Quantitative analysis of β1,6GlcNAc-branched N-glycans on β4 integrin in cutaneous squamous cell carcinoma

α6β4 integrin plays pivotal roles in cancer progression in several types of cancers. Our previous study using N-glycan-manipulated cell lines demonstrated that defects in N-glycans or decreased β1,6GlcNAc-branched N-glycans on β4 integrin suppress β4 integrin-mediated cancer cell adhesion, migration, invasion, and tumorigenesis. Furthermore, immunohistochemical analysis has shown that colocalization of β1,6GlcNAc-branched N-glycans with β4 integrin was observed in cutaneous squamous cell carcinoma (SCC) tissue. However, until now there has been no direct evidence that β1,6GlcNAc-branched N-glycans are upregulated on β4 integrin in cutaneous SCC. In the present study, we performed an ELISA analysis of β1,6GlcNAc-branched N-glycans on β4 integrins as well as β4 integrins in cell lysates from human normal skin and cutaneous SCC tissues. The SCC samples showed a 4.9- to 7.4-fold increase in the ratio of β1,6GlcNAc-branched N-glycans to β4 integrin compared with normal skin samples. These findings suggest that the addition of β1,6GlcNAc-branched N-glycans onto β4 integrin was markedly elevated in cutaneous SCC tissue compared to normal skin tissue. The value of β1,6GlcNAc-branched N-glycans on β4 integrin may be useful as a diagnostic marker associated with cutaneous SCC tumor progression.     

早老蛋白1功能缺失性突变导致家族性阿尔茨海默病的研究进展

阿尔茨海默病（AD）是一种衰老相关的神经退行性疾病，主要病理特征是大脑中存在异常聚集的β淀粉样蛋白（Aβ）。AD可分为家族性和散发性，其中早老蛋白1（ PS1）是家族性AD最主要的风险基因， PS1突变占已知致家族性AD突变的80%以上。PS1是构成γ-分泌酶的催化亚基，后者负责加工Aβ前体蛋白（APP）生成Aβ。虽然新型 PS1突变日渐被报道，但其诱发家族性AD的分子机制仍无定论。由于90%的 PS1突变降低γ-分泌酶活性，学术界提出了 PS1功能缺失性突变假说，认为 PS1突变通过显性负效应导致PS1功能下降或缺失是诱发家族性AD的关键。近年，大量实验研究支持了该假说。首先， PS1功能缺失性突变通过干扰γ-分泌酶在APP上的切割位点促进长链Aβ生成，进而增加Aβ ₄₂/Aβ ₄₀比率；其次， PS1功能缺失性突变可破坏神经细胞内质网中的钙离子稳态以及通过阻断神经细胞自噬活性导致APP加工产物的异常聚集；再者， PS1功能缺失性突变可通过干扰神经元的内吞和转胞吞作用诱发神经元萎缩以及通过激活神经免疫细胞（星形胶质细胞和小胶质细胞）增强神经炎症；最后， PS1功能缺失性突变降低糖酵解和乳酸输出，破坏机体对神经元的能量供应。本文总结了 PS1功能缺失性突变诱发家族性AD的分子机制，并对今后潜在的研究方向进行了探讨。     

α-细辛醚、β-细辛醚改善Aβ 25-35 诱导的 PC12细胞损伤及机制

目的：探究α-细辛醚、β-细辛醚对β淀粉样蛋白活性片段Aβ _25-35诱导的PC12细胞损伤模型的保护作用及相关作用机制。 方法：采用Aβ _25-35诱导PC12细胞建立Aβ毒性损伤细胞模型。将PC12细胞分为空白对照组、模型对照组、α-细辛醚组（0.5、1.0、1.5 μg/mL）、β-细辛醚组（6.3、12.5、25.0 μg/mL）、血管活性肠肽（VIP）组，并设VIP拮抗剂对照。采用细胞计数试剂盒（CCK-8）法检测细胞存活率；流式细胞术检测细胞凋亡率；酶联免疫吸附试验检测炎症因子白介素（IL）-1、IL-10、肿瘤坏死因子（TNF）-α，氧化因子诱生型一氧化氮合酶（iNOS）、一氧化氮（NO）和凋亡因子caspase-3、p53水平；蛋白质印迹法检测细胞c-Jun氨基端激酶（JNK）、p38丝裂原活化蛋白激酶（p38MAPK）蛋白表达。 结果：与模型对照组比较，α-细辛醚组、β-细辛醚组和VIP组细胞存活率增加，细胞凋亡率下降，凋亡因子caspase-3、p53和炎症因子IL-1、TNF-α水平下降，IL-10水平升高，氧化因子iNOS和NO水平下降，c-Jun氨基端激酶（JNK）、p38MAPK蛋白表达减少（均 P<0.05）。VIP拮抗剂干预后，β-细辛醚组细胞存活率下降，细胞凋亡率增加，凋亡因子caspase-3、p53和炎症因子IL-1、TNF-α水平升高，IL-10水平下降，氧化因子iNOS和NO水平升高，JNK、p38MAPK蛋白表达增加（均 P<0.05）；α-细辛醚组无显著变化（均 P>0.05）。 结论：α-细辛醚、β-细辛醚对Aβ _25-35诱导的PC12细胞损伤模型具有保护作用，β-细辛醚可通过促进VIP的分泌，调控JNK/MAPK通路从而抑制炎症因子、氧化因子水平，改善PC12细胞凋亡；α-细辛醚作用机制与VIP分泌水平无明显联系。     

