Methodical and pre-analytical characteristics of a multiplex cancer biomarker immunoassay

AIM: To test the methodical and pre-analytical performance of a new multiplex cancer biomarker panel using magnetic beads. METHODS: The MILLIPLEX? MAP Human Circulating Cancer Biomarker Magnetic Bead Panel 1 comprises the tumor markers carcinoembryonic antigen, alpha-fetoprotein, total prostate-specific antigen, cancer antigen 15-3, cancer antigen 19-9, cancer antigen 125, cytokeratine 19-fragment, β-human chorionic gonadotropin, human epididymis protein 4, osteopontin, prolactin, the cell death and angiogenesis markers soluble Fas, soluble Fas-ligand, tumor necrosis factor related apoptosisinducing ligand, vascular endothelial growth factor andthe immunological markers interleukin-6(IL-6), IL-8, tumor necrosis factor-α, transforming growth factor α, fibroblast growth factor-2, macrophage migration inhibitory factor, leptin, hepatocyte growth factor, and stem cell factor. We determined intra- and inter-assay imprecision as well as dilution linearity using quality controls and serum pools. Furthermore, the stability of the 24 biomarkers examined in this panel was ascertained by testing the influence of different storage temperatures and time span before centrifugation.RESULTS: For all markers measured in the synthetic internal quality controls, the intra-assay imprecision ranged between 2.26% and 9.41%, while for 20 of 24 measured markers in the physiological serum pools, it ranged between 1.68% and 12.87%. The inter-assay imprecision ranged between 1.48%-17.12% for 23 biomarkers in synthetic, and between 4.59%-23.88% for 18 biomarkers in physiological quality controls. Here, single markers with very low concentration levels had increased imprecision rates. Dilution linearity was acceptable(70%-130% recovery) for 20 biomarkers. Regarding pre-analytical influencing factors, most markers were stable if blood centrifugation was delayed or if serum was stored for up to 24 h at 4 ℃ and 25 ℃ after centrifugation. Comparable results were obtained in serum and plasma for most markers. However, great changes were observed for single markers.CONCLUSION: MILLIPLEX? MAP Human Circulating Cancer Biomarker Magnetic Bead Panel 1 assay is a stable and precise method for detection of most biomarkers included in the kit. However, single markers have to be interpreted with care.

Methods to produce induced pluripotent stem cell-derived mesenchymal stem cells: Mesenchymal stem cells from induced pluripotent stem cells

Mesenchymal stem cells(MSCs)have received significant attention in recent years due to their large potential for cell therapy.Indeed,they secrete a wide variety of immunomodulatory factors of interest for the treatment of immune-related disorders and inflammatory diseases.MSCs can be extracted from multiple tissues of the human body.However,several factors may restrict their use for clinical applications:the requirement of invasive procedures for their isolation,their limited numbers,and their heterogeneity according to the tissue of origin or donor.In addition,MSCs often present early signs of replicative senescence limiting their expansion in vitro,and their therapeutic capacity in vivo.Due to the clinical potential of MSCs,a considerable number of methods to differentiate induced pluripotent stem cells(iPSCs)into MSCs have emerged.iPSCs represent a new reliable,unlimited source to generate MSCs(MSCs derived from iPSC,iMSCs)from homogeneous and well-characterized cell lines,which would relieve many of the above mentioned technical and biological limitations.Additionally,the use of iPSCs prevents some of the ethical concerns surrounding the use of human embryonic stem cells.In this review,we analyze the main current protocols used to differentiate human iPSCs into MSCs,which we classify into five different categories:MSC Switch,Embryoid Body Formation,Specific Differentiation,Pathway Inhibitor,and Platelet Lysate.We also evaluate common and method-specific culture components and provide a list of positive and negative markers for MSC characterization.Further guidance on material requirements to produce iMSCs with these methods and on the phenotypic features of the iMSCs obtained is added.The information may help researchers identify protocol options to design and/or refine standardized procedures for large-scale production of iMSCs fitting clinical demands.

