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关键词： FAM50A   抗体制备   基因敲除   纤毛   胰腺   糖尿病  

摘要： 目的 利用CRISPR/Cas9基因编辑技术构建50序列相似的家庭成员A(FAM50A)基因敲除的小鼠胰岛素瘤胰岛β细胞系Beta-TC-6,并制备特异性识别FAM50A的多克隆抗体。方法 设计2条靶向FAM50A基因的向导RNA(sgRNA),然后构建表达亮蓝荧光蛋白(BFP)的重组质粒用于基因敲除。将构建成功的重组质粒转染入Beta-TC-6细胞,并筛选出BFP阳性单细胞进行克隆扩增。对扩增后的单克隆细胞采用Sanger测序进行基因型鉴定,并使用Western blot检测FAM50A蛋白的表达。将纯化的人源FAM50A重组蛋白免疫新西兰兔以制备多克隆抗体,并利用已构建的基因敲除细胞系验证其特异性。结果成功筛选并鉴定出1株FAM50A基因敲除的单克隆细胞系,Sanger测序证实其靶向位点存在碱基缺失。Western blot检测结果显示,该细胞系中无FAM50A蛋白表达。所制备的多克隆抗体能够识别野生型Beta-TC-6细胞中的鼠源FAM50A蛋白及hTERT-RPE1细胞中过表达的人源FAM50A-GFP融合蛋白,但在FAM50A敲除细胞中未检测到信号。结论 成功建立了FAM50A基因敲除的Beta-TC-6细胞模型,且成功制备了FAM50A的多克隆抗体。这些成果为后续研究提供了有力工具。

关键词： 腹膜透析相关性腹膜炎   扶肾方   腹腔驻留巨噬细胞   氧化磷酸化   细胞因子  

摘要： [目的]基于腹腔驻留巨噬细胞(PRM)探讨扶肾方对脂多糖(LPS)诱导的腹膜透析相关性腹膜炎(PDAP)小鼠的影响及作用机制。[方法]将36只c57BL/6J雄性小鼠按随机数字表法随机分为6组:正常组、1.5%PDS组、1.5%PDS+扶肾方低剂量(FS-L)组、1.5%PDS+扶肾方高剂量(FS-H)组、1.5%PDS+LPS组、1.5%PDS+LPS+FS-H组,每组6只。观察小鼠一般状态,采用流式细胞仪检测小鼠PRM的比例、吞噬功能及一氧化氮(NO)的生成;使用酶联免疫吸附实验(ELISA)检测各组小鼠腹腔灌洗液中白介素-6(IL-6)、白介素-10(IL-10)和肿瘤坏死因子-α(TNF-α)。[结果]与PD组比较,LPS组小鼠PRM比例显著下降(P<0.001),吞噬功能明显减弱(P<0.001),NO生成显著增多(P<0.001),腹腔灌洗液中IL-10和TNF-α也显著增多(P<0.001)。与LPS组比较,给药小鼠PRM比例、吞噬功能均有一定的恢复(P<0.01),NO显著下降(P<0.001),IL-6、TNF-α显著减少((P<0.001),IL-10明显增多(P<0.05)。[结论]扶肾方可以显著改善PDAP小鼠的炎症状态,可能与通过减少PRM的NO合成,促进氧化磷酸化,来调节高糖环境下腹腔的免疫稳态有关。

