循环肿瘤细胞在女性实体器官肿瘤中的研究进展

循环肿瘤细胞(circulating tumor cells,CTCs)指的是从实体的肿瘤或转移的病灶进入外周血液循环的恶性肿瘤细胞。自发现以来,随着其检验技术日趋成熟,循环肿瘤细胞(CTCs)日渐成为肿瘤学炙手可热的研究对象。因为它将通过外周血的检验来实现监测肿瘤的发生、发展、转移、复发等情况,相对于肿瘤实体活检,"液体活检"不仅让患者易于接受,更有利于医务工作者监测病情变化。本文综述了循环肿瘤细胞(CTCs)的检测方法并综述了循环肿瘤细胞在女性实体肿瘤--乳腺癌、卵巢癌、宫颈癌、子宫内膜癌中的研究进展。其中着重介绍了其在早期乳腺癌及复发转移性乳腺癌中的重大意义以及在评价治疗效果中的分子学特征。实践表明,循环肿瘤细胞(CTCs)与HE-4、CA125的联合应用在评估卵巢癌化疗敏感性中也具有重要的临床意义。     

循环肿瘤细胞在外周血中的存活与转移机制

肿瘤转移是恶性肿瘤发展的一个重要阶段,也是导致患者死亡的主要原因。实体恶性肿瘤病灶(原发或转移灶)的肿瘤细胞因自发性脱落或外部因素释放入血形成循环肿瘤细胞(circulating tumor cells,CTCs)。CTCs是肿瘤患者出现复发转移的最直接原因。外周血中的CTCs会因血流剪切力作用、失巢凋亡以及免疫细胞杀伤作用死亡,但仍有小部分的CTCs可以存活下来并发生转移。因此,研究CTCs在外周血中的存活转移机制对肿瘤患者的诊治具有重要意义,本文对CTCs在外周血中存活转移机制的最新研究进展进行了综述,并对其未来发展进行展望。     

微流控芯片在分选富集循环肿瘤细胞中的应用

循环肿瘤细胞(circulating tumor cells, CTCs)是指从原发肿瘤部位脱落,进入外周血循环的肿瘤细胞,对其准确检测有利于早期癌症转移的诊断与治疗。然而,血液中CTCs的量极少,检测前要先对其进行分选富集。在众多的分选富集方法中,微流控芯片技术成本低、通量高、样品需求量小,可达到特异、灵敏、高捕获率的效果。该文综述了近年来使用微流控芯片分选富集CTCs的方法,同时,分析了各种方法的优缺点,并对未来的发展趋势进行了探讨。     

微流控芯片技术在循环肿瘤细胞分离中的研究进展

循环肿瘤细胞(circulating tumor cells,CTCs)是指从原发肿瘤或转移灶脱落、发生上皮-间质转化进入患者外周血血液循环的恶性肿瘤细胞.CTCs在肿瘤研究和临床诊断上的作用逐渐得到认可,外周血中CTCs存在与否以及数量多少不但可以用于肿瘤的早期诊断,还可以用于评估肿瘤预后、监测肿瘤的转移和复发.微流控芯片作为一个高通量、小型化的细胞实验平台,已被应用于CTCs的分选当中.本文综述了用于CTCs捕获的微流控芯片系统的最新研究进展,着重介绍各类芯片的捕获原理、芯片结构和捕获效率,最后对微流控芯片技术在CTCs分选中的应用前景进行了展望.     