用Midi MACS方法从白血病患者分选CD34^+/CD123^+细胞

本研究的目的是应用MidiMACS方法从急性髓系白血病(AML)患者骨髓细胞分选CD34+/CD123+细胞。首先,用淋巴细胞分离液从AML患者骨髓细胞分离单个核细胞(BMMNC),然后再通过MidiMACS方法相继分选出CD34+细胞,以及从CD34+细胞中分选出CD123+细胞并通过流式细胞术分析分选后细胞的富集度和回收率。结果显示:经过第一次分选后,CD34+细胞的富集度高达98.73%,平均为95.6%,其回收率最高可达到84.6%,平均为51%;第二次分选后,CD34+/CD123+细胞的富集度高达99.23%,平均约为83%;相对于分选前BMMNC而言,CD34+/CD123+细胞的回收率为34%,但相对于第一次分选后的CD34+细胞而言,其回收率为56%。结论:MidiMACS方法可用于AML患者骨髓CD34+/CD123+细胞的分选,分选后细胞的富集度和回收率与文献报道的通过FACS方法所获得的结果相类似。

疏肝健脾法对非小细胞肺癌患者化疗期间肿瘤标志物的影响

目的:探讨疏肝健脾法对非小细胞肺癌(NSCLC)患者化疗期间肿瘤标志物水平的影响。方法:选取NSCLC患者100例,采用随机数字表法分为观察组、对照组,各50例。对照组患者给予常规化疗方案,观察组患者在对照组基础上联合疏肝健脾法。比较两组患者临床疗效,同时评估中医证候积分、血清肿瘤标志物[癌胚抗原(CEA)、癌抗原125(CA125)]、免疫功能指标[抗原分化簇4受体(CD4^(+))、抗原分化簇8受体(CD8^(+))、CD4^(+)/CD8^(+)]。最后比较两组患者不良反应发生情况。结果:观察组患者疾病控制率96.00%高于对照组84.00%,差异有统计学意义(P<0.05)。观察组患者治疗后食少纳呆、体倦乏力、神疲懒言、腹痛绵绵、舌质淡、脉细弱积分低于对照组,差异有统计学意义(均P<0.05)。观察组患者治疗后血清CEA、CA125指标低于对照组,差异有统计学意义(P<0.05)。观察组患者治疗后CD4^(+)、CD4^(+)/CD8^(+)高于对照组,CD8^(+)指标低于对照组,差异有统计学意义(均P<0.05)。观察组、对照组不良反应总发生率分别为16.00%、28.00%,组间比较差异无统计学意义(P>0.05)。结论:疏肝健脾法用于NSCLC患者化疗期间不仅能有效改善患者临床症状、体征,改善患者血清肿瘤标志物、免疫功能,安全性较理想。

求解非定常不可压湍流的SMAC隐式方法

基于简化标记和单元(Simplified marker and cell,SMAC)方法,发展一种在任意曲线坐标系中求解三维非定常不可压湍流Reynolds时均方程的隐式数值方法。控制方程包括以逆变速度为变量的动量方程、压力Poisson方程和k-ε湍流模型方程,控制方程的离散在三维标记和单元(Marker and cell,MAC)交错网格系统中进行。为提高方程数值计算的稳定性,动量方程、k方程和ε方程对流项离散均采用Chakravarthy-Osher总变差衰减(Total variation diminishing,TVD)格式。动量方程、k方程和ε方程离散后的代数方程组采用循环三对角阵算法(Cyclic tridiagonal matrix algorithm,CTDMA)方法进行求解,Poisson方程离散后的代数方程组采用Tschebyscheff超线性松弛(successive linear over relaxation,SLOR)方法交替方向迭代求解。用该方法自编程序对简化后的射流放水阀内非定常流场进行数值模拟,计算结果和试验结果吻合。

Large Eddy Simulation for Plunge Breaker and Sediment Suspension

Breaking waves are a powerful agent for generating turbulence that plays an important role in many fluid dynamical processes, particularly in the mixing of materials. Breaking waves can dislodge sediment and throw it into suspension, which will then be carried by wave-induced steady current and tidal flow. In order to investigate sediment suspension by breaking waves, a numerical model based on large-eddy-simulation (LES) is developed. This numerical model can be used to simulate wave breaking and sediment suspension. The model consists of a free-surface model using the surface marker method combined with a two-dimensional model that solves the flow equations. The turbulence and the turbulent diffusion are described by a large-eddy-simulation (LES) method where the large turbulence features are simulated by solving the flow equations, and a subgrid model represents the small-scale turbulence that is not resolved by the flow model , A dynamic eddy viscosity subgrid scale stress model has been used for the present simulation. By applying this model to Stokes' wave breaking problem in the surf zone, we find that the model results agree very well with experimental data. By use of this model to simulation of the breaking process of a periodic wave, it can be found that the model can reproduce the complicated flow phenomena, especially the plunging breaker. It reflects the dynamic structures of roller or vortex in the plunging breaker, and when the wave breaks, many strong vortex structures will be produced in the inner surf zone where the concentration of suspended sediment can thereby become relatively high.

