基因重组TNFα衍生物TRSP10的高效制备及其对DU145细胞抑制作用研究

利用基因工程技术表达能够促使肿瘤细胞DU145凋亡的肿瘤坏死因子α(TNFα)的衍生物TRSP10,并在体外研究其对DU145细胞的抑制效应。以重叠延伸PCR方法合成TRSP10基因序列,并插入高效表达的质粒载体p KYB-MCS的NdeⅠ和SapⅠ酶切位点之间,优化融合蛋白诱导表达的条件,建立了从载体构建到重组菌表达、制备的工艺技术条件。MTT法检测TRSP10对前列腺癌细胞DU145增殖的抑制作用。实验结果表明:重组菌ER2566诱导表达可溶性融合蛋白的最佳条件是诱导剂IPTG浓度为0.8 mmol/L、诱导表达温度37℃、诱导表达时间8h。利用IMPACT系统及HPLC技术纯化制备TRSP10,得到产物纯度达到96%,质谱鉴定确定其分子质量为3.59k Da,与理论值相符;体外细胞学研究结果表明,TRSP10对前列腺癌细胞DU145有明显的抑制作用,在5,10,20,40μmol/L TRSP10及10μmol/L TNFα阳性对照处理后48h抑制率分别达到11.40%,22.97%,33.26%,48.35%及42.50%。     

山药水浸提物对alpha- 葡萄糖苷酶抑制作用的初步研究

目的:通过研究山药水浸提物对α-葡萄糖苷酶的抑制作用,初步探索山药对2型糖尿病的预防和治疗作用。方法:对山药进行热水浸提,采用1:5的料液比,浸提温度90℃,时间2小时,超滤获得山药水浸提物,利用α-葡萄糖苷酶对淀粉降解反应建立酶活检测体系,通过检测酶反应产物分析山药水浸提物对α-葡萄糖苷酶的抑制作用,并计算IC50。结果:山药水浸提物对α-葡萄糖苷酶的抑制率大于46%,IC50值为1.9 g/ml。结论:山药水浸提物对α-葡萄糖苷酶的抑制作用提示山药可以作为肥胖或者2型糖尿病患者有效的一种药食同源食材,本实验结果为2型糖尿病高危人群及患者合理选择膳食结构提供新的科学依据。     

猪骨骼肌MyHC-Ⅱb基因上调表达的分子机制

哺乳动物骨骼肌由各种不同类型的肌纤维镶嵌而成,不同类型肌球蛋白重链的表达是造成不同类型肌纤维的主要原因.目前已知的肌球蛋白重链家族包含8种亚型,其中长白猪骨骼肌My HC-Ⅱb的表达量显著高于中国地方猪,然而造成这种差异的分子机制未见报道.本研究用荧光定量PCR证明了长白猪背最长肌中My HC-Ⅱb m RNA的表达量显著高于莱芜猪(P=0.013).删除实验结果表明,从转录起始位点上游-1024 bp删除到-187 bp之后,My HC-Ⅱb表达量显著下降,分析发现,在这段启动子区域内存在3个E-box序列;分别突变这3个E-box序列后,My HC-Ⅱb启动子驱动的荧光素酶活性显著下降(P=0.036).另外,在My HC-Ⅱb上游启动子区?1398 bp处发现一个GT的突变,所检测的64头莱芜猪在该位点全部为GG型,65头长白猪中13头为GG型,16头为TT型,36头为GT型.在C2C12细胞系中的转染实验结果显示,G突变为T之后有增加My HC-Ⅱb表达的趋势.Western blot的结果表明,转录因子Myo D在两猪种间表达差异不显著(P=0.136),而Myf-5在长白猪中的表达量极显著高于其在莱芜猪中的表达量(P=0.0036).这些数据表明,Myf-5是造成猪My HC-Ⅱb基因m RNA上调表达的重要因素之一.     

Clinical,Virological and Immunological Features from Patients Infected with Re-Emergent Avian-Origin Human H7N9 Influenza Disease of Varying Severity in Guangdong Province

BackgroundThe second wave of avian influenza H7N9 virus outbreak in humans spread to the Guangdong province of China by August of 2013 and this virus is now endemic in poultry in this region.MethodsFive patients with H7N9 virus infection admitted to our hospital during August 2013 to February 2014 were intensively investigated. Viral load in the respiratory tract was determined by quantitative polymerase chain reaction (Q-PCR) and cytokine levels were measured by bead-based flow cytometery.ResultsFour patients survived and one died. Viral load in different clinical specimens was correlated with cytokine levels in plasma and broncho-alveolar fluid (BALF), therapeutic modalities used and clinical outcome. Intravenous zanamivir appeared to be better than peramivir as salvage therapy in patients who failed to respond to oseltamivir. Higher and more prolonged viral load was found in the sputum or endotracheal aspirates compared to throat swabs. Upregulation of proinflammatory cytokines IP-10, MCP-1, MIG, MIP-1α/β, IL-1β and IL-8 was found in the plasma and BALF samples. The levels of cytokines in the plasma and viral load were correlated with disease severity. Reactivation of herpes simplex virus type 1(HSV-1) was found in three out of five patients (60%).ConclusionExpectorated sputum or endotracheal aspirate specimens are preferable to throat swabs for detecting and monitoring H7N9 virus. Severity of the disease was correlated to the viral load in the respiratory tract as well as the extents of cytokinemia. Reactivation of HSV-1 may contribute to clinical outcome.     

