大黄附子汤对重症急性胰腺炎小鼠肠道微皱褶细胞变化的影响

目的研究大黄附子汤对重症急性胰腺炎(SAP)小鼠肠道微皱褶细胞(M细胞)变化的影响，为大黄附子汤临床防治SAP提供理论基础。 方法选取40只健康SPF级C57BL/6J雄性小鼠，随机分为4组(每组10只)：空白对照组、大黄附子汤对照组、SAP组、大黄附子汤治疗组，其中SAP组和大黄附子汤治疗组分别以3.5 g/kg剂量腹腔注射20% L-精氨酸，空白对照组和大黄附子汤对照组注射等剂量的等渗盐水；于造模后12、24、36 h，空白对照组和SAP组给予等渗盐水0.2 mL，大黄附子汤对照组和治疗组给予大黄附子汤0.2 mL灌肠，均于造模完成48 h后处死取材，使用ELISA检测血清淀粉酶、内毒素、IL-1β及TNF-α含量，取回肠及胰腺组织行HE染色、评分，免疫组化染色检测M细胞特异性蛋白GP2并评分。 结果(1)一般情况：对照组腹腔注射后小鼠一般情况好，精神状态良好，能正常进水，四肢活动自如，行动未受影响；SAP组小鼠腹腔注射后一般情况差，精神萎靡，反应迟钝，行动迟缓，呼吸急促，拱背收腹，饮水减少。(2)SAP组淀粉酶、内毒素、IL-1β及TNF-α水平较对照组明显升高(P<0.05)；与SAP组进行对比，大黄附子汤治疗组血清淀粉酶、内毒素、IL-1β及TNF-α水平出现显著下降(P<0.05)。(3)HE染色：SAP组胰腺及回肠组织坏死严重，可见大量白细胞浸润。大黄附子汤治疗组胰腺及回肠组织轻度坏死，可见少量中性粒细胞等白细胞浸润。(4)免疫组化染色：SAP组与对照组相比GP2表达降低(P<0.05)；相较于SAP组，大黄附子汤治疗组GP2的表达水平升高(P<0.05)。 结论大黄附子汤治疗可改善作为肠道免疫应答起始的M细胞数量与功能，增强肠道免疫应答，减轻肠免疫屏障损伤，减少内毒素入血，改善SAP症状。     

温阳解郁颗粒调节BDNF/TrkB/ERK信号通路保护皮质酮诱导损伤型海马神经细胞的作用机制研究＊

目的 观察温阳解郁颗粒（Wenyang Jieyu granule，WYJY）对皮质酮（Corticosterone，CORT）诱导损伤型小鼠海马神经细胞（TH22 cell）的保护作用，基于脑源性神经营养因子（Brain derived neurotrophic factor， BDNF）/酪氨酸激酶B（Tyrosine kinase B， TrkB）/细胞外信号调节蛋白激酶（Extra cellular regulated protein kinases， ERK）信号通路探讨WYJY保护海马神经细胞的作用机制。方法 体外构建小鼠海马神经细胞皮质酮诱导损伤模型，以不同浓度的WYJY和氟西汀（Fluoxetine，FXT）含药血清作用于模型细胞，细胞增殖-毒性检测（Cell Counting Kit-8， CCK-8）法分析细胞活性，倒置显微镜下观察给药前后细胞形态结构的改变，采用蛋白免疫印迹法（Western Blot）、实时荧光定量PCR（Quantitative real-time PCR， qPCR）法检测神经细胞内凋亡因子（BCL2-Associated X， Bax）、抗凋亡因子（B-cell lymphoma-2， Bcl-2）、BDNF、Trkb、ERK以及丝氨酸/苏氨酸激酶（Phospho-p90RSK， RSK）、环磷腺苷效应元件结合蛋白（cAMP-response element binding protein， CREB）蛋白表达水平以及相关基因的表达水平。结果 在浓度为459.5 μmol·L^-1的CORT作用24 h后，HT22细胞的活性抑制率达到50%，在此条件作用下细胞形态结构损伤明显，凋亡程度严重，细胞上清中BDNF的含量显著减少（P<0.05），细胞内凋亡相关因子Bax/Bcl-2的比值明显升高（P<0.01），BDNF、Trkb、ERK、RSK、CREB磷酸化蛋白表达水平和mRNA表达水平明显降低（P<0.01）；以5%的浓度为2.85 g·kg^-1的WYJY和10%的FXT含药血清作用于受损的HT22细胞后，HT22细胞存活率明显提升（P<0.01），细胞结构的损伤明显改善，细胞凋亡程度减轻，细胞外BDNF的含量显著升高（P<0.05），细胞内Bax/Bcl-2比值显著下调（P<0.01），BDNF、Trkb、ERK、RSK、CREB磷酸化蛋白表达水平和mRNA表达水平显著提升（P<0.05，P<0.01）。结论 温阳解郁颗粒可有效保护高浓度CORT造成的小鼠海马神经细胞损伤。调控BDNF/Trkb/ERK通路，放大CREB信号传导，影响Bcl-2、BDNF水平，可能是其保护海马神经元，发挥抗抑郁疗效的重要机制。     