MAC方法及其在铸造充型模拟中的应用

介绍了一种基于MAC的半拉格朗日方法,用于模拟铸造充型过程中的金属流动。结合拉格朗日和欧拉两种方法的优点,将流体视为具有某些物理性质的大量粒子的集合,绕过了纯欧拉角度下的复杂自由界面跟踪问题。对于流体内部,采用投影法求解不可压缩N-S方程。通过将粒子携带的信息映射到网格上,在背景网格上求解扩散方程,避免了拉格朗日方法中场描述的不便。引入水平集方法描述边界。对比计算分析了该方法和广泛应用的VOF方法的两个实例。结果表明,该方法能产生尖锐、不连续的流体界面,更接近铸造过程,尤其适合于充型速度快的铸造过程。

固本解毒祛瘀法联合化疗治疗晚期非小细胞肺癌临床研究

目的:观察固本解毒祛瘀法联合化疗治疗晚期非小细胞肺癌(NSCLC)的临床疗效。方法:将78例晚期NSCLC患者按照随机奇偶法分为对照组与研究组各39例。对照组接受化疗治疗,研究组在对照组基础上联合固本解毒祛瘀法治疗。比较2组临床疗效、不良反应发生率,并比较2组治疗前后血清肿瘤标志物、免疫因子水平。结果:治疗后,研究组治疗总有效率69.23%,高于对照组46.15%(P<0.05)。治疗前,2组血清神经元特异性烯醇化酶(NSE)、癌胚抗原(CEA)以及癌抗原(CA125)水平比较,差异无统计学意义(P>0.05);治疗后,2组NSE、CEA、CA125水平均下降(P<0.05),且研究组低于对照组(P<0.05)。治疗前,2组CD3^(+)、CD4^(+)、CD8^(+)、CD4^(+)/CD8^(+)比较,差异无统计学意义(P>0.05);治疗后,2组CD3^(+)、CD4^(+)、CD4^(+)/CD8^(+)均升高(P<0.05),CD8^(+)均下降(P<0.05),且研究组CD3^(+)、CD4^(+)、CD4^(+)/CD8^(+)高于对照组(P<0.05),CD8^(+)低于对照组(P<0.05)。治疗期间,研究组恶心呕吐、白细胞降低、血小板减少、肝功能损害等不良反应发生率均明显低于对照组(P<0.05)。结论:固本解毒祛瘀法联合化疗治疗晚期NSCLC可有效降低血清肿瘤标志物水平,提高机体免疫机能,安全性高,疗效显著。

两串列方柱绕流的干扰数值研究

用数值方法研究了绕流两串列方柱的气动力及其相互干扰 ,使用了经改进的标志网格 (MAC)方法对相同尺寸的方柱在不同间距比下的流场进行了数值模拟 .结果表明 :实验中观测到的两串列方柱的阻力不随两方柱的间距比连续变化 ,而是在临界间距比时出现不连续跳跃的现象得到了成功的再现 ,数值计算所得的阻力值与风洞实验值吻合得比较好 .