Spectrum of UGT1A1 Variations in Chinese Patients with Crigler-Najjar Syndrome Type II

Crigler–Najjar Syndrome type II (CNS-II) is an autosomal recessive hereditary condition of unconjugated hyperbilirubinemia without hemolysis, with bilirubin levels ranging from 102.6 μmol/L to 342 μmol/L. CNS-II is caused by a deficiency of UDP-glucuronyl transferase (UGT), which is encoded by the UDP-glucuronyl transferase 1A1 gene (UGT1A1). In East Asian populations, the compound homozygous UGT1A1 G71R and Y486D variants are frequently observed in cases with bilirubin levels exceeding 200 μmol/L. In this study, we investigated the spectrum of UGT1A1 variations in Chinese CNS-II patients. We sequenced the enhancer, promoter, and coding regions of UGT1A1 in 11 unrelated Chinese CNS-II patients and 80 healthy controls. Nine of these patients carried variations that are here reported for the first time in CNS-II patients, although they have been previously reported for other types of hereditary unconjugated hyperbilirubinemia. These individual variations have less influence on UGT activity than do the compound homozygous variation (combination of homozygous G71R variant and Y486D variant). Therefore, we propose that the spectrum of UGT1A1 variations in CNS-II differs according to the bilirubin levels.     

Surgical Complications of Primary Rhegmatogenous Retinal Detachment: A Meta-Analysis

Background

To investigate the surgical complications of scleral buckling (SB) and pars plana vitrectomy (PPV) performed on primary rhegmatogenous retinal detachment (RRD) and to discover which surgical procedures bring fewer complications.

Methods

An electronic literature search using the PubMed database, ISI Web of Knowledge and the Cochrane Central Register of Controlled Trials to identify randomized controlled trials and observational studies comparing SB with PPV on primary RRD. Outcome measures included intra-operative complications and early and late post-operative complications.

Results

During the operation, significantly less subretinal hemorrhage occurred in the PPV group than in the SB group (OR = 4.71; 95%CI, 1.33–16.64; p = 0.02) and the hypotony incidence was significantly higher in the SB group (OR = 18.24; 95%CI, 2.37–140.44; p = 0.005); however, the occurrence of iatrogenic breaks was significantly lower in the SB group (OR = 0.05; 95%CI, 0.01–0.21; p<0.0001). In the early stage of post-operation, significantly higher incidence of choroidal detachment was identified in the SB group than in the PPV group (OR = 10.19; 95%CI, 2.36–44.09; p = 0.002); patients undergoing SB had significantly higher odds of residual subretinal fluid (OR = 14.71; 95%CI, 1.84–117.32; p = 0.01); the occurrence of high intraocular pressure was significantly lower in the SB group (OR = 0.46; 95%CI, 0.23–0.89; p = 0.02); and no significant difference was shown in the incidence of epithelia defect (p = 0.37) between the two groups. In the late stage of post-operation, the incidence of diplopia/extraocular muscle dysfunction was significantly higher in the SB group (OR = 4.04; 95%CI, 1.30–12.52; p = 0.02); and significantly less cataract was observed in the SB group (OR = 0.20; 95%CI, 0.14–0.30; p<0.00001); no significant difference was found in the incidences of cystoid macular edema (p = 0.65), macular pucker (p = 0.52), post-operative proliferative vitreoretinopathy (p = 0.73) and epiretinal membrane (p = 0.47) in other late post-operative complications.

Conclusions

This meta-analysis suggests that PPV could be considered as potential surgical management on primary RRD.     

Hypoxia Precondition Promotes Adipose-Derived Mesenchymal Stem Cells Based Repair of Diabetic Erectile Dysfunction via Augmenting Angiogenesis and Neuroprotection