Use of unidirectional and spherical porous β-tricalcium phosphate in opening-wedge high tibial osteotomy: a case series

Introduction: Unidirectional porous β-tricalcium phosphate (UDPTCP) consists of a novel porous artificial bone that is structurally different from conventional artificial bone comprised of spherical porous β-tricalcium phosphate (SPTCP).Case presentation: We present our first four clinical cases of opening-wedge high tibial osteotomy (OWHTO) using UDPTCP and SPTCP together. The patients’ mean age was 54.5 ± 5.9 years, and the mean observation period was 20.8 ± 2.8 months. In OWHTO, two wedge shaped pieces of UDPTCP and SPTCP were cut to fit the gap and implanted parallel to each other in the anterior and posterior parts, respectively. We evaluated the correction loss and bone remodeling for UDPTCP and SPTCP over time using radiography and computed tomography, and evaluated the clinical outcomes.Conclusion: There was no correction loss reported in any case, and early bone remodeling was observed with UDPTCP. All patients achieved satisfactory clinical results with no adverse events.     

Development of novel-nanobody-based lateral-flow immunochromatographic strip test for rapid detection of recombinant human interferon α2b

Recombinant human interferon α2b (rhIFNα2b) is widely used as an antiviral therapy agent for the treatment of hepatitis B and hepatitis C. The current identification test for rhIFNα2b is complex. In this study, an anti-rhIFNα2b nanobody was discovered and used for the development of a rapid lateral flow strip for the identification of rhIFNα2b. RhIFNα2b was used to immunize an alpaca, which established a phage nanobody library. After five steps of enrichment, the nanobody I22, which specifically bound rhIFNα2b, was isolated and inserted into the prokaryotic expression vector pET28a. After subsequent purification, the physicochemical properties of the nanobody were determined. A semiquantitative detection and rapid identification assay of rhIFNα2b was developed using this novel nanobody. To develop a rapid test, the nanobody I22 was coupled with a colloidal gold to produce lateral-flow test strips. The developed rhIFNα2b detection assay had a limit of detection of 1 μg/mL. The isolation of I22 and successful construction of a lateral-flow immunochromatographic test strip demonstrated the feasibility of performing ligand-binding assays on a lateral-flow test strip using recombinant protein products. The principle of this novel assay is generally applicable for the rapid testing of other commercial products, with a great potential for routine use in detecting counterfeit recombinant protein products.     

Serum S100β as a predictor of severity and outcomes for mixed subtype acute ischaemic stroke

INTRODUCTIONSerum S100β levels are mostly used for predicting outcomes of large-vessel stroke. Its application to mixed subtypes of acute ischaemic stroke (AIS) has been limited.METHODSPatients with mixed subtypes of AIS who were aged over 18 years and presented within 24 hours of stroke onset were consecutively enrolled. Serum S100β levels at presentation (S100β_b) and 72 hours (S100β_72hrs), and corresponding National Institutes of Health Stroke Scale (NIHSS_b and NIHSS_72hrs, respectively) scores were assessed. Stroke outcomes were evaluated using the modified Rankin Scale (mRs) at 30 days (mRs₃₀) and 90 days (mRs₉₀). Correlations between S100β_b and S100β_72hrs, as well as differences between the two (∆S100β) and the corresponding NIHSS, mRs₃₀ and mRs₉₀ scores, were evaluated (p < 0.05).RESULTS35 patients were eligible for analysis. On univariate analysis, stroke outcomes had a significant association with S100β_b, S100β_72hrs, NIHSS_b, NIHSS_72hrs and ∆S100β. Both S100β_b and S100β_72hrs correlated with corresponding NIHSS values (ρ_b = 0.51, p < 0.001; ρ_72hrs = 0.74, p < 0.001), mRs₃₀ (ρ_b = 0.58, p < 0.001; ρ_72hrs = 0.72, p < 0.001) and mRs₉₀ (ρ_b = 0.51, p = 0.002; ρ_72hrs = 0.68, p < 0.001). Correlations existed between ∆S100β and mRs₃₀ (ρ = 0.74, p < 0.001) and mRs₉₀ (ρ = 0.71, p < 0.001). Practical cut-off points for unfavourable outcomes (mRs 3–6) were S100β_72hrs > 0.288 µg/L (sensitivity 92.3%, specificity 86.4%) and ∆S100β > 0.125 µg/L (sensitivity 100%, specificity 81.8%).CONCLUSIONHigh serum S100β is associated with unfavourable outcomes for mixed subtype AIS. Cut-off values of S100β_72hrs and ∆S100β were optimal for predicting unfavourable stroke outcomes.     

