造血干/祖细胞的体外扩增培养研究

利用旋转壁式生物反应器(Rotating wall vessel,RWV)体外培养脐带血干细胞,使其大量扩增,以满足临床应用对造血干/祖细胞的数量与质量要求。从脐带血分离得到的单个核细胞(Mononuclear cells,MNC)在T-flask中培养24h,之后接种到RWV反应器中,培养200h。每24h细胞计数,测量培养基的pH和渗透压变化;在144h和197h测CD34 细胞含量并做CFU-GM半固体培养。有核细胞(Nucleated cells,NC)与CD34 细胞在第197h,分别扩增了435.5±87.6倍和32.7±15.6倍,CFU-GM(Colony-forming unit-granulocyte/macrophage)细胞扩增了21.7±4.9倍。整个培养过程中,RWV反应器中的pH和渗透压都保持在造血细胞最佳的扩增条件内,pH基本保持在7.2~7.4之间,渗透压基本保持在290~310mmol/kg之间。由于旋转壁式生物反应器(RWV)结构上的特殊性,可以保证细胞在悬浮流动的状态下生长,很好地模拟了脐带中的造血微环境,使脐带血造血干细胞在该反应器中短期内得到大量扩增。     

国际学员三站式强化内镜黏膜下剥离术动物模拟培训的实践

目的　探索家猪离体胃模型在对国际学员进行内镜黏膜下剥离术（endoscopic submucosal dissection,ESD）培训中的应用价值。方法　对15名国际学员进行为期20 d的ESD培训,第一阶段为理论知识学习,完成消化内镜基础及动物模拟培训调查问卷。第二阶段将其随机分为离体动物模型组（A组,８人）和临床观察学习组（B组,7人）。A组在临床观察学习期间,同时按照规范操作步骤进行离体动物模型培训课程。培训结束时考核两组学员ESD操作情况,并再次完成问卷。应用SPSS 20.0进行t检验和卡方检验。结果　A组与B组ESD总操作时间差异有统计学意义（P＜0.05）,注射时间、标记时间差异没有统计学意义（P＞0.05）,切开时间及黏膜剥离时间差异有统计学意义。A 组与B组的ESD操作速度差异无统计学意义。两组切除标本大小差异无统计学意义。两组均有1例为分块切除病灶,其余为整块切除。在操作过程中各发生1例穿孔。同时,对比离体动物模型组学员培训前后操作情况,发现培训后ESD总操作时间、切开时间明显提高。结论　以理论知识为基础结合家猪离体胃动物模型培训的内镜培训具有较高的教学参考价值,能够使各国学员熟悉并初步掌握ESD的基础理论知识及操作方法,帮助各国学员日后在临床开展ESD操作。     

体外肝切除自体肝移植在复杂肝切除中的应用

目的 评估体外肝切除自体肝移植在巨大肝癌患者复杂肝切除中的临床价值.方法 回顾性分析2008年1月至2010年5月首都医科大学附属北京朝阳医院收治的4例巨大原发性肝癌患者的临床资料.肿瘤最大直径10 ～ 18 cm,病灶不同程度地累及了第一、二、三肝门.患者难以耐受常规肝切除,均行体外肝切除自体肝移植.结果 4例患者顺利完成手术,手术时间690 ～840 min,无肝期250～300 min,术中出血量400～1400 ml,术中无肝期未行门、腔静脉转流术.4例患者在体外肝切除后行下腔静脉或肝静脉及门静脉修复成型,均应用成型异体血管来延长剩余肝脏肝上腔静脉以利于腔静脉吻合及第一肝门的重建.本组患者1例术后肝功能正常,1例出现腹腔出血再次手术止血,1例发生肝功能不全,1例出现肝肾功能不全于术后5d放弃治疗而死亡.3例术后生存的患者术后1～2个月间剩余肝脏均发生不同程度的代偿增生.术后生存的3例患者中2例分别于术后8、9个月发现肺部多发转移瘤,分别于术后13个月及15个月死亡.随访截至2012年4月,1例患者无瘤生存37个月.结论 体外肝切除自体肝移植为复杂肝切除的巨大肝癌患者提供了技术上的可行性,术后肝功能代偿不全及近期肿瘤的复发是限制该手术发展的主要问题.     