The aim of the present study was to examine whether hypoxia preconditioning could improve therapeutic effects of adipose derived mesenchymal stem cells (AMSCs) for diabetes induced erectile dysfunction (DED). AMSCs were pretreated with normoxia (20% O₂, N-AMSCs) or sub-lethal hypoxia (1% O₂, H-AMSCs). The hypoxia exposure up-regulated the expression of several angiogenesis and neuroprotection related cytokines in AMSCs, including vascular endothelial growth factor (VEGF) and its receptor FIK-1, angiotensin (Ang-1), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), stromal derived factor-1 (SDF-1) and its CXC chemokine receptor 4 (CXCR4). DED rats were induced via intraperitoneal injection of streptozotocin (60 mg/kg) and were randomly divided into three groups—Saline group: intracavernous injection with phosphate buffer saline; N-AMSCs group: N-AMSCs injection; H-AMSCs group: H-AMSCs injection. Ten rats without any treatment were used as normal control. Four weeks after injection, the mean arterial pressure (MAP) and intracavernosal pressure (ICP) were measured. The contents of endothelial, smooth muscle, dorsal nerve in cavernoursal tissue were assessed. Compared with N-AMSCs and saline, intracavernosum injection of H-AMSCs significantly raised ICP and ICP/MAP (p<0.05). Immunofluorescent staining analysis demonstrated that improved erectile function by MSCs was significantly associated with increased expression of endothelial markers (CD31 and vWF) (p<0.01) and smooth muscle markers (α-SMA) (p<0.01). Meanwhile, the expression of nNOS was also significantly higher in rats receiving H-AMSCs injection than those receiving N-AMSCs or saline injection. The results suggested that hypoxic preconditioning of MSCs was an effective approach to enhance their therapeutic effect for DED, which may be due to their augmented angiogenesis and neuroprotection.     

The Role of Toll-Like Receptor 9 in Chronic Stress-Induced Apoptosis in Macrophage

Emerging evidence implied that chronic stress has been exerting detrimental impact on immune system functions in both humans and animals. Toll-like receptors (TLRs) have been shown to play an essential role in modulating immune responses and cell survival. We have recently shown that TLR9 deficiency protects against lymphocyte apoptosis induced by chronic stress. However, the exact role of TLR9 in stress-mediated change of macrophage function remains unclear. The results of the current study showed that when BALB/c mice were treated with restraint stress (12 h daily for 2 days), the number of macrophages recruited to the peritoneal cavity was obviously increased. Results also demonstrated that the sustained effects of stress elevated cytokine IL-1β, TNF-α and IL-10 production yet diminished IFN-γ production from macrophage, which led to apoptotic cell death. However, TLR9 deficiency prevented the chronic stress-mediated accumulation of macrophages. In addition, knocking out TLR9 significantly abolished the chronic stress-induced imbalance of cytokine levels and apoptosis in macrophage. TLR9 deficiency was also found to reverse elevation of plasma IL-1β, IL-10 and IL-17 levels and decrease of plasma IFN-γ level under the condition of chronic stress. These results indicated that TLR9-mediated macrophage responses were required for chronic stress-induced immunosuppression. Further exploration showed that TLR9 deficiency prevented the increment of p38 MAPK phosphorylation and reduction of Akt/Gsk-3β phosphorylation; TLR9 deficiency also attenuated the release of mitochondrial cytochrome c into cytoplasm, caused upregulation of Bcl-2/Bax protein ratio, downregulation of cleavage of caspase-3 and PARP, as well as decreased TUNEL-positive cells in macrophage of stressed mice. Collectively, our studies demonstrated that deficiency of TLR9 maintained macrophage function by modulating macrophage accumulation and attenuating macrophage apoptosis, thus preventing immunosuppression in restraint-stressed mice.     

Combining Serum Cystatin C with Total Bilirubin Improves Short-Term Mortality Prediction in Patients with HBV-Related Acute-On-Chronic Liver Failure

Background & Aims

HBV-related acute-on-chronic liver failure (HBV-ACLF) is a severe liver disease which results in a high mortality in China. To early predict the prognosis of the patients may prevent the complications and improve the survival. This study was aimed to develop a new prognostic index to estimate the survival related to HBV-ACLF.

Methods

Consecutive patients with HBV-ACLF were included in a prospective observational study. Serum Cystatin C concentrations were measured by using the particle-enhanced immunonephelometry assay. All of the patients were followed for at least 3 months. Cox regression analysis was carried out to identify which factors were predictive of mortality. The area under the receiver operating characteristic curve (AUC) was used to evaluate the efficacy of the variates for early predicting mortality.

Results

Seventy-two patients with HBV-ACLF were recruited between January 2012 and January 2013. Thirty patients died (41.7%) during 3-months followed up. Cox multivariate regression analysis identified serum cystatin C (CysC) and total bilirubin (TBil) were independent factors significantly (P < 0.01) associated with survival. Our results further showed that new prognostic index (PI) combining serum CysC with TBil was a good indicator for predicting the mortality of patients with HBV-ACLF. Specifically, the PI had a higher accuracy than the CTP, MELD, or MELD-Na scoring for early prediction short-term survival of HBV-ACLF patients with normal levels of serum creatinine (Cr). The survival rate in low risk group (PI < 3.91) was 94.3%, which was markedly higher than those in the high-risk group (PI ≥ 3.91) (17.4%, P < 0.001).

Conclusion

We developed a new prognostic index combining serum CysC with TBil which early predicted the short-term mortality of HBV-ACLF patients.