Occurrence and distribution of extended-spectrum β-lactamase in clinical Escherichia coli isolates at Ho Teaching Hospital in Ghana

ObjectiveThis study determined the occurrence and distribution of Extended Spectrum β-Lactamase (ESBL) genotypes of E. coli isolates in Ho Teaching Hospital, Ghana.DesignA cross-sectional study.SettingA single centre study was conducted at Ho Teaching Hospital of Ghana.ParticipantsPatients who visited Ho Teaching Hospital Laboratory with the request for culture and susceptibility testing.Main outcome measureEscherichia coli were isolated, and Extended-Spectrum β-Lactamase genes were detected.ResultsOf the 135 isolates, 56(41.5%,95% CI: 33.1% – 50.3%) were ESBL producers. More males, 14(58.3%), produced ESBL than females, 42(37.8%). The ESBL prevalence was highest among the elderly who were 80 years and above 3(100.0%), with the least prevalence among patients within 50–59 years and 0–9 years age bracket, representing 4(25.0%) and 3(27.3%), respectively. The total prevalence of ESBL was marginally higher among out-patients (41.8% 95% CI: 31.9% – 52.2%) compared to in-patients [40.5% 95% CI: 24.8% – 57.9]. BlaTEM-1 was the predominant ESBL genotype obtained from 83.9% (47/56) of the confirmed ESBL producing isolates, with the least being TOHO-1 4(7.1%). The co-existence of 2 different ESBL genes occurred in 19(33.9%) of the isolates. The single and quadruple carriage were 16(28.6%) and 3(5.4%), respectively. The highest co-existence of the ESBL genotypes was recorded for blaTEM-1 and blaCTXM-1 15(26.8%), followed by blaTEM-1, blaCTXM-1 and blaSHV-73 [12(21.4%)].ConclusionThe high prevalence of ESBL-producing E. coli isolates with multiple resistant gene carriage is a threat to healthcare in the study area.FundingThis research received no external funding.     

Hepatocyte nuclear factor 4α in the pathogenesis of non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is emerging as the most common chronic liver disease worldwide. It refers to a range of liver conditions affecting people who drink little or no alcohol. NAFLD comprises non-alcoholic fatty liver and non-alcoholic steatohepatitis (NASH), the more aggressive form of NAFLD. NASH is featured by steatosis, lobular inflammation, hepatocyte injury, and various degrees of fibrosis. Although much progress has been made over the past decades, the pathogenic mechanism of NAFLD remains to be fully elucidated. Hepatocyte nuclear factor 4α (HNF4α) is a nuclear hormone receptor that is highly expressed in hepatocytes. Hepatic HNF4α expression is markedly reduced in NAFLD patients and mouse models of NASH. HNF4α has been shown to regulate bile acid, lipid, glucose, and drug metabolism. In this review, we summarize the recent advances in the understanding of the pathogenesis of NAFLD with a focus on the regulation of HNF4α and the role of hepatic HNF4α in NAFLD. Several lines of evidence have shown that hepatic HNF4α plays a key role in the initiation and progression of NAFLD. Recent data suggest that hepatic HNF4α may be a promising target for treatment of NAFLD.     