Design and validation of a clinical-scale bioreactor for long-term isolated lung culture

The primary treatment for end-stage lung disease is lung transplantation. However, donor organ shortage remains a major barrier for many patients. In recent years, techniques for maintaining lungs ex vivo for evaluation and short-term (<12 h) resuscitation have come into more widespread use in an attempt to expand the donor pool. In parallel, progress in whole organ engineering has provided the potential perspective of patient derived grafts grown on demand. As both of these strategies advance to more complex interventions for lung repair and regeneration, the need for a long-term organ culture system becomes apparent. Herein we describe a novel clinical scale bioreactor capable of maintaining functional porcine and human lungs for at least 72 h in isolated lung culture (ILC). The fully automated, computer controlled, sterile, closed circuit system enables physiologic pulsatile perfusion and negative pressure ventilation, while gas exchange function, and metabolism can be evaluated. Creation of this stable, biomimetic long-term culture environment will enable advanced interventions in both donor lungs and engineered grafts of human scale.     

新型腔内射频消融导管消融离体猪肝

目的 观察新型腔内射频消融导管对离体猪肝(肝实质、栓子模型)的消融效果。方法 应用EMcision Habib腔内射频消融导管及RITA射频发生器对新鲜离体猪肝肝实质及栓子模型进行消融,输出功率分别为5 W、10 W、15 W和20 W,消融时间分别为60 s、90 s和120 s,观察消融灶组织凝固形态及范围。结果 消融肝实质时,输出功率为10 W、延长消融时间(90 s延长至120 s),输出功率为15 W、延长消融时间(60 s延长至90 s)以及消融时间为60 s和90 s、增加输出功率(15 W增加至20 W)获得的消融灶长径增加(P均<0.05),而宽径增加不明显(P均>0.05)。消融栓子模型时,输出功率为10 W、延长消融时间(90 s延长至120 s),以及消融时间为60 s、增加输出功率(15 W增加至20 W)均可增加消融灶长径(P均<0.05),而宽径增加不明显(P均>0.05)。肉眼见所有消融灶附着处血管管壁颜色均与邻近血管管壁无差异。结论 采用EMcision Habib腔内射频导管消融离体猪肝可出现明确的消融范围,且对管道壁无明显损伤。     

Liver transplantation with grafts obtained after cardiac death-current advances in mastering the challenge

The scarcity of donor livers has increased the interest in donation after cardiac death(DCD) as an additional pool to expand the availability of organs. However, the initial results of liver transplantation with DCD grafts have been suboptimal due to an increased rate of complications, as well as decreased graft survival. These challenges have led to many developments in DCD donation outcome, as well as basic and translational research. In this article we review the unique characteristics of DCD donors, nuances of DCD organ procurement, the effect of prolonged warm and cold ischemia times, and discuss major studies that compared DCD to donation after brain death liver transplantation, in terms of outcomes and complications. We also review the different methods of donor treatment that has been applied to ameliorate DCD organ outcome, and we discuss the role of machine perfusion techniques in organ reconditioning. We discuss the two major perfusionmodels, namely, hypothermic machine perfusion and normothermic machine perfusion; we compare both methods, and delineate their major differences.     

Ev vivo organ culture as potential prioritization tool for breast cancer targeted therapy

The growing use of genomic testing presents new treatment options but also new dilemmas. We describe here a heavily-pretreated metastatic triple negative breast cancer patient who failed to respond to conventional treatment. Genomic analyses were performed that discovered several targetable alterations (e.g. FGFR1, CDK6, INSR) and created a clinical challenge – which target to target first? Our solution to this relatively common scenario was using ex-vivo organ culture (EVOC) system to prioritize treatment directed toward the best molecular target. EVOC enabled the trial of several potent targeted agents (Everolimus, Linsitinib, Palbociclib, AZD4547) and allowed semi-quantitative measurement of tumor response. The best response was to FGFR inhibitor, AZD4547. Consequently, the most accessible FGFR inhibiting agents (Pazopanib, then Nintedanib) were administered and some response was achieved. This report provides a potential rationale for utilizing EVOC system to predict tumor response to targeted therapy when multiple targets are proposed.