miR-513a-5p慢病毒载体的构建及其对人骨肉瘤细胞放疗敏感性的影响

目的:构建miR-513a-5p慢病毒过表达载体，转染人骨肉瘤细胞株，观察miR-513a-5p对人骨肉瘤细胞放疗敏感性的影响。方法:PCR法扩增人miR-513a-5p基因，克隆入pLentis-CMV-GFP-MCS-PGK-PURO载体获得重组质粒pLentis-miR513a，双酶切鉴定并测序后将正确的重组质粒和对照质粒转染293FT细胞制备慢病毒，分别转染骨肉瘤HOS和U2OS细胞，qRT-PCR法及荧光显微镜鉴定转染结果。克隆形成实验、MTT法检测miR-513a-5p高表达HOS和U2OS细胞在X射线照射下细胞存活情况。结果:双酶切及测序结果确定成功构建miR-513a-5p慢病毒载体pLentis-miR513a。qRT-PCR结果提示，转染骨肉瘤细胞株后miR-513a-5p表达显著升高。克隆形成实验结果显示miR-513a-5p高表达后骨肉瘤细胞在X射线照射下细胞增殖减慢。MTT结果提示miR-513a-5p高表达骨肉瘤细胞经X射线照射后细胞存活减少。结论:成功构建了miR-513a-5p慢病毒载体，建立了高效稳定表达miR-513a-5p的骨肉瘤细胞株，高表达miR-513a-5p能显著增加X射线照射后骨肉瘤细胞的放疗敏感性。     

PEG-rhG-CSF预防卵巢癌、宫颈癌化疗中性粒细胞缺乏的临床研究

目的:探讨应用聚乙二醇化重组人粒细胞集落刺激因子(PEG-rhG-CSF)对卵巢癌、宫颈癌化疗患者粒细胞减少的影响，并观察不良反应。方法:选择本院2015年4月至2017年6月卵巢癌、宫颈癌患者136例，随机分为对照组（68例）和研究组（68例），对照组应用rhG-CSF，研究组应用PEG-rhG-CSF，两组均化疗两个周期，评估患者两个周期不同时间血常规绝对中性粒细胞计数值（ANC）水平、ANC不同数量发生率和持续时间以及不良反应。结果:研究组患者第一个化疗周期第5 d、第7 d、第10 d和第14 d 及第二个化疗周期第3 d、第5 d、第7 d、第10 d和第14 d血ANC分别为（3.19±0.32）×109/L、（1.93±0.35）×109/L、（2.08±0.39）×109/L、（2.11±0.36）×109/L和（2.79±0.40）×109/L、（2.44±0.33）×109/L、（1.68±0.34）×109/L、（1.71±0.37）×109/L、（1.92±0.34）×109/L，均高于对照组，差异具有统计学意义(P＜0.05)。研究组患者第一个和第二个化疗周期ANC＜1.5×109/L、ANC＜1.0×109/L和ANC＜0.5×109/L发生率分别为23.53%（16/68）、8.82%（6/68）、2.94%（2/68）和29.41%（20/68）、10.29%（7/68）、5.88%（4/68），均低于对照组，差异具有统计学意义(P＜0.05)。研究组患者第一个和第二个化疗周期ANC＜1.5×109/L、ANC＜1.0×109/L和ANC＜0.5×109/L持续时间分别为（3.50±0.18）d、（2.17±0.23)d、（2.06±0.29）d和（4.08±0.22）d、（4.62±0.29）d、（4.11±0.24）d，均短于对照组，差异具有统计学意义(P＜0.05)。研究组中、重度不良反应总发生率为10.29%（7/68），低于对照组23.53%（16/68），差异有统计学意义(P＜0.05)。结论:应用PEG-rhG-CSF能够有效预防卵巢癌、宫颈癌化疗患者粒细胞减少，不良反应显著降低，值得临床推广应用。     