宫内发育迟缓出生后追赶生长大鼠脂肪组织 LRP6/ β -catenin通路表达变化

目的：探讨宫内发育迟缓出生后追赶生长（CG-IUGR）大鼠脂肪组织低密度脂蛋白受体相关蛋白6（LRP6）/β-联蛋白（β-catenin）通路表达变化。方法：随机将SD孕鼠分为两组，一组孕鼠全程限食喂养，生产后通过减少喂养子鼠数量，建立子鼠CG-IUGR模型（CG-IUGR组）；另一组孕鼠正常喂养，生产的子鼠作为对照组。为排除性别因素的干扰，CG-IUGR组和对照组均选取雄性子鼠。CG-IUGR组和对照组在12周龄时进行葡萄糖耐量试验，并取肾周脂肪组织标本，通过苏木素-伊红染色观察脂肪结构，共聚焦显微镜下观察脂肪组织LRP6、β-catenin及胰岛素受体底物1（IRS-1）的免疫活性，蛋白质印迹法检测LRP6、β-catenin、IRS-1蛋白表达。结果：CG-IUGR组在60 min时血糖浓度及曲线下面积均大于对照组（均 P<0.05），表明CG-IUGR组葡萄糖耐量受损。CG-IUGR组脂肪细胞面积较对照组增大，脂肪组织中LRP6、β-catenin和IRS-1免疫活性较对照组降低（均 P<0.05）。     

TRIM21可抑制肝癌细胞的侵袭能力：基于泛素化途径降解β-catenin

目的探讨TRIM21基因在肝癌侵袭表型中的作用及机制。方法利用siRNA敲低肝癌细胞MHCC 97H，HCC LM3中的TRIM21、β-catenin基因，将肝癌细胞分为对照组：转染对照siRNA；TRIM21敲低组：转染siTRIM21 RNA；CTNNB1敲低组：转染siCTNNB1 RNA；双敲低组：共转染siTRIM21 RNA及siCTNNB1 RNA。利用Western blotting验证敲除效率及相关通路的激活或抑制情况。通过Transwell侵袭实验检测肿瘤细胞的侵袭性。通过尾静脉注射敲低TRIM21的HCC LM3细胞构建裸鼠肺转移模型以观测其肺转移能力。在HEK 293细胞中过表达TRIM21，将HEK 293细胞分为对照组：加入转染试剂；过表达组：转染flagTRIM21。并利用Co-IP检测TRIM21蛋白与β-catenin的相互作用及泛素化水平。生信分析TCGA数据库中CTNNB1^highTRIM21^high和CTNNB1^highTRIM21^low两种亚型肝癌的预后。结果敲低TRIM21可以显著增强肝癌细胞MHCC 97H和HCC LM3的侵袭能力（P < 0.01，P < 0.05）和HCC LM3细胞的肺转移能力（P < 0.01）。机制研究发现，TRIM21可通过泛素化蛋白酶体途径降解β-catenin以减少其入核，进而降低肝癌细胞的的侵袭性（P < 0.0001，P < 0.05）。由于TRIM21表达水平降低使肝癌细胞中Wnt信号通路激活，最终使CTNNB1^highTRIM21^high亚型肝癌患者的生存预后明显优于CTNNB1^highTRIM21^low亚型肝癌患者（P < 0.05）。结论TRIM21能通过泛素化途径降解β-catenin来抑制肝癌细胞的侵袭性，这可能导致了CTNNB1^highTRIM21^low肝癌患者预后较差。     

Rapid and sensitive analysis of trace β-blockers by magnetic solid-phase extraction coupled with Fourier transform ion cyclotron resonance mass spectrometry

A rapid and sensitive method for analyzing trace β-blockers in complex biological samples, which involved magnetic solid-phase extraction (MSPE) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), was developed. Novel nanosilver-functionalized magnetic nanoparticles with an interlayer of poly(3,4-dihydroxyphenylalanine) (polyDOPA@Ag-MNPs) were synthesized and used as MSPE adsorbents to extract trace β-blockers from biological samples. After extraction, the analytes loaded on the polyDOPA@Ag-MNPs were desorbed using an organic solvent and analyzed by FTICR-MS. The method was rapid and sensitive, with a total detection procedure of less than 10 min as well as limits of detection and quantification in the ranges of 3.5–6.8 pg/mL and 11.7–22.8 pg/mL, respectively. The accuracy of the method was also desirable, with recoveries ranging from 80.9% to 91.0% following the detection of analytes in human blood samples. All the experimental results demonstrated that the developed MSPE-FTICR-MS method was suitable for the rapid and sensitive analysis of trace β-blockers in complex biological samples.     