关键词： 细粒棘球蚴抗原B   巨噬细胞极化   STAT6   PPAR-γ  

摘要： 探讨天然细粒棘球蚴抗原B (nAgB)通过信号转导子和转录激活子6 (STAT6)/过氧化物酶体增殖物激活受体-γ (PPAR-γ)信号通路体外对RAW264.7巨噬细胞极化的调控作用。方法无菌条件下提取并纯化nAgB。RAW264.7细胞加入0、1、4、16、64μg/ml浓度的nAgB，分别于0、6、12、24 h、48 h后用CCK8法检测细胞活力，并于24 h后用EdU染色法检测细胞增殖能力。RAW264.7细胞分为对照组、M1诱导组、M2诱导组、nAgB组、nAgB干预M1诱导组和nAgB干预M2诱导组6个组：nAgB组、nAgB干预M1诱导组和nAgB干预M2诱导组分别加入1μg/mlnAgB；干预1 h后，M1诱导组和nAgB干预M1诱导组分别加入500 ng/ml脂多糖LPS和100 ng/mlγ-干扰素（IFN-γ）,M2诱导组和nAgB干预M2诱导组分别加入100 ng/ml白细胞介素4 (IL-4)和100 ng/mlIL-13。收集各组细胞和上清液，RT-qPCR检测细胞极化标志物和STAT6、PPAR-γ的mRNA相对转录水平，Western blotting检测相关蛋白的相对表达水平，ELISA法检测相关细胞因子的含量。CCK-8检测结果表明，培养24 h时，1、 4、 16、 64μg/mlnAgB干预的细胞活力（1.41±0.09、 1.62±0.08、 1.78±0.04、 1.90±0.04）均高于0μg/ml(1.07±0.05)(t=8.67、14.57、27.43、31.87，均P <0.01)。EdU检测结果表明，1、4、16、64μg/mlnAgB干预的细胞增殖能力（0.65±0.01、 0.78±0.02、 0.89±0.02、 0.99±0.02）均高于0μg/ml (0.47±0.02)(t=16.21、20.58、33.47、39.43，均P <0.01)。RT-qPCR结果显示，nAgB干预M1诱导组细胞的iNOS、IL-6和IL-1βmRNA相对转录水平（3.23±0.92、1.56±0.58、19.87±0.35）均低于M1诱导组（10.62±1.68、3.97±0.25、24.40±0.03）(t=6.69、6.62、22.07，均P <0.01),nAgB干预M2诱导组细胞的Arg-1mRNA相对转录水平（29.30±2.92）高于M2诱导组（14.94±0.77）(t=8.23,P <0.01);nAgB组细胞的STAT6和PPAR-γmRNA相对转录水平（59.12±3.03、7.82±0.50）均高于对照组（26.38±1.89、3.71±0.17）(t=15.90、13.40，均P <0.01),nAgB干预M1诱导组细胞（40.73±2.91、4.19±0.88）均高于M1诱导组（17.93±1.90、1.76±0.08）(t=11.37、4.75，均P <0.01),nAgB干预M2诱导组细胞（140.50±5.64、11.67±0.80）均高于M2诱导组（37.55±5.92、6.87±0.28）(t=21.82、9.84，均P <0.01)。Westernblotting结果显示，nAgB干预M1诱导组细胞的iNOS蛋白相对表达水平（0.60±0.02）低于M1诱导组（1.02±0.03）(t=21.86,P <0.01),nAgB干预M2诱导组细胞的CD206蛋白相对表达水平（1.03±0.04）高于M2诱导组（0.84±0.02）(t=7.78,P <0.01);nAgB组细胞的P-STAT6和PPAR-γ蛋白相对表达水平（0.76±0.03、0.77±0.02）均高于对照组（0.55±0.05、0.37±0.00）(t=6.11、40.16,P <0.05、0.01),nAgB干预M1诱导组细胞（0.60±0.01、0.42±0.04）均高于M1诱导组（0.39±0.05、0.18±0.01）(t=6.64、10.06，均P <0.01), nAgB干预M2诱导组细胞（1.12±0.11、 0.94±0.02）均高于M2诱导组（0.86±0.05、 0.66±0.00）(t=3.71、28.18，均P <0.01)。ELISA检测结果显示，nAgB组细胞上清中TGF-β1和IL-10的含量[（70.27±4.57）、（167.00±29.27） pg/ml]均高于对照组[（29.87±2.24）、（50.17±8.99） pg/ml](t=13.76、6.61，均P <0.01);nAgB干预M1诱导组中TNF-α和IL-1β的含量[（523.20±6.72）、（387.80±3.84） pg/ml]均低于M1诱导组[（995.70±9.92

关键词： mRNA   vaccines   driver antigens   Merkel cell carcinoma   IL-7   T cell memory   adjuvants   patient samples   human translation   Merkel cell polyomavirus  

摘要： Although mRNA technologies have reinvigorated cancer vaccine development, the identification of strong antigens with consistent tumor cell expression and generation of durable antigen-specific CD8+ T cell memory remain key challenges. We identified the Merkel cell carcinoma (MCC) large T antigen (LTA) as an optimal vaccine target, essential for tumor cell survival and immunogenic in a cancer with high unmet clinical need. We developed an mRNA vaccine to MCC-LTA in murine studies and patient samples. We showed that antigen loss develops rapidly and causes resistance in mouse models when immunogenic, but non-essential antigens are targeted. To improve T cell response durability, we co-encoded LTA and IL-7, co-localizing proliferative and memory signals spatially and temporally with antigen exposure. IL-7-containing mRNA vaccines improved antigen-specific T cell expansion, memory differentiation, and tumor control. We propose that the principles of antigen essentiality and memory signal co-encoding may be adapted to improve the efficacy of mRNA therapeutics.

