.使人感兴趣的是该分子既可广泛介导多种组织来源的肿瘤细胞发生凋亡而基本不影响正常细胞的功能,也可引起许多对于FasL和TNF α有抗性的细胞凋亡[1 ,2 ] .因此,有可能成为一种新的抗肿瘤药物.     

Construction and characterization of two versions of bifunctional EGFP–sTRAIL fusion proteins

The extracellular portion (amino acids 95–281 or 114–281) of the human tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) was genetically linked to the C terminus of the fluoresce-enhanced green fluorescent protein variant (EGFP) to generate two versions of EGFP–sTRAIL fusion proteins, designated EGFP–sTR95 and EGFP–sTR114, respectively. The two versions of EGFP–sTRAIL fusion proteins both induce extensive apoptosis in lymphoid as well as nonlymphoid tumor cell lines. In addition, the two versions of fusion proteins retain similar fluorescence spectra to those of EGFP and have shown the specific binding to TRAIL receptor-positive cells; thus, the stained cells could be analyzed with flow cytometry. Hence, the two versions of fusion proteins represent a readily obtainable source of biologically active sTRAIL that may prove useful in exploit fully the characteristics of both the soluble TRAIL and its receptor system.     

不同亚型脂联素与非酒精性脂肪性肝病的关系

目的:探究总脂联素(total adiponectin,total APN)和高分子量脂联素(high-molecular-weight adiponectin,HMW APN)与非酒精性脂肪性肝病(Nonalcoholic Fatty Liver Disease,NAFLD)的关系。方法:连续纳入50名男性健康男性及50名非酒精性脂肪性肝病男性患者,收集患者临床资料及其他临床生化数据,通过ELISA法检测总脂联素、支链氨基酸及可溶性晚期糖基化终末产物含量,Western blot法测定高分子量、中分子量和低分子量脂联素水平,进一步分析其相关性。结果:与对照组的健康受试者相比,NAFLD患者的总脂联素和三种不同形式的脂联素水平均显著降低。在NAFLD患者中,总脂联素与身高(R=-0.270, P=0.032)和羧甲基赖氨酸(R=-0.259, P=0.040)显著负相关;高分子量脂联素与空腹血糖(R=0.350, P=0.016)显著正相关,与丙氨酸氨基转移酶(R=-0.321, P=0.029)和天门冬氨酸氨基转移酶(R=-0.295, P=0.045)显著负相关。结论:总脂联素和三种不同形式的脂联素水平均与NAFLD呈显著负相关。