羟尼酮可阻止大鼠肝星状细胞活化：基于抑制TGF-β1通路蛋白的磷酸化

目的探讨羟尼酮对CCl4诱导的大鼠肝纤维化的作用及相关机制。方法66只雄性SD大鼠按随机数字法分为对照组10只、模型组20只、羟尼酮（100 mg/kg）组12只、羟尼酮（250 mg/kg）组12只、吡非尼酮（250 mg/kg）组12只，采用CCl₄皮下注射进行肝纤维化造模，羟尼酮、吡非尼酮予以灌胃给药，处死大鼠后，留取血清检测肝功能，利用肝脏组织检测羟脯氨酸的含量，肝组织进行HE染色和Sirius Red染色，对炎症和纤维化程度进行评分。将人肝星状细胞系LX-2分组：对照组、TGF-β1组、羟尼酮组、吡非尼酮组，经过TGF-β1刺激和羟尼酮、吡非尼酮处理后，Western blot分别检测α-SMA、I型胶原蛋白和磷酸化Smad3、磷酸化p38、磷酸化Erk1/2、磷酸化Akt等磷酸化蛋白。结果动物实验显示羟尼酮组大鼠肝功能值有改善，肝组织羟脯氨酸含量明显减少，肝组织纤维化程度显著降低，且均具有统计学意义（P < 0.05）。细胞实验发现随羟尼酮浓度的增高，ɑ-SMA和I型collagen蛋白表达水平降低；TGF-β1刺激细胞后，TGF-β信号转导通路中Smad3、P38、ERK、Akt等蛋白的磷酸化水平随着作用时间的延长而增加，但在相同作用时间下，羟尼酮组细胞TGF-β信号转导通路蛋白磷酸化的表达水平低于TGF-β1组细胞（P < 0.05）。结论羟尼酮可抑制TGF-β信号转导通路蛋白的磷酸化，从而阻止TGF-β1介导的肝星状细胞活化，可能是其缓解CCl₄诱导的大鼠肝纤维化机制之一。     

吡非尼酮通过下调TGF-β/Smad通路中TGF-β3的表达抑制兔Tenons囊成纤维细胞增殖

目的探讨吡非尼酮在抗青光眼滤过术后抑制瘢痕形成的可能机制。方法体外培养兔眼Tenons囊成纤维细胞(RYTF)；设计6组不同浓度的吡非尼酮作为实验组、1组未加入吡非尼酮的作为对照组及1组调零组，采用CCK-8检测法初步确定吡非尼酮的起始作用浓度及最适作用浓度；采用免疫荧光法检测经起始、最适作用浓度吡非尼酮作用后RYTF中TGF-β3，Ⅰ、Ⅲ型胶原蛋白与对照组相比的荧光染色表达情况；通过Western blot法检测经起始、最适作用浓度吡非尼酮作用后TGF-β3，CollagenⅠ、CollagenⅢ因子与对照组相比的蛋白表达情况；通过RT-PCR法检测经起始、最适作用浓度吡非尼酮作用后TGF-β3、CollagenⅠ、CollagenⅢ与对照组相比的mRNA表达变化。结果吡非尼酮抑制RYTF增殖的起始作用浓度和最适作用浓度分别为0.1 mg和0.27 mg；与对照组相比，经起始、最适作用浓度吡非尼酮作用24 h后，实验组RYTF中TGF-β3、CollagenⅠ、Collagen Ⅲ的荧光表达均少于对照组（P < 0.05）；与对照组相比，经起始、最适作用浓度吡非尼酮作用24 h后，实验组RYTF中TGF-β3、CollagenⅠ、CollagenⅢ的蛋白表达量均少于对照组（P < 0.05）；与对照组相比，经起始、最适作用浓度吡非尼酮作用24h后，实验组RYTF中TGF-β3、CollagenⅠ、Collagen Ⅲ的基因相对表达量均少于对照组（P < 0.05）。结论吡非尼酮对体外培养RYTF增殖有明显抑制作用，且呈浓度依赖关系；吡非尼酮抑制RYTF增殖的机制可能是通过影响RYTF中TGF-β/Smad途径上TGF-β3效应因子的基因和蛋白表达。     