Biomarkers of in vivo limbal stem cell function

PurposeTo evaluate in vivo parameters as biomarkers of limbal stem cell function and to establish an objective system that detects and stage limbal stem cell deficiency (LSCD).MethodsA total of 126 patients (172 eyes) with LSCD and 67 normal subjects (99 eyes) were included in this observational cross-sectional comparative study. Slit-lamp biomicroscopy, in vivo laser scanning confocal microscopy (IVCM), and anterior segment optical coherence tomography (AS-OCT) were performed to obtain the following: clinical score, cell morphology score, basal cell density (BCD), central corneal epithelial thickness (CET), limbal epithelial thickness (LET), total corneal nerve fiber length (CNFL), corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD), and tortuosity coefficient. Their potential correlations with the severity of LSCD were investigated, and cutoff values were determined.ResultsAn increase clinical score correlated with a decrease in central cornea BCD, limbal BCD, CET, mean LET, maximum LET, CNFL, CNFD, CNBD, and tortuosity coefficient. Regression analyses showed that central cornea BCD, CET and CNFL were the best parameters to differentiate LSCD from normal eyes (Coef = 3.123, 3.379, and 2.223; all p < 0.05). The rank correlation analysis showed a similar outcome between the clinical scores and the central cornea BCD (ρ = 0.79), CET (ρ = 0.82), and CNFL (ρ = 0.71). A comprehensive LSCD grading formula based on a combination of these parameters was established.ConclusionsA comprehensive staging system combining clinical presentation, central cornea BCD, CET, and CNFL is established to accurately and objectively diagnose LSCD and stage its severity.     

Tumor-induced erythroid precursor-differentiated myeloid cells mediate immunosuppression and curtail anti-PD-1/PD-L1 treatment efficacy

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核因子κB受体活化因子配体和肿瘤坏死因子α经炎性牙周膜干细胞外泌体促进破骨细胞分化

目的　慢性牙周炎表现的病理性骨吸收非常常见，牙周膜干细胞（PDLSCs）的外泌体（Exo）对骨吸收的作用和影响尚不明确，本研究分析了该Exo蛋白组分对破骨细胞分化的影响。方法　分别从正畸前拔牙患者和慢性牙周炎患者的牙周膜组织中提取PDLSCs，通过流式细胞术检测表面标记物；通过差速离心分别提取2种细胞的Exo，即Exo-WT和Exo-CP，并通过Western blot、透射电子显微镜（TEM）、蛋白浓度检测、纳米粒径追踪检测Exo特征；通过蛋白质谱检测2种Exo的蛋白组分；通过酶联免疫吸附（ELISA）验证了差异表达蛋白肿瘤坏死因子α（TNF-α）、核因子κB受体活化因子配体（RANKL）、白细胞介素（IL）-1α、转化生长因子β（TGF-β）、骨形态发生蛋白2（BMP-2）表达水平；将10、100、1 000 μg·mL^-1的Exo-CP或Exo-WT加入RAW264.7培养基中并于5 d时通过实时荧光定量聚合酶链反应（RT-qPCR）、Western blot、抗酒石酸酸性磷酸酶（TRAP）染色检测破骨分化相关指标表达情况；采用SPSS 24.0软件对实验数据进行统计学分析。结果　Exo-CP富集的差异表达蛋白主要与破骨细胞分化的TNF信号通路、核转录因子κB（NF-κB）信号通路相关；ELISA实验证实了Exo-CP中高表达TNF-α、RANKL、IL-1α，低表达TGF-β1、BMP-2（P<0.05）；Exo-CP作用于RAW264.7，显著提高了细胞的破骨分化相关基因及蛋白的表达水平，TRAP染色可见分化的破骨细胞，且呈现浓度依赖性，100、1 000 μg·mL^-1浓度Exo-CP对破骨细胞分化的促进作用显著高于10 μg·mL^-1浓度组（P<0.05）。结论　慢性牙周炎的病理性骨吸收可能由炎性PDLSCs所分泌的Exo通过促进破骨细胞分化引起，Exo中主要的作用蛋白可能为RANKL和TNF-α，本研究为慢性牙周炎骨吸收的发病机制提供了新视角。     