肺癌循环肿瘤细胞的单细胞EGFR基因突变检测

循环肿瘤细胞（Circulating tumor cells,CTCs）是从肿瘤原发病灶脱落并侵入外周血循环的肿瘤细胞。由于CTCs存在较大的异质性,其与癌症发展转移密切相关,但目前尚缺乏有效的CTCs单细胞异质性检测方法。鉴于此,本文发展了在单细胞层面对CTCs进行基因突变的检测方法并用于单个肺癌CTC的EGFR（Epidermal growth factor receptor）基因突变检测。首先用集成式微流控系统完成血液中稀有CTCs的捕获,接着将CTCs释放入含有多个微孔的微阵列芯片中,得到含有单个CTC的微孔,通过显微操作将单个CTC转入PCR管内完成单细胞基因组的放大,并进行单细胞的EGFR基因突变检测。以非小细胞肺癌细胞系A549、NCI-H1650和NCI-H1975为样本,通过芯片与毛细管修饰、引物扩增条件（复性温度、循环次数）的优化,结果显示在复性温度59℃、30个循环次数的条件下,引物扩增效果最优。利用该方法成功地对非小细胞肺癌（Non-small cell lung cancer, NSCLC）患者的血液样本进行了测试。从患者2 mL血液中获取5个CTCs,分别对其EGFR基因的第18、19、20、21外显子进行测序,发现该患者CTCs均为EGFR野生型。研究结果证明此检测方法可以灵敏地用于单个CTC基因突变的检测,在临床研究上具有重要的指导意义。     

捕获循环肿瘤细胞的纳米系统

循环肿瘤细胞(circulating tumor cells, CTCs)是引起癌症转移的重要因素。检测CTCs对于癌症早期诊断、治疗监控和预后判定具有重要意义。由于癌症患者血液中CTCs数量稀少,CTCs检测困难,研究具有高CTCs捕获效率与捕获纯度的CTCs捕获系统是近年来研究的热点。纳米系统因为具有比表面积大、易于修饰、粒径形态可控、生物相容性良好等优势,在CTCs捕获中展现了良好的应用前景。基于此,本文概述了用于CTCs捕获的代表性纳米系统,并对纳米系统与微流控技术结合捕获CTCs的研究进展进行了介绍。     

循环肿瘤细胞在肿瘤治疗中的研究进展

循环肿瘤细胞(circulating tumor cells,CTCs)由原发肿瘤和转移肿瘤灶中的癌细胞播散而来,对CTCs的定量和定性检测可用于肿瘤的早期诊断、转移复发监测、药效分析和生存期预后等方面,在肿瘤预防与治疗中具有重要应用价值。CTCs在肿瘤临床中的应用是当今非常活跃的研究领域,探索新的特异性CTCs分子标志,并研发更加灵敏、高效和特异的CTCs分析新技术,也成为目前研究的热点。本文简要综述了CTCs用于肿瘤防治的研究进展。     

靶向循环肿瘤细胞治疗策略在抗肿瘤转移中的应用

转移是肿瘤治疗的主要挑战，90%的肿瘤相关死亡病例与肿瘤转移有关。循环肿瘤细胞(circulating tumor cells,CTCs)是肿瘤转移形成的关键，与肿瘤转移的形成过程密切相关。因此，靶向CTCs的治疗策略成为目前抗肿瘤转移研究的热点。基于此，本文从CTCs的产生、CTCs的播散和CTCs的远端定植三个阶段综述了CTCs参与肿瘤转移的机制，并分别从调节肿瘤转移相关基因表达和抑制EMT过程抑制肿瘤细胞脱落产生CTCs、激活自身免疫细胞和仿生细胞膜或特异性分子修饰纳米制剂在血液循环捕获消除CTCs、抑制CTCs黏附并穿过血管内皮细胞和破坏PMN处适宜肿瘤细胞生长的微环境等方面总结了控制肿瘤转移的研究进展，以期为肿瘤转移预防和治疗提供新的思路。     