紫草素通过抑制PKM2/PHD3/HIF-1α通路诱导肝癌细胞凋亡

目的探讨紫草素诱导人肝癌细胞SMMC-7721死亡的可能机制。方法体外培养人肝癌细胞（SMMC-7721）和正常肝细胞（L-02），实验分为对照组和紫草素给药组（4、8、16 μmol/L）。采用3-（4，5-二甲基噻唑-2）-2，5-二苯基四氮唑溴盐3-（4, 5-二甲基噻唑-2-yl）-2, 5-二苯基四氮唑溴盐，MTT法检测细胞活力，试剂盒检测三磷酸腺苷（ATP）和乳酸水平，免疫共沉淀和免疫荧光双染实验明确M2型丙酮酸激酶（PKM2）、脯氨酰酸羟化酶3（PHD3）、缺氧诱导因子1α（HIF-1α）三者之间的作用关系及蛋白表达情况；Annexin V/PI检测细胞凋亡；Western blot观察PKM2、PHD3、HIF-1α及凋亡蛋白Bax、cleaved caspase-3、Bcl-2表达水平；采用小干扰核糖核酸（siRNA）干扰法建立PKM2低表达的人肝癌SMMC-7721细胞，Western blot检测PKM2低表达对人肝癌SMMC-7721细胞中的PHD3、HIF-1α蛋白表达水平的影响。结果紫草素对SMMC-7721和L-02细胞的半抑制浓度（IC₅₀）分别是8.041 μmol/L与31.75 μmol/L，与对照组相比紫草素能抑制肝癌SMMC-7721细胞中PKM2、HIF-1α和PHD3蛋白表达及PKM2、HIF-1α入核（P < 0.05）。免疫共沉淀和免疫荧光双染实验证实，紫草素可以抑制PKM2/PHD3/HIF-1α复合体形成（P < 0.05），并且抑制有氧糖酵解产物乳酸和ATP含量（P < 0.05）。与对照组相比，PKM2敲低后PHD3和HIF-1α表达明显降低（P < 0.05）；与未处理组相比，紫草素处理后凋亡率明显升高且凋亡蛋白Bax和cleaved caspase-3表达升高，Bcl-2表达下降（P < 0.05）。结论紫草素靶向PKM2调控PHD3/HIF-1α复合物，从而抑制肝癌细胞的有氧糖酵解，破坏其供能途径，导致肝癌细胞凋亡。     

TNF-α通过唾液酸化促进小鼠破骨细胞分化

目的探究TNF-α促进破骨细胞分化的作用机制。方法利用4月龄野生型小鼠与同龄TNF-α转基因鼠进行体内研究，分别收集双侧膝骨关节样本后，进行CT扫描分析骨量差异，TRAP染色及免疫荧光染色分析破骨细胞分化程度及唾液酸表达水平。体外培养破骨细胞前体细胞系RAW264.7分为3组：RAW对照组（不含药物的分化培养基处理）；RAW实验组（含有TNF-α的分化培养基处理）；RAW药物组（含有TNF-α和唾液酸酶的分化培养基处理）。体外培养3 d后进行qPCR检测，TRAP染色，唾液酸免疫荧光共定位染色分析。同时，分离培养野生型小鼠（BM对照组）和TNF-α转基因鼠[BM实验组（不含药物的分化培养基处理），BM药物组（含有唾液酸酶的分化培养基处理）]原代骨髓源巨噬细胞，3 d后进行TRAP染色及唾液酸染色。结果TRAP染色显示，TNF-α转基因鼠膝关节软骨下骨中破骨细胞数量较野生型小鼠显著增多（P < 0.001）；其骨量/体积（BV/TV）、骨小梁数量（Tb.N）、骨小梁厚度（Tb.Th）则显著降低（P < 0.001）；而TNF-α转基因鼠软骨下骨中唾液酸与TRAP荧光共定位阳性细胞数也显著高于野生型小鼠（P=0.004）。RAW实验组唾液酸表达平均荧光强度（P < 0.001）及破骨细胞形成率（P < 0.001）均显著高于RAW对照组，而RAW药物组唾液酸表达平均荧光强度、破骨细胞形成率以及TRAP基因转录水平显著低于RAW实验组（P < 0.001）。BM实验组较BM对照组唾液酸表达平均荧光强度、破骨细胞形成率均显著提高（P < 0.001），而BM药物组较BM实验组唾液酸表达平均荧光强度、破骨细胞形成率均显著降低（P < 0.001）。结论TNF-α可通过提高破骨细胞的唾液酸化水平进而促进破骨细胞的分化和功能。     

地黄对阿尔茨海默病小鼠行为学的影响及其对血脑屏障的保护作用

目的：探讨中药地黄对阿尔茨海默病模型小鼠日常活动能力和恐惧记忆能力的行为学影响及其对血脑屏障的保护作用。方法：38只4月龄APP/PS1双转基因小鼠随机分为模型对照组（给予等渗氯化钠溶液）、地黄小剂量组（给予地黄5 g·kg ^–1·d ^–1）和地黄大剂量组（给予地黄12 g·kg ^–1·d ^–1），连续灌胃给药3个月，每日给药1次。分别采用筑巢实验和条件恐惧实验检测小鼠日常活动能力和恐惧记忆能力；硫黄素T染色检测小鼠皮层和海马CA1区β淀粉样蛋白（Aβ）沉积；免疫荧光双重标记染色检测小鼠皮层和海马CA1区血管内皮完整性及血脑屏障渗出情况。 结果：与模型对照组比较，地黄小、大剂量组日常活动能力均明显改善（均 P<0.01）；地黄大剂量组恐惧记忆能力明显提高（ P<0.01）；地黄大剂量组皮层和海马CA1区Aβ沉积量明显减少，面积占比明显减小，地黄小剂量组海马CA1区Aβ沉积面积占比也减小（均 P<0.05）；地黄小、大剂量组大脑皮层CD34阳性面积占比显著增加，纤维蛋白原阳性面积占比显著减小（均 P<0.05）；地黄大剂量组海马CA1区CD34阳性面积占比显著增加，纤维蛋白原阳性面积占比显著减小（均 P<0.05）。 结论：中药地黄可以提高模型小鼠的日常活动能力及恐惧记忆功能，减少脑内Aβ沉积，且大剂量时效果更为显著；其机制可能与降低血脑屏障通透性、保护血脑屏障完整性有关。     