免疫磁性活化细胞分选方法优化及纯化后细胞的生物学特性检测

目的:改良常规免疫磁性活化细胞分选(MACS)的分离纯化方法,提高分选后细胞的纯度并确保细胞仍具有良好活性及功能。方法:以干细胞抗原-1(Sca-1)标记的Sca-1+造血干/祖细胞(Sca-1+HSC/HPC)为例,分别用改良后和常规MACS法分选出小鼠骨髓细胞Sca-1+细胞,流式细胞术检测两种分选方法获得Sca-1+细胞纯度;计算阳性细胞回收率;台酚蓝染色检测分选细胞存活百分率;CCK-8检测细胞增殖,混合造血祖细胞(CFU-Mix)体外培养评价分选Sca-1+细胞分化能力。结果:改良MACS法分选获得的Sca-1+细胞纯度达93%以上(常规MACS法仅为87%),阳性细胞的回收率可达73%(常规法仅为62.3%);细胞存活率、细胞增殖和CFU-Mix集落形成能力两种分选方法无明显差别。结论:改良法能明显提高细胞分选纯度及回收率,细胞活性和功能保持良好,值得各种细胞免疫磁性分选参考采用。

非均匀介质热蠕变流动的数值求解

针对非均匀介质中热蠕变流动问题,给出了有限单元方法与网格-粒子方法联合求解新技术,即有限单元方法求解欧拉网格节点上的未知量,分布于单元内部作为物质成分标记的粒子反映变形过程.有限元法求解动量方程和连续性方程时引入了速度场和压力场等阶插值的压力场稳定的Petrov-Galerkin方法,求解能量方程时采用了流线迎风Petrov-Galerkin方法,网格-粒子算法中采用双线性插值与有限单元插值函数对应.有限单元计算与网格-粒子计算相对独立,两种方法计算的数据通过有限单元节点传递.同时,实现了三角形单元的算法和程序,解决了复杂结构条件下不规则网格计算的问题.通过经典方腔热对流问题验证了程序,给出了不规则形态块体沉降算例,并分析了数值解的稳定性.

振荡流底部边界层运动的数值研究

将大涡模拟法简化为二维形式 ,利用 SGS格子涡模型封闭二维 Navier- Storkes方程水流运动方程组 ,得到平底振荡流边界层立面二维水流数值模型 ,控制方程采用 SMAC法求解 .计算结果与水槽实验的实测资料比较表明 ,该模型能够模拟振荡流边界层流动特性 .并进一步讨论了振荡流边界的紊动特性沿垂线分布和随相位变化的情况 .

多肿瘤标志物诊断模型对老年非小细胞肺癌胸膜转移的临床诊断研究

目的比较不同肿瘤标志物对老年非小细胞肺癌(NSCLC)胸膜转移的诊断价值,并筛选最佳诊断效能标志物及标志物组合。方法选取我院内科胸腔镜诊断为NSCLC胸膜转移的老年病人60例为肺癌组,另选取同期各类良性胸腔积液病人49例为对照组。检测2组病人胸腔积液中的癌胚抗原(CEA)、细胞角质蛋白19片段(CYFRA21-1)、神经烯醇化酶(NSE)、糖类抗原(CA)125、CA199、CA153的表达水平。采用Logistic逐步回归法纳入2组间差异有统计学意义的肿瘤标志物并建立函数诊断模型,进行预测分类、串联诊断试验及绘制受试者工作特征曲线等方法筛选出诊断效能最佳的标志物及标志物组合。结果2组间CEA、CYFRA21-1、NSE、CA125、CA199、CA153的表达水平差异均有统计学意义(P<0.05或P<0.01)。经Logistic多元回归分析筛选出评分较高的5种标志物串联组成的诊断模型为:CEA+NSE+CYFRA21.1、CYFRA21.1+NSE+CA125、CEA+CYFRA21.1+CA199、CEA+NSE+CA125+CA199及NSE+CYFRA21.1+CA125+CA199,其中CYFRA21.1+NSE+CA125具有更高的诊断效能。结论胸腔积液肿瘤标志物检测对NSCLC的胸膜转移有定性诊断价值,由标志物串联组成的诊断模型对胸膜转移恶性胸腔积液有临床辅助诊断价值,可为老年晚期肺癌病人提供无创、特异的诊断方法。