重组碱性成纤维细胞生长因子通过Notch1信号通路对糖尿病大鼠视网膜神经节细胞的保护作用

目的　探讨重组碱性成纤维细胞生长因子(recombinant basic fibroblast growth factor,rbFGF)通过Notch1信号通路对糖尿病大鼠视网膜神经节细胞(retinal ganglion cell，RGC)的保护作用。方法　雄性SD大鼠40只，随机分成对照组、糖尿病组、rbFGF组、rbFGF+DAPT组(DAPT为Notch1通路的特异性拮抗剂)，每组10只。后三组大鼠采用单次腹腔注射链脲佐菌素(streptozotocin,STZ) 诱导糖尿病模型。模型诱导成功后，rbFGF组给予rbFGF 10 μL(200 U)玻璃体内注射给药，rbFGF+DAPT组在给予rbFGF基础上加用DAPT(10 μmol·L^-1)。注射12周后，HE染色检测RGC密度，免疫组织化学染色检测神经元再生相关蛋白GAP-43、Notch1蛋白表达，Western blot检测GAP-43、Notch1蛋白及凋亡相关蛋白Caspase-3相对表达量。结果　与对照组RGC密度(433.49±6.02)个·mm^-2，GAP-43荧光强度(8.96±0.26)%、Notch1阳性率(45.04±0.46)%及Caspase-3 (27.91±0.63) %蛋白表达相比，糖尿病组RGC密度(328.35±6.43)mm^-2，Notch1荧光强度(31.66±0.40)%蛋白表达明显降低，GAP-43荧光强度(13.66±0.52)%、Caspase-3 (48.91±0.64)%蛋白表达明显增加(均为P<0.05)；而与糖尿病组相比，rbFGF组RGC密度(425.30±7.98)个·mm^-2，Notch1阳性率(41.76±0.62)% 及GAP-43荧光强度(33.05±0.37)%蛋白表达明显增加，Caspase-3 (28.86±0.71)%蛋白表达明显降低(均为P<0.05)；而rbFGF+DAPT组RGC密度(324.91±8.22)个·mm^-2，GAP-43荧光强度(14.27±0.64)%、Notch1阳性率(30.40±0.82)%及Caspase-3 (47.63±0.68)%蛋白表达均无明显变化(均为P>0.05)。结论　rbFGF可上调糖尿病状态下视网膜GAP-43蛋白表达，下调Caspase-3蛋白表达，进而提高RGC的存活，其机制可能与激活Notch1信号通路有关。     

Safety and Efficacy of Repeated Thrombolysis with Alteplase in Early Recurrent Ischemic Stroke: A Systematic Review

Background and Aim: The current American Heart Association guidelines for the management of acute ischemic stroke advise against the use of intravenous (IV) alteplase in patients with recurrent stroke occurring within 90 days of their index event. Following these guidelines strictly, patients having early recurrent ischemic stroke would be unable to avail of this reperfusion strategy that has been proven to confer superior clinical outcomes. While some registry-based studies have demonstrated the safety of IV alteplase in this subgroup of patients, data on the repeated use of the drug are lacking. Thus, we aim to determine the safety and efficacy of repeated thrombolysis in patients with early recurrent ischemic strokes. Methods: The following electronic databases were searched for relevant studies: the Cochrane Central Register for Controlled Trials by The Cochrane Library, MEDLINE by PubMed, Health Research and Development Information Network, Scopus, and ClinicalTrials.gov. Data on symptomatic intracranial hemorrhage, 90-day clinical outcomes, systemic hemorrhage and allergic reactionswere synthesized. Results: Ten articles with 33 patients in total were included in our review. One patient developed symptomatic intracranial hemorrhage after the second reperfusion attempt and subsequently died from pneumonia. Another died from spontaneous rupture of previously unidentified infrarenal aortic aneurysm. Six of the 13 patients with available follow-up data had good clinical outcomes (Modified Rankin Score 0-2). There were no allergic reactions and other drug-related adverse events noted. Conclusions: Repeated IV alteplase can be safe and efficacious in patients who have early recurrent ischemic stroke. Larger studies, trials, or registry-based data are needed to ascertain the encouraging findings of our review.     

Structural and functional characterization of β2‐glycoprotein I domain 1 in anti‐melanoma cell migration

We previously found that circulating β₂‐glycoprotein I inhibits human endothelial cell migration, proliferation, and angiogenesis by diverse mechanisms. In the present study, we investigated the antitumor activities of β₂‐glycoprotein I using structure‐function analysis and mapped the critical region within the β₂‐glycoprotein I peptide sequence that mediates anticancer effects. We constructed recombinant cDNA and purified different β₂‐glycoprotein I polypeptide domains using a baculovirus expression system. We found that purified β₂‐glycoprotein I, as well as recombinant β₂‐glycoprotein I full‐length (D12345), polypeptide domains I‐IV (D1234), and polypeptide domain I (D1) significantly inhibited melanoma cell migration, proliferation and invasion. Western blot analyses were used to determine the dysregulated expression of proteins essential for intracellular signaling pathways in B16‐F10 treated with β₂‐glycoprotein I and variant recombinant polypeptides. Using a melanoma mouse model, we found that D1 polypeptide showed stronger potency in suppressing tumor growth. Structural analysis showed that fragments A and B within domain I would be the critical regions responsible for antitumor activity. Annexin A2 was identified as the counterpart molecule for β₂‐glycoprotein I by immunofluorescence and coimmunoprecipitation assays. Interaction between specific amino acids of β₂‐glycoprotein I D1 and annexin A2 was later evaluated by the molecular docking approach. Moreover, five amino acid residues were selected from fragments A and B for functional evaluation using site‐directed mutagenesis, and P11A, M42A, and I55P mutations were shown to disrupt the anti‐melanoma cell migration ability of β₂‐glycoprotein I. This is the first study to show the therapeutic potential of β₂‐glycoprotein I D1 in the treatment of melanoma progression.     