远隔缺血预适应对脑缺血再灌注损伤大鼠缺血半暗带区PERK/p-eIF2α通路及自噬的影响

目的 研究远隔缺血预适应(remote ischemic preconditioning, RIPC)对局灶性脑缺血再灌注大鼠缺血半暗带区自噬相关蛋白Beclin1、LC3B以及内质网分子伴侣GRP78、内质网应激(endoplasmic reticulum stress, ERS)相关通路PERK/p-eIF2α的影响,探讨RIPC保护脑缺血损伤作用的相关机制。方法 将24只健康雄性Sprague-Dawley大鼠采用抽签法随机分为3组:假手术(Sham)组、大脑中动脉梗塞(middle cerebral artery occlusion, MCAO)组和RIPC+MCAO组。每组8只大鼠。采用改良线栓法制备大鼠MCAO再灌注模型,于缺血90 min后拔出线栓再灌注24 h。其中RIPC+MCAO组在MCAO之前,给予大鼠连续3 d的RIPC(采用夹闭双侧股动脉10 min开放10 min,每天3个循环的方法)。免疫荧光双标染色法检测再灌注大鼠缺血半暗带区自噬相关蛋白Beclin1、LC3B以及GRP78、p-eIF2α的表达。结果 1)Sham组大鼠脑内偶见Beclin1阳性细胞。MCAO组大鼠再灌注24 h缺血半暗带区Beclin1表达水平比Sham组显著升高(P<0.05)。给予RIPC的脑缺血再灌注大鼠半暗带区Beclin1的表达水平比MCAO组显著减少(P<0.05)。Beclin1与神经元标志物NeuN共定位。2)MCAO组大鼠再灌注24 h,脑缺血半暗带区GRP78分别与Beclin1和LC3B免疫荧光染色共定位。3)MCAO组大鼠再灌注24 h,脑缺血半暗带区Beclin1与p-eIF2α免疫荧光染色共定位。Sham组大鼠未见Beclin1和p-eIF2α双标阳性细胞,MCAO组大鼠再灌注24 h半暗带区可见大量Beclin1和p-eIF2α双标阳性细胞。给予RIPC后,缺血大鼠半暗带区Beclin1和p-eIF2α双标阳性细胞数目比MCAO组明显减少(P<0.05)。结论 RIPC可能通过抑制ERS相关通路PERK/p-eIF2α,减少神经元中自噬相关蛋白产生,避免神经元过度激活自噬,发挥对脑缺血再灌注损伤的保护作用。     

黄芪多糖抑制IL-1β表达及改善脓毒症小鼠心功能研究

目的 探讨黄芪多糖对脓毒症小鼠心肌成纤维细胞IL-1β表达水平及心功能的影响。方法 取2~3月龄C57BL/6小鼠,设立对照组。以腹腔注射脂多糖(lipopolysaccharidi,LPS)建立脓毒症模型,设立脓毒症组和黄芪多糖(astragalus polysaccharides,APS)处理组,6 h后酶联免疫吸附法(ELISA)检测小鼠外周血IL-1β蛋白表达,24 h后超声心动图检测小鼠心脏功能。取对照组C57BL/6小鼠,体外分离培养小鼠心肌成纤维细胞,设立对照组、LPS处理组和黄芪多糖低、中、高浓度(1、10、100 mg/L)+LPS处理组。采用ELISA检测细胞上清液中IL-1β,采用免疫印迹法检测Pro-IL-1β及IL-1β蛋白。结果 脓毒症小鼠心脏功能较对照组明显减退(P<0.05),黄芪多糖处理组较未处理的脓毒症小鼠心脏功能明显改善(P<0.05)。脓毒症组外周血IL-1β蛋白较对照组明显升高(P<0.05),黄芪多糖处理组的脓毒症小鼠IL-1β明显降低(P<0.05); LPS或联合三磷酸腺苷(ATP)诱导的心肌成纤维细胞与对照组相比,LPS组(LPS 1 ng/mL)诱导心肌成纤维细胞Pro-IL-1β及IL-1β蛋白明显增加(P<0.05),而黄芪多糖处理组较LPS组相比,Pro-IL-1β及IL-1β蛋白表达明显减少(P<0.05)。结论 黄芪多糖对脓毒症小鼠心肌成纤维细胞IL-1β蛋白表达有抑制作用,改善脓毒症引起的心脏功能障碍。     