自体骨髓间充质干细胞移植对兔角膜碱烧伤炎症反应的抑制作用

背景 眼表碱烧伤可引起角膜溃疡及角膜新生血管形成,导致角膜混浊,目前尚无有效的疗法.已有研究认为间充质干细胞（MSCs）移植具有修复角膜损伤的作用,但其在体内对角膜碱烧伤治疗的作用机制尚不清楚. 目的 研究兔自体骨髓间充质干细胞（BM SCs）对碱烧伤角膜的抗炎机制.方法 抽取2～3月龄日本大白兔骨髓,经体外分离培养得到BMSCs并进行传代,取第3代细胞用于实验,倒置相差显微镜下观察培养细胞的形态,采用流式细胞仪检测细胞表面标志物的表达,对细胞进行生物学鉴定.采用角膜贴敷NaOH滤纸法在24只日本大白兔的右眼建立中度角膜碱烧伤模型,然后将造模成功的实验兔应用随机数字表法随机分为2个组,于碱烧伤后1h分别在模型眼结膜下注射同种自体BMSCs悬液300μl（细胞密度5×106/μ1）（BMSCs组）或等容积PBS（PBS组）,每组各12只.分别于移植后3、14和28 d于裂隙灯显微镜下观察角膜的混浊度变化并进行评分,采用组织病理学方法检查角膜组织中炎症反应程度并进行多形核中性粒细胞（PMNs）计数;采用免疫组织化学染色法定位并检测角膜组织中基质金属蛋白酶-2（MMP-2）的表达.结果 体外分离培养的兔BMSCs贴壁生长,呈长梭形,细胞形态均一,传代后其形态无明显改变.细胞表面黏附分子标志物CD29和CD90阳性表达的细胞比例分别为99.18％和97.94％,而造血细胞标志物CD34和CD31的阳性表达分别为0.74％和0.15％.造模后兔右眼出现角膜混浊、上皮脱落、角膜基质水肿和新生血管形成,移植后14d和28 d BMSCs组混浊度评分分别为2.37±0.52和2.25±0.50,均明显低于PBS组的3.00±0.53和3.25±0.50,差异均有统计学意义（t=2.376、2.828,均P＜0.05）;移植后14 d和28 d BMSCs组角膜中PMNs数目分别为（34.17±1.85）/12个视野和（25.64±3.86）/12个视野,明显少于PBS组的（42.70±1.54）/12个视野和（32.67±1.42）/12个视野,差异均有统计学意义（t=10.021、4.832,均P=0.000）;移植后14 d和28 d BMSCs组角膜组织中MMP-2蛋白的阳性表达量（A值）分别为0.388±0.016和0.384±0.006,明显低于PBS组的0.438±0.006和0.412±0.005,差异均有统计学意义（t=10.205、13.514,均P=0.000）.结论 角膜碱烧伤后早期行自体BMSCs移植可减少PMNs浸润,减轻角膜的炎症反应,下调或抑制MMP-2在受损角膜处的表达,抑制基质胶原纤维的降解,从而减少碱烧伤后角膜溃疡的发生。

用积分法评价7项血清肿瘤标志物对非小细胞肺癌的诊断价值

目的探讨用积分法评价血清细胞角蛋白片段21-1(CYFRA21-1)、癌胚抗原(CEA)、组织多肽特异性抗原(TPS)、糖类抗原CA125、CA153、CA199、和铁蛋白(FER)等7项肿瘤标志物对非小细胞肺癌(NSCLC)的诊断价值。方法采用免疫化学发光法、增强化学发光酶联免疫分析法和免疫放射分析法分别检测70例NSCLC患者、53例肺部良性疾病和72名正常对照者血清中的CYFRA21-1、CEA、TPS、CA125、CA153、CA199、FER,根据这些肿瘤标志物单项诊断NSCLC的有效性分别积分为4、3、32、、21、、1分,计算总积分。用总积分评价7项肿瘤标志物对NSCLC诊断的临床应用价值,并与NSCLC的TNM分期进行相关性分析和组织学分型比较。结果单项指标诊断NSCLC的灵敏度在32.9%-65.7%间,特异性在64.0%-96.8%,有效性在62.1%-85.1%间,积分法诊断NSCLC的灵敏度为82.8%,特异性为96.8%,诊断有效性91.8%。总积分水平与NSCLC患者的TNM分期呈显著正相关(r=0.68,P<0.001)。结论在对NSCLC患者用CYFRA21-1、CEA、TPS、CA125、CA153、CA153、和FER进行组合检查时,用积分法评价可显著提高对NSCLC的诊断价值。