Is weight-based IV dosing of trastuzumab preferable to SC fixed-dose in some patients? A systematic scoping review

Trastuzumab, a key treatment for HER2-positive breast cancer, is available in weight-based IV and fixed-dose (600 mg) SC formulations. While the Phase 3 HannaH trial indicated non-inferiority of the SC formulation, there is some concern that the target plasma concentration may not be reached in overweight/obese patients whereas low-body-weight patients may be at risk of toxicity.This scoping review evaluated whether overweight/obese patients are at risk of below-target exposure with fixed-dose SC trastuzumab, whether low-body-weight patients are at risk of increased toxicity, especially cardiotoxicity, and whether IV and SC trastuzumab are equivalent in terms of treatment-emergent adverse events (TEAEs) (e.g. infections). Thirty-seven publications that met the eligibility criteria were included.Body weight is not an important determinant of exposure to trastuzumab at steady state (i.e. pre-dose cycle 8); however, real-world evidence suggests that the target concentration (20 μg/mL) may not be reached with the first SC dose in overweight/obese patients. There is no evidence that low-body-weight patients are at increased risk of cardiotoxicity with SC trastuzumab, although this may be confounded by the higher rate of cardiovascular comorbidities in overweight patients. In Phase 3 trials, SC trastuzumab was associated with higher rates of ISRs, ADAs and SAEs, the latter often requiring hospitalization and occurring during adjuvant treatment when patients are not burdened by chemotherapy.The route of administration of trastuzumab (IV vs SC) in different treatment settings should be discussed with the patient, taking into account the risks and benefits associated with each route.     

恩度联合多西他赛序贯腹腔灌注治疗胃癌伴恶性腹水的临床观察

目的:回顾性分析恩度联合多西他赛序贯腹腔灌注治疗胃癌伴恶性腹水的疗效及不良反应。方法:收集72例胃癌伴腹水患者，曾接受过二线及以上方案化疗，观察组35例，采用恩度45 mg联合多西他赛35 mg/m2 d1，d5序贯腹腔灌注1周期；对照组37例，采用多西他赛35 mg/m2 d1，d5腹腔灌注1周期，统计患者腹水控制有效率、KPS改善率、腹水控制时间及不良反应。结果:治疗组中腹水控制有效率71.43%；对照组中腹水控制有效率48.65%，P=0.049，两组具有统计学差异；治疗组中KPS改善率77.14%，对照组中KPS改善率54.05%，P=0.04，两组具有统计学差异；观察组中腹水控制时间8~90天，中位控制时间44天，对照组中腹水控制时间5~66天，中位控制时间28天，两组控制时间比较，P=0.048，具有统计学差异；III级以上不良反应发生率低，无治疗相关性死亡，两组不良反应比较，P>0.05，无统计学差异。结论:恩度联合多西他赛序贯腹腔灌注治疗体力状况（performance status,PS）评分较差的胃癌伴恶性腹水患者，腹水控制较好，能明显提高患者生活质量，未见明显不良反应。     

Idarucizumab for Intravenous Thrombolysis and Endovascular Thrombectomy in Acute Stroke: A Case Report

BackgroundNon–vitamin K antagonist oral anticoagulants (NOACs), such as dabigatran, are widely used to prevent ischemic stroke in patients with nonvalvular atrial fibrillation. Nonetheless, stroke occurs in 1–2% of patients, and the use of NOACs may increase the bleeding risk for patients who are receiving acute treatment of intravenous thrombolysis (IVT) or endovascular thrombectomy (EVT). Idarucizumab, a monoclonal antibody developed to bind dabigatran, has been proven safe and effective for patients with uncontrolled bleeding or for patients planning to receive emergent procedures. It is now accepted that patients taking dabigatran with recurrent stroke may benefit from IVT after idarucizumab. However, there are limited data regarding idarucizumab use in patients planning to have EVT.Case ReportWe present the case of a male patient taking dabigatran who had a stroke and who was treated with idarucizumab followed by combined IVT and EVT. The patient had immediate recanalization of the occluded vessel and near total recovery of function after 3 months.Why Should an Emergency Physician be Aware of This?Our case report supports the evidence that patients presenting with acute ischemic stroke (AIS) despite being under dabigatran therapy should be evaluated for reversal by idarucizumab which can contribute to the eligibility for IVT as well as EVT. It has also been proved to provide better outcomes for patients with AIS. The availabilities of specific reversal agents for NOACs will probably alter the current management of patients with AIS.     

Molecular approaches to allergen‐specific immunotherapy: Are we so far from clinical implementation?

Conventional allergen‐specific immunotherapy (AIT), based on administrations of allergen extracts, represents up to now the unique protocol for the desensitization of allergic patients. Whereas the effectiveness of AIT was evidenced for the treatment of allergic rhinitis and allergic asthma, such strategy remains experimental for food allergies up to now. However, important issues are commonly associated with AIT as the quality of natural allergen extracts, the long duration and adverse side‐effects which negatively affect successful desensitization together with the patient compliance. The rapid progression of molecular allergology made possible the quest of safer, shorter and more effective immunotherapeutic approaches. The aim of this review was to provide an update on these different innovative recombinant derivatives including their efficacy but also their limitations. Despite promising preclinical and early clinical studies, the absence of convincing data in large phase III trials precludes so far the translation of these immunotherapeutic candidates into the clinic.