Methodological advances in the design of peptide-based vaccines

The tremendous advances in genomics, recombinant DNA technology, bioengineering and nanotechnology, in conjunction with the development of high-end computations, have been instrumental in the process of rational design of peptide-based vaccines. The use of peptide vaccines was limited owing to their inherent instability when systemically administered; however, advanced formulation techniques have been developed for their systemic delivery, thereby overcoming their degradation, clearance, cellular uptake and off-target effects. With the rise of sophisticated immunological predictors and experimental techniques, several methodological advances have occurred in this field. This review examines contemporary methods to identify and optimize epitopes, engineer their immunogenic properties and develop their safe and efficient delivery into the host.     

飞秒激光辅助超声乳化联合Ahmed青光眼引流阀植入术治疗合并难治性青光眼的白内障

目的：评估飞秒激光辅助超声乳化联合Ahmed青光眼引流阀植入术治疗合并难治性青光眼的白内障的有效性和安全性。

方法：回顾性病例对照研究。2019-10/2021-10入院合并难治性青光眼的白内障患者53例53眼，依据自愿选择分为飞秒激光辅助白内障超声乳化(FLACS)组26例26眼和常规白内障超声乳化(CPCS)组27例27眼。两组分别行FLACS和CPCS联合Ahmed青光眼引流阀植入术。比较两组患者术中超声乳化能量释放量(CDE)、有效超声时间(EPT)的差异和术前与术后抗青光眼药物数量的变化，以及术后观察不同时期(1d，1wk，1、3mo)在提高最佳矫正视力(BCVA)，降低眼压、角膜内皮细胞损伤程度和手术并发症及成功率状况。

结果：FLACS组术中CDE和EPT明显低于CPCS组(t=8.50、5.16； P<0.01、=0.001)。两组术后抗青光眼药物较术前均明显减少(t=9.12、7.76； P=0.011、0.016)，但两组间无差异(t=1.79，P=0.082)。两组术后BCVA均较术前改善，眼压均较术前降低(P<0.05)。FLACS组在术后早期(1d，1wk)BCVA的改善较CPCS组更显著(t=9.74、8.49； P=0.008、0.012)，但在术后1、3mo的BCVA改善程度并无不同(t=0.62、0.44； P=1.415、2.021)。CPCS组在术后随访不同时期的角膜内皮细胞损伤较FLACS组更明显(P<0.05)。术后随访的不同时期FLACS组和CPCS组在控制眼压方面无差异(F_组间=0.64，P_组间=0.421)。FLACS组的手术并发症发生率27%(7/26)较CPCS组89%(24/27)低(χ²=20.95，P<0.01)，其中角膜水肿(8% vs 41%)、前囊撕裂(0 vs 11%)在FLACS组中明显低于CPCS组，后囊破裂(0 vs 7%)、玻璃体脱出(0 vs 4%)及人工晶状体偏位(0 vs 7%)也均发生在CPCS组。但两组的治疗总成功率相近(P=28.718)。

结论：飞秒激光辅助超声乳化联合Ahmed青光眼引流阀植入术可充分发挥联合手术的精准微创可控优势，帮助合并难治性青光眼的白内障患者有效降低眼压及更早获得视力恢复。     

Comparison of P25 and nanobelts on Kruppel-like factor-mediated nitric oxide pathways in human umbilical vein endothelial cells

Recently, we reported that titanium dioxide (TiO₂) materials activated endothelial cells via Kruppel-like factor (KLF)-mediated nitric oxide (NO) dysfunction, but the roles of physical properties of materials are not clear. In this study, we prepared nanobelts from P25 particles and compared their adverse effects to human umbilical vein endothelial cells (HUVECs). TiO₂ nanobelts had belt-like morphology but comparable surface areas as P25 particles. When applied to HUVECs, P25 particles or nanobelts did not induce cytotoxicity, although nanobelts were much more effective to increase intracellular Ti element concentrations compared the same amounts of P25 particles. Only nanobelts significantly induced THP-1 adhesion onto HUVECs. Consistently, nanobelts were more significant to induce the expression of intracellular adhesion molecule-1 (ICAM1) and the release of soluble ICAM-1 (sICAM-1), indicating that nanobelts were more potent to induce endothelial activation in vitro. As the mechanisms for endothelial activation, both P25 and nanobelts reduced the generation of intracellular NO as well as the expression of NO regulators KLF2 and KLF4. Combined, the results from this study indicated that the different morphologies of P25 particles and nanobelts only changed their internalization into HUVECs but showed minimal impact on KLF-mediated NO signaling pathways.