脂肪肉瘤干细胞表面标志物研究

脂肪肉瘤中存在一种具有自我更新、扩增能力,并具有癌变倾向的异质性细胞,这种细胞被称为脂肪肉瘤干细胞。脂肪肉瘤干细胞对脂肪肉瘤的发生、发展有着十分重要的作用。明确脂肪肉瘤干细胞的特异标志物可使其准确分离,并对其特性及通路的研究有一定作用。本文对脂肪肉瘤干细胞标志物的研究进展进行概述,旨在为脂肪肉瘤的早期诊断、改善治疗方法、提高患者的生存及预后提供可借鉴的理论依据。

头颈部肿瘤干细胞的表面标志物及其分选

越来越多的研究显示,头颈部肿瘤干细胞很可能是头颈部肿瘤形成、复发、转移和耐药的根源。本文就头颈部肿瘤干细胞表面特异分子标志物,头颈部肿瘤干细胞的分选等研究进展作一综述。

Large Eddy Simulation for Wave Breaking in the Surf Zone

In this paper, the large eddy simulation method is used combined with the marker and cell method to study the wave propagation or shoaling and breaking process. As wave propagates into shallow water, the shoaling leads to the increase of wave height, and then at a certain position, the wave will be breaking. The breaking wave is a powerful agent for generating turbulence, which plays an important role in most of the fluid dynamic processes throughout the surf zone, Such as transformation of wave energy, generation of near-shore current and diffusion of materials. So a proper numerical model for describing the turbulence effect is needed. In this paper, a revised Smagorinsky subgrid-scale model is used to describe the turbulence effect. The present study reveals that the coefficient of the Smagorinsky model for wave propagation or breaking simulation may be taken as a varying function of the water depth and distance away from the wave breaking point. The large eddy simulation model presented in this paper has been used to study the propagation of the solitary wave in constant water depth and the shoaling of the non-breaking solitary wave on a beach. The model is based on large eddy simulation, and to track free-surface movements, the Tokyo University Modified Marker and Cell (TUMMAC) method is employed. In order to ensure the accuracy of each component of this wave mathematical model, several steps have been taken to verify calculated solutions; with either analytical solutions or experimental data. For non-breaking waves, very accurate results are obtained for a solitary wave propagating over a constant depth and on a beach. Application of the model to cnoidal wave breaking in the surf zone shows that the model results are in good agreement with analytical solution and experimental data. From the present model results, it can be seen that the turbulent eddy viscosity increases from the bottom to the water surface in surf zone. In the eddy viscosity curve, there is a turn-point obviously, dividing water depth into two parts, in the upper part, the eddy viscosity becomes very large near the wave breaking position.

免疫标志物及AB/PAS黏液染色在子宫颈癌分型中的应用研究

目的探讨免疫标志物细胞角蛋白7(cytokeratin 7,CK7)、低分子质量细胞角蛋白CAM5.2、癌胚抗原(carcino-embryonic antigen,CEA)、癌基因p63、高分子质量细胞角蛋白34βE12及阿利新蓝-过碘酸雪夫氏(alcian blue-periodic acid schiff,AB/PAS)黏液染色在子宫颈癌分型中的意义。方法采用免疫组织化学En Vision法检测CK7、CAM5.2、CEA、p63、34βE12在59例子宫颈癌中的表达,结合AB/PAS黏液染色,分析上述免疫标志物及特殊染色与子宫颈癌分型的关系。结果 CK7、CAM5.2、CEA在子宫颈腺癌(6例)、腺鳞癌(4例)的腺癌细胞质中均有100.0%的表达,低分化鳞癌(34例)中CK7、CAM5.2阳性率分别为52.9%、32.4%,而中及高分化鳞癌(15例)中不表达。CEA在鳞癌中有不同程度表达,中及高分化鳞癌仅在角化区域表达,低分化鳞癌中阳性率44.1%;p63在腺癌中不表达,在鳞癌中100.0%表达;34βE12在腺癌及鳞癌中均有100.0%表达,鳞癌中强度高,腺癌中较弱;AB/PAS在子宫颈腺癌、腺鳞癌、低分化鳞癌中发生率分别为83.3%(5/6)、75.0%(3/4)、5.9%(2/34),但表达程度很弱。结论 CK7、CAM5.2、p63、34βE12联合检测对子宫颈癌的分型有重要意义。AB/PAS黏液染色对子宫颈癌有一定的鉴别作用。