自发性高血压大鼠永久性脑缺血模型建立的研究

目的在比较自发性高血压大鼠(SHR)与同龄无高血压Wistar大鼠永久性大脑中动脉阻塞(pMCAO)后脑缺血损伤情况并初步分析其可能机制。方法雄性SHR和Wistar大鼠各30只分别随机分为:pMCAO模型6 h组、假手术6 h组、pMCAO模型24 h组、假手术24 h组和正常组(均n=6)。采用线栓法制作pMCAO模型,术后6、24 h对大鼠进行神经功能学评分后处死,制作脑冠状切片。术后6 h处死大鼠脑部切片行尼氏染色后在组织学层面上观察神经元损伤情况;术后24 h处死大鼠脑部切片行尼氏染色后计算脑梗死体积和水肿程度百分比。正常组脑部切片经苏木精-伊红染色后计算脑部小血管壁/腔比。结果术后6和24 h,不同品系大鼠神经功能学评分差异无统计学意义(P0.05);术后6 h尼氏染色示SHR神经元损伤重于Wistar大鼠。术后24 h SHR脑梗死体积百分比[(28.05±2.38)%]大于Wistar大鼠[(25.23±1.33)%],差异有统计学意义(P0.05)。两品系大鼠之间脑水肿程度差异无显著性。脑部小血管壁/腔比SHR[(11.46±3.74)%]较Wistar大鼠[(8.73±1.73)%]增大(P0.05)。结论 pMCAO术后SHR的脑缺血损伤程度重于Wistar大鼠,可能与高血压引起的脑侧支循环血管壁增厚、僵硬,自我调节能力降低有关。     

肺腺癌对一代EGFR-TKIs原发耐药的危险因素分析

目的:分析对一代表皮生长因子受体酪氨酸激酶抑制剂(epidermal growth factor receptor-tyrosine kinase inhibitors,EGFR-TKIs)原发耐药的EGFR突变肺腺癌患者的临床特征,为预测肺腺癌患者是否对一代EGFR-TKIs原发耐药提供依据。方法:收集2014年01月至2019年04月于本院住院,一线使用一代EGFR-TKIs且随访时间超过6个月的EGFR敏感突变(19Del/21L858R)肺腺癌患者,根据疗效纳入原发性耐药组(NR=40)和敏感组(NS=237),比较两组患者的临床、影像特征及实验室指标之间的差异,分析对一代EGFR-TKIs原发耐药的危险因素。结果:EGFR敏感突变患者的原发性耐药发生率为14.4%。原发性耐药组与敏感组患者相比,二者在吸烟指数(P=0.004)及淋巴结转移(P=0.03)的差异有统计学意义。血清神经元特异性烯醇化酶(neuron specific enolase,NSE)≥10.725 ng/mL、肿瘤直径≥3.55 cm的患者更易对一代EGFR-TKIs耐药(P<0.05),各因素AUC值分别为0.615、0.716。联合NSE+肿瘤直径两项指标时AUC为0.735(95%CI:0.665~0.804),联合NSE+肿瘤直径+吸烟指数三项指标时AUC为0.751(95%CI:0.679~0.822),均优于单项指标。多因素Logistics回归分析证实,血清NSE浓度、肿瘤直径及吸烟指数是预测EGFR敏感突变患者对一代EGFR-TKI原发耐药的独立影响因素(P<0.05)。结论:吸烟指数≥400、病灶直径≥3.55 cm、血清NSE浓度≥10.725 ng/mL的患者更易对一代EGFR-TKIs原发耐药。单因素对预测EGFR突变患者是否对一代EGFR-TKIs原发耐药准确性较低,综合上述三项指标预测效果更好。     

Necroptotic TNFα-Syndecan 4-TNFα Vicious Cycle as a Therapeutic Target for Preventing Temporomandibular Joint Osteoarthritis