基于物理过滤与原位杂交技术的骨肉瘤循环肿瘤细胞检测及临床意义

目的:探寻一种有效地从骨肉瘤患者外周血中富集并鉴定循环肿瘤细胞的方法。方法:利用基于物理过滤与原位杂交结合的技术对骨肉瘤患者外周血循环肿瘤细胞分离并鉴定。采用直径8μm纳米滤膜截留外周血中体积较大的白细胞及肿瘤细胞,利用多重RNA原位杂交技术检测CD45、EpCAM、CK8、CK18、CK19、vimentin及twist基因表达,并根据结果对滤膜截留下的细胞进行鉴定并分型。结果:本研究所使用的基于物理过滤与原位杂交技术的循环肿瘤细胞检测方法可以高效地从骨肉瘤患者外周血中富集骨肉瘤循环肿瘤细胞,该方法富集细胞的效率超过90%。15例健康志愿者中1例志愿者检测结果阳性。20例纳入研究的骨肉瘤患者中19例患者外周血中检测出CTC,CTC计数范围为0-20。肿瘤转移患者外周血CTC计数为11.33±5.88,肿瘤未转移患者外周血CTC计数为4.36±2.98,差异具有统计学意义(P=0.0022)。肿瘤转移患者外周血间质型CTC比例高于肿瘤未转移患者(P=0.0031)。结论:利用基于物理过滤与原位杂交结合的技术可以有效地检测骨肉瘤患者外周血循环肿瘤细胞。CTC检测结果可以作为辅助判断肿瘤转移情况的辅助指标。     

循环肿瘤细胞在乳腺癌预后评价中的应用和意义

循环肿瘤细胞(CTCs)是导致肿瘤转移的主要原因,它在外周血中的含量与乳腺癌的转移、治疗和预后有密切的关系.本文对于CTCs的概念、检测方法、以及作为肿瘤转移预测指标的优势进行了综述.并对其在临床上的运用前景进行了展望.     

基于转录组测序分析甜菜夜蛾卵巢细胞离体培养成系进程

【目的】本研究旨在分析甜菜夜蛾Spodoptera exigua蛹卵巢细胞建立细胞系的整个过程,探究细胞由体内到体外培养过程中其基因表达在转录水平的变化,为昆虫体外培养模型的建立提供理论基础。【方法】利用Illumina Hiseq测序平台对甜菜夜蛾蛹卵巢细胞离体培养过程中各阶段的细胞分别进行转录组测序,对获得注释的差异表达基因及其相关信号通路进行分析;通过荧光定量PCR对部分细胞周期相关基因(cycd和cdk4)、调控基因(cdc20,apc1,skp2和mad1)、增殖相关分子标志物(mcm4和pcna)在甜菜夜蛾卵巢细胞离体培养过程中的转录进行验证。【结果】甜菜夜蛾蛹卵巢细胞离体培养过程包括5个阶段:解剖获得离体的卵巢组织,卵巢组织贴壁培养后游离出原代细胞,细胞转化重新具备增殖能力,成功首次传代,以及能够连续传代15代以上建立细胞系。上述5个阶段的细胞经转录组测序、数据组装后共获得46 796条unigenes序列,组装得到序列长度完整性好;转录本unigenes序列拼接长度分布合理,样本碱基Q30均在94%以上。通过KEGG数据库获得注释的unigenes有1 473条,参与细胞过程紧密相关的20条信号通路,其中有92条unigenes在细胞周期信号通路中获得注释。聚类分析表明,在体内处于快速发育状态的卵巢细胞与同样处于增殖状态的细胞系基因表达模式非常接近。原代细胞由短暂停滞生长至成簇细胞的转化关键期,筛选到差异表达基因619个,cdk4在离体培养期表达量显著降低,cycd在细胞转化关键期之后表达量显著升高,cdc20,apc1,skp2和mad1在卵巢组织和细胞系的表达量显著高于原代细胞、转化关键期细胞和首次传代细胞的。从原代细胞至传代后,cycd的表达显著升高8.7倍,显著高于mcm4和pcna的变化水平。【结论】甜菜夜蛾卵巢细胞离体培养过程中5个阶段的细胞转录组测序获得的序列质量符合数据分析的基本要求。筛选获得了甜菜夜蛾蛹卵巢细胞经离体培养过程中的差异表达基因。原代细胞逆转增殖可能与cdk4,cycd,skp2和mad1等细胞周期调控基因表达有关。另外,cycd可作为原代细胞具备传代能力的标志物。     

Induction of differentiation of human stem cells ex vivo: Toward large-scale platelet production