关键词： hemophagocytic lymphohistiocytosis   sIL-2R   sCD163   pediatric reference intervals   biomarker stability  

摘要： Objectives: Hemophagocytic lymphohistiocytosis (HLH) is a severe condition requiring timely diagnosis to initiate immunomodulatory therapy. Soluble interleukin-2 receptor (sIL-2R) and soluble CD163 (sCD163) are biomarkers crucial for making this diagnosis. This study aims to establish pediatric reference intervals (RIs) for serum concentrations of sIL-2R and sCD163, and to evaluate the pre-analytic stability of sIL-2R. Methods: Serum samples from 243 healthy children (aged 0-18 years) were analyzed to determine RIs for sIL-2R and sCD163. Samples were processed according to stringent pre-analytic protocols, with additional stability studies evaluating sIL-2R under various conditions, including temperature fluctuations and freeze-thaw cycles. The concentration of sCD163 was additionally determined in a protein reference material. RIs were calculated using non-parametric quantile regression, and subgroups were analyzed for potential partitioning by age, sex, and inflammation status (C-reactive protein (CRP) >8 mg/L). Results: Discrete and continuous RIs were established for serum sIL-2R and sCD163 in healthy children, with distinct age-dependent variations observed for sIL-2R. Stability studies confirmed sIL-2R's robustness under common pre-analytic conditions. Results were consistent across samples obtained from general practitioners and hospital clinics, enhancing their transferability. Conclusions: This study provides comprehensive pediatric RIs for serum sIL-2R and sCD163 to support clinical decision-making in HLH and related inflammatory disorders. The stability of both sIL-2R and sCD163 under diverse conditions supports their reliable use in clinical settings. These findings may facilitate broader clinical implementation, pending on local validation and standardization.

关键词： 益胃消癌汤   胃癌   体液免疫   肿瘤标志物   血管内皮生长因子   重组人Dickkopf相关蛋白-1  

摘要： 目的 探讨益胃消癌汤联合化疗治疗胃癌患者的近期疗效及对体液免疫、肿瘤标志物、血管内皮生长因子（vascular endothelial growth factor，VEGF）及重组人Dickkopf相关蛋白-1（recombinanthumanDickkopf-associatedprotein1，DKK-1）水平的影响。方法 选取2023年1月—2024年6月河南省鄢陵县中医院收治的68例胃癌患者，采用随机数表法分为研究组（34例）和对照组（34例）。对照组接受奥沙利铂联合卡培他滨方案化疗（capecitabineplusoxaliplatin，XELOX）方案化疗，研究组在此基础上加用益胃消癌汤。对比两组患者治疗前后近期疗效、体液免疫[免疫球蛋白A（immunoglobulinA，IgA）、免疫球蛋白G（immunoglobulinG，IgG）、免疫球蛋白M（immunoglobulinM，IgM）]、肿瘤标志物[癌胚抗原（carcinoembryonicantigen，CEA）、糖类抗原19-9（carbohydrateantigen19-9，CA19-9）、糖类抗原125（carbohydrateantigen125，CA125）]、血清VEGF及DKK-1水平的变化。结果 治疗后，研究组近期疗效明显优于对照组（P<0.05）。与治疗前相比，治疗后，两组患者体液免疫指标均有所改善，但研究组IgA、IgG、IgM水平高于对照组（P<0.05）。治疗后，研究组CEA、CA19-9、CA125下降幅度大于对照组（P<0.05）。研究组VEGF及DKK-1水平显著低于对照组（P<0.05）。结论 益胃消癌汤联合化疗可提高胃癌患者的近期疗效，改善体液免疫功能，降低肿瘤标志物水平，能够有效抑制VEGF和DKK-1表达，值得推广。