Temporomandibular joint osteoarthritis (TMJOA) is a chronic degenerative disease for which the underlying mechanism still remains unclear. Compared with apoptosis and autophagy, necroptosis causes greater harm to tissue homeostasis by releasing damage-associated molecular patterns (DAMPs). However, the role of necroptosis and downstream key DAMPs in TMJOA is unknown. Here, rodent models of TMJOA were established by the unilateral anterior crossbite (UAC). Transmission electron microscopy (TEM) and immunohistochemistry of receptor interacting protein kinase 3 (RIPK3)/phosphorylation of mixed lineage kinase domain-like protein (pMLKL) were conducted to evaluate the occurrence of necroptosis in vivo. The therapeutic effects of blocking necroptosis were achieved by intra-articularly injecting RIPK3 or MLKL inhibitors and using RIPK3 or MLKL knockout mice. In vitro necroptosis of condylar chondrocyte was induced by combination of tumor necrosis factor alpha (TNFα), second mitochondria-derived activator of caspases (SMAC) mimetics and carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (z-VAD-fmk). The possible DAMPs released by necroptotic chondrocytes were screened by quantitative proteomics and blocked by specific antibody. Translucent cytosol, swollen organelles, and ruptured cell membranes, features of necroptosis, were frequently manifested in chondrocytes at the early stage of condylar cartilage degeneration in TMJOA, which was accompanied by upregulation of RIPK3/pMLKL. Inhibiting or knocking out RIPK3/MLKL significantly prevented cartilage degeneration. DAMPs released by necroptotic condylar chondrocytes, such as syndecan 4 (SDC4) and heat shock protein 90 (HSP90), were verified. Furthermore, blocking the function of SDC4 significantly attenuated the expression of TNFα in cartilage and synovium, and accordingly increased cartilage thickness and reduced synovial inflammation. Thus, the necroptotic vicious cycle of TNFα-SDC4-TNFα contributes to cartilage degeneration and synovitis, and can serve as a potential therapeutic target for treating TMJOA. © 2022 American Society for Bone and Mineral Research (ASBMR).     

针刺辅助治疗难治性肠易激综合征临床试验方案关键点设计的思考＊

肠易激综合征（Irritable bowel syndrome，IBS）是临床常见病、多发病，其治疗方法丰富，但部分患者疗效欠佳，发展成难治性IBS。目前国内外关于针灸治疗难治性IBS的临床随机对照试验尚不多见。本文立足试验方案设计的“PICOS”原则，从研究对象及诊断标准、干预措施、对照措施、结局指标四个方面入手，重点探讨针刺辅助治疗难治性肠易激综合征临床试验设计的关键要点。从选择特色优势病种、明确诊断标准、制定符合临床实际的干预方案、运用符合目标的安慰针刺、结合研究设计和目的选定结局指标几个角度，阐述试验相关环节设计的原因和思考。     

纯化痘病毒筛选的常用方法

痘病毒是自然界普遍存在的DNA病毒，能够高效表达外源基因，诱导较强的细胞免疫和体液免疫，受到基因治疗学者的广泛关注。痘病毒发生同源重组的概率较低，所以有效地筛选和纯化痘病毒十分重要。目前常用的筛选重组痘病毒方法有基于CRISPR进行筛选；根据报告基因GFP、LacZ、GPT等进行筛选；利用TK^-和Brdu结合荧光进行筛选；利用药物抗性基因与荧光或显色基因融合，即可在药物筛选的同时观察荧光或显色基因表达情况。本文旨在对目前常用的痘病毒的筛选纯化方法进行综述。     

Sequential Therapy Based on Evolvement of Patterns: A New Model for Treatment of Alzheimer’s Disease

In order to solve the problem of long-term (>9 months) efficacy in the treatment of Alzheimer''s disease (AD) by conventional therapy (CT), a staged and multiply-targeted sequential therapy based on the evolvement of patterns (STEP) was developed. Its main innovations include: (1) the time order of evolution of patterns defined by Chinese medicine (CM) in AD was found, that is, "the orderly pattern evolution starting from Shen (Kidney) deficiency, progressing to phlegm, stasis and fire, and worsening to severe toxin as well as functional collapse"; (2) the cascade hypothesis of Shen deficiency in AD and its sequential therapy based on Shen-reinforcing was proposed, that is, "reinforcing Shen in the early stage and throughout the whole process, resolving phlegm, activating blood and purging fire in the middle stage, detoxifying and replenishing vitality to stop the collapse in the advanced stage", and through meta-analysis, clinical drug use was optimized, thus the leap from "inferential selection" to "evidence-based selection" was realized; (3) the STEP regimen combined with CT maintained cognitive and behavioral stability in AD patients for at least 12 months, with cognitive enhancement and behavioral synergy after 9 months, and cognitive benefit was superior to CT at 9, 12, 15, 18, 21, and 24 months, respectively. The 2-year cognitive improvement rate was increased by 25.64% (P=0.020) and the cognitive deterioration rate was decreased by 48.71% (P=0.000). Among them, the cognitive and functional benefits of Shen-reinforcing therapy for very early AD (350 cases) for 1 year were better than the placebo (P<0.001), and the dementia conversion rate was reduced by 8.85% (P=0.002). The behavioral symptomatic relief of patients with vascular dementia received fire-purging therapy (540 cases) was superior to those received CT (P=0.016). These data suggested that the STEP regimen has synergistic effects on CTs at least in terms of cognitive benefit, and the earlier the use, the greater the benefit will have. Therefore, the STEP regimen should be considered as one of the clinical options, particularly for the dearth of effective pharmaceutical or immunological interventions that are currently available for AD.