Platelet transfusion is one of the most reliable strategies to cure patients suffering from thrombocytopenia or platelet dysfunction. With the increasing demand for transfusion, however, there is an undersupply of donors to provide the platelet source. Thus, scientists have sought to design methods for deriving clinical-scale platelets ex vivo. Although there has been considerable success ex vivo in the generation of transformative platelets produced by human stem cells (SCs), the platelet yields achieved using these strategies have not been adequate for clinical application. In this review, we provide an overview of the developmental process of megakaryocytes and the production of platelets in vivo and ex vivo, recapitulate the key advances in the production of SC-derived platelets using several SC sources, and discuss some strategies that apply three-dimensional bioreactor devices and biochemical factors synergistically to improve the generation of large-scale platelets for use in future biomedical and clinical settings.     

TGF-β signalling-related markers in cancer patients with bone metastasis

We measured transforming growth factor (TGF)-β-dependent biomarkers in plasma and in peripheral blood mononuclear cells (PBMCs) to identify suitable pharmacodynamic markers for future clinical trials with TGF-β inhibitors. Forty-nine patients with bone metastasis were enrolled in the study, including patients with breast (n=23) and prostate cancer (n=15). Plasma TGF-β1 levels were elevated in more than half of the cancer patients (geometric mean 2.63 ng ml^-1) and positively correlated with increased platelet factor 4 (PF4) levels, parathyroid-related protein (PTHrP), von Willebrand Factor (vWF) and interleukin (IL)-10. PBMC were stimulated ex vivo to determine the individual biological variability of an ex vivo assay measuring pSMAD expression. This assay performed sufficiently well to allow its future use in a clinical trial of a TGF-β inhibitor.     

Prospects for the therapeutic development of umbilical cord blood-derived mesenchymal stem cells

Umbilical cord blood (UCB) is a primitive and abundant source of mesenchymal stem cells (MSCs). UCB-derived MSCs have a broad and efficient therapeutic capacity to treat various diseases and disorders. Despite the high latent self-renewal and differentiation capacity of these cells, the safety, efficacy, and yield of MSCs expanded for ex vivo clinical applications remains a concern. However, immunomodulatory effects have emerged in various disease models, exhibiting specific mechanisms of action, such as cell migration and homing, angiogenesis, anti-apoptosis, proliferation, anti-cancer, anti-fibrosis, anti-inflammation and tissue regeneration. Herein, we review the current literature pertaining to the UCB-derived MSC application as potential treatment strategies, and discuss the concerns regarding the safety and mass production issues in future applications.     

PI3Kgamma is the dominant isoform involved in migratory responses of human T lymphocytes: effects of ex vivo maintenance and limitations of non-viral delivery of siRNA

Use of mice in which individual PI3K isoforms have been deleted or mutated by gene targeting, has determined that PI3Kγ provides a key migratory signal for T lymphocyte migration. Since PI3Kγ can be a dispensable signal for directional migration of human T cells, we have adopted a pharmacological and siRNA strategy to assess the contribution of individual PI3K isoforms to chemokine-stimulated migration of human T cells. The broad spectrum PI3K isoform inhibitor Ly294002 inhibits CXCL12-stimulated migration of freshly isolated T lymphocytes. Use of second generation inhibitors that can discriminate between individual PI3K isoforms, revealed that PI3Kγ was the major contributor to CXCL12-induced migration and PI3K/Akt signaling (as assessed by S6 phosphorylation). Non-viral delivery of siRNA targeting class I (PI3Kγ), class II (PI3KC2 and PI3KC2β) and class III PI3Ks, followed by 3 days ex vivo culture, reduces the levels of isoform mRNA, but is insufficient to impact on cell migration responses. However, ex vivo maintenance of T cells alone, independently of siRNA treatment, resulted in the migratory response of T cells toward CXCL12 becoming insensitive to Ly294002. Remarkably, random migration remains sensitive to Ly294002. This study therefore, highlights that the migratory response of freshly isolated human T cells is dependent on PI3K signals that are provided predominantly by PI3Kγ. However, the role of PI3K in cell migration is context-dependent and diminishes during ex vivo maintenance.     