摘要： Although immunotherapy has demonstrated clinical efficacy in advanced gastric cancer (AGC), its therapeutic benefits are limited to a subset of patients. This study aimed to evaluate circulating cytokines as potential prognostic biomarkers to enhance the precision of clinical decision-making in PD-1 inhibitor-based immunotherapy for AGC. A total of 269 patients with advanced gastric cancer were enrolled according to predefined inclusion criteria. Among them, 117 patients received anti-PD-1 immunotherapy combined with chemotherapy (immunochemotherapy), while 152 received chemotherapy alone. Plasma levels of 12 cytokines were quantified using flow cytometry, and appropriate statistical analyses were performed. Compared with healthy controls, plasma concentrations of IL-6, IL-8, and IL-10 were significantly elevated in AGC patients. Furthermore, levels of these cytokines were markedly higher in the immunochemotherapy no response group compared to both the chemotherapy-alone group and response group. Multivariate analysis confirmed that IL-6, IL-8, and IL-10 were independent risk factors for poor treatment outcomes in patients receiving immunochemotherapy. Notably, the combined measurement of these three cytokines provided superior diagnostic accuracy compared to individual markers. The present study preliminarily demonstrates that IL-6, IL-8, and IL-10 cytokines may serve as potential predictive biomarkers for treatment outcomes in patients with AGC receiving anti-PD-1 immunochemotherapy. These easily accessible blood biomarkers are predictive and rapid and may play a key role in identifying individuals who may benefit from PD-1 blockade immunotherapy.

关键词： 抑制素多克隆抗体   C57BL/6J雌性小鼠   超数排卵   卵细胞数   两细胞胚胎数  

摘要： 目的 使用猪抑制素多肽偶联钥孔血蓝蛋白（keyhole limpet hemocyanin,KLH）免疫家兔制备抑制素多克隆抗体（anti-inhibin-serum, AIS）并探究其对C57BL/6J小鼠的超数排卵的影响。方法 将人工合成的猪抑制素多肽偶联钥孔血蓝蛋白（KLH）免疫家兔，制备抑制素多克隆抗体。将抗体联合孕马血清促性腺激素（pregnant mare serum gonadotropin,PMSG）刺激雌性C57BL/6J小鼠，48 h后使用人绒毛膜促性腺激素（human chorionic gonadotropin,hCG）进行超数排卵，检测C57BL/6J小鼠超数排卵的卵细胞数以及体外受精24 h后的两细胞胚胎数。结果 使用AIS联合PMSG的超数排卵效果优于常规PMSG联合h CG超数排卵方法，此种方法超数排卵产生的卵细胞数量相比较于普通超数排卵方法可以提高1.64倍（P<0.05）。将纯化AIS进行SDS-PAGE电泳分析，电泳结果显示，在50kDa及25kDa处出现了清晰可见的目的条带，符合抗体重链和轻链的分布，纯化效果良好。结论 本研究结果显示，使用猪的抑制素多克隆抗体作为改良的超数排卵试剂可以提高雌性C57BL/6J小鼠超数排卵效果，在后续的市场应用上有较大应用意义。

关键词： 急性淋巴细胞白血病   化疗   胃肠道功能障碍   中性粒细胞/淋巴细胞比值   血小板/淋巴细胞比值   单核细胞/淋巴细胞比值  

摘要： 目的:探讨外周血中性粒细胞/淋巴细胞比值(NLR)、血小板/淋巴细胞比值(PLR)、单核细胞/淋巴细胞比值(MLR)对急性淋巴细胞白血病(ALL)患儿化疗期间胃肠道功能障碍的预测价值。方法:选取2020年12月至2024年12月河北医科大学第四医院儿科收治的243例ALL患儿,均接受1～2个疗程化疗,化疗前检测NLR、PLR、MLR。化疗期间发生胃肠道功能障碍92例。采用Logistic回归分析NLR、PLR、MLR与ALL患儿化疗期间胃肠道功能障碍的关系,采用ROC曲线分析NLR、PLR、MLR预测胃肠道功能障碍的价值。结果:胃肠道功能障碍组NLR、PLR、MLR均高于无胃肠道功能障碍组(P<0.05)。回归分析结果显示,NLR、PLR、MLR与ALL患儿化疗期间胃肠道功能障碍风险呈正关联,OR(95%CI)分别为1.724(1.072～2.774)、1.638(1.013～2.650)、1.915(1.102～3.327)。NLR、PLR、MLR和三者联合预测ALL患儿化疗期间胃肠道功能障碍的AUC(95%CI)为0.739(0.676～0.803)、0.721(0.658～0.785)、0.764(0.702～0.827)和0.890(0.850～0.931),三者联合的AUC较大(P<0.05)。结论:NLR、PLR、MLR联合可预测ALL患儿化疗期间胃肠道功能障碍。