VIPR1启动子甲基化促进转录因子AP-2α下调VIPR1的表达并促进肝细胞癌的生长

目的探讨血管活性肠肽受体1（VIPR1）在肝细胞癌（HCC）组织中低表达的转录调控机制和生物学功能。方法肝癌细胞系Hep3B和Huh7常规培养，实验分为VIPR1启动子野生型组、启动子突变载体1组、启动子突变载体2组和启动子突变载体1+ 2组。双荧光素酶报告基因实验检测AP-2α表达对VIPR1启动子活性的影响；DNA甲基化转移酶抑制剂（DAC）处理肝癌细胞，焦磷酸测序法检测VIPR1启动子甲基化水平的变化；染色质免疫共沉淀检测AP-2α与VIPR1启动子的结合能力；Western blot检测AP-2α的敲减作用以及VIPR1的表达；Western blot检测两种细胞株中VIPR1的差异表达；qPCR和Western blot检测VIPR1过表达和敲减效果；流式细胞术检测细胞周期和细胞凋亡；CCK8实验检测细胞增殖。建立裸鼠移植瘤模型，检测LV-NC组和LV-VIPR1组中肿瘤的体积和质量。结果与VIPR1启动子野生型组相比，启动子突变载体1+2与AP-2α表达质粒共转染后，荧光素酶活性明显恢复（P < 0.05）；DAC处理后，VIPR1启动子甲基化水平降低，并且随着甲基化程度减弱，与VIPR1启动子结合的AP-2α显著减少（P < 0.01）；敲减AP-2α后，VIPR1表达上升；Huh7细胞株中VIPR1表达低于Hep3B细胞株，在两种细胞株中成功构建了VIPR1过表达和敲减模型，VIPR1过表达增加了G2/M期的细胞数量（P < 0.01），凋亡细胞显著增加（P < 0.001），细胞增殖受到抑制（P < 0.001），而VIPR1敲低则相反。体内实验表明VIPR1过表达组的肿瘤体积减小（P < 0.001），肿瘤量降低（P < 0.05）。结论HCC中VIPR1的启动子甲基化促进了转录因子AP-2α的结合，并且抑制了VIPR1表达，而VIPR1过表达可使细胞周期阻滞在G2/M期，促进细胞凋亡，抑制细胞增殖和肿瘤生长。     

产后抑郁与初乳中转化生长因子-β水平的关系：90例巢氏队列研究

目的探究产后抑郁（PPD）与初乳转化生长因子-β（TGF-β）水平的关系。方法于2020年12月~2021年9月在广东省某家三级综合医院建立的母婴队列中招募产妇。产后第2天下午，采用爱丁堡产后抑郁量表（EPDS）对产妇进行筛查，评分≥10分者为产后抑郁产妇（PPD暴露组），并按1∶1配比（年龄±5岁、分娩方式相同）正常产妇（PPD非暴露组）。产后第3天上午，采集初乳标本并采用酶联免疫法测定其TGF-β水平。分析比较两组产妇初乳TGF-β水平，并探究其与EPDS评分的相关性。结果共招募90例产妇。PPD暴露组产妇初乳TGF-β1、TGF-β2及TGF-β3的水平分别为684.03（321.22，859.25）pg/mL，（5116.50±1747.04）pg/mL及（147.84±48.68）pg/mL；而PPD非暴露组产妇初乳TGF-β1、TGF-β2及TGF-β3的水平分别为745.67（596.00，964.22）pg/mL，（4912.40±1516.80）pg/mL及（168.21±48.15）pg/mL。PPD暴露组产妇初乳TGF-β1（P=0.026）和TGF-β3（P= 0.049）水平显著低于正常产妇。Spearman相关分析显示初乳TGF-β1（r=-0.23，P=0.03）及TGF-β3（r=-0.25，P=0.02）水平与EPDS评分呈负相关。结论初乳TGF-β1和TGF-β3水平降低与PPD有关，提示我们应加强孕产期PPD的早期筛查与早期干预，降低其对母乳成分的影响，从而促进婴幼儿健康发育。