Effects of pretreatment with a xanthine oxidase inhibitor on free radical levels during carotid endarterectomy

Objective: Free radicals contribute to the tissue damage caused by ischaemia-reperfusion. The aim of the present study was to investigate whether preoperative antioxidant therapy (allopurinol) affects free radical levels in cerebral venous blood in connection with surgery for carotid artery stenosis.

Materials and methods: Twenty-five patients were randomised into the study. Thirteen were controls and 12 were pretreated with allopurinol the day before surgery. Before, during and after surgery, blood samples were drawn from the ipsilateral jugular vein. Radical levels were measured using the spin trap technique ex vivo using OXANOH as the spin trap. Multivariate statistics were used with Principal Component Analysis and Partial Least Square regression analysis.

Results: Radical levels increased with diabetes, high leukocyte count, high creatinine and a high degree of contralateral stenosis. Radical levels decreased with high age, blood pressure, collateral circulation as well as operation for left-side carotid artery stenosis. After pretreatment with allopurinol, several of the relationships noted in the control group were eliminated, i.e. leukocyte count, side of operation, Betapred pretreatment and collateral circulation.

Conclusions: Radical levels can be determined in connection with surgery for carotid artery stenosis using an ex vivo spin trap method. With preoperative antioxidant therapy the relationships between enhanced radical levels and clinical data, as seen in control subjects, disappeared. This might indicate a beneficial effect of preoperative pretreatment with antioxidants.     

Estrogen-like effect of a Cimicifuga racemosa extract sub-fraction as assessed by in vivo, ex vivo and in vitro assays

Black cohosh (Cimicifuga racemosa) is used in the treatment of painful menstruation and menopausal symptoms. Data about the nature of the active compounds and mechanism(s) of action are still controversial, chiefly with respect to its estrogenic activity.

This work aimed to assess the possible estrogenic activity of a commercial dry hydro-alcoholic extract of C. racemosa and its hydrophilic and lipophilic sub-fractions on in vivo, ex vivo, and in vitro assays.

In a yeast estrogen screen, only the lipophilic sub-fraction was able to activate the human estrogen receptor , with a lower potency but comparable efficacy to that of 17 β-estradiol.

Neither the total extract nor the lipophilic sub-fraction showed an in vivo uterotrophic effect in 21-day-old rats. Uterine tissues obtained ex vivo from C. racemosa treated animals were generally much less sensitive to oxytocin, prostaglandin F_2, and bradykinin than tissues obtained from estradiol valerate treated rats.

The lipophilic sub-fraction, instead, induced a dose-dependent inhibitory activity on the in vitro response to oxytocin, prostaglandin F_2, and bradykinin of uterine horns from naïve 28-day-old rats, with a potency rate close to 1:30 of that of 17 β-estradiol.

Reported results confirm the effectiveness of C. racemosa in menstrual distress and further emphasize the possibility that lipophilic constituents bind to an as yet not identified estrogen receptor, likely inversely involved in inflammation.     

Flavonoids protect against oxidative damage to LDL in vitro: use in selection of a flavonoid rich diet and relevance to LDL oxidation resistance ex vivo?

The ability of a range of dietary flavonoids to inhibit low-density lipoprotein (LDL) oxidation in vitro was tested using a number of different methods to assess oxidative damage to LDL. Overall quercetin was the most effective inhibitor of oxidative damage to LDL in vitro. On this basis, a diet enriched with onions and black tea was selected for a dietary intervention study that compared the effect on the Cu²⁺ ion-stimulated lag-time of LDL oxidation ex vivo in healthy human subjects of a high flavonoid diet compared with a low flavonoid diet. No significant difference was found in the Cu²⁺ ion-stimulated lag-time of LDL oxidation ex vivo between the high flavonoid and low flavonoid dietary treatments (48 ± 1.6 min compared to 49 ± 2.1 min).