冠心病患者循环内皮祖细胞与冠脉病变轻重程度的临床分析

目的:探讨冠心病患者循环内皮祖细胞与冠脉病变轻重程度的临床意义,为冠心病的防治提供科学依据。方法:选择经过冠状动脉造影检查确诊为冠心病的患者35例和冠脉造影检查阴性的患者40例。分别采集患者入院当天和入院48 h空腹血15 mL,进行内皮祖细胞(EPCSs)的分离培养,观察细胞培养1周内细胞形态;流式细胞术测定CD133含量。结果:EPCSs在不同培养天数中依次呈现小圆形、椭圆形、短梭形以及长梭型形状。冠心病组CD133细胞计数与冠脉病变程度(单支、2~3支、3支以上)进行Sperm an秩相关分析,发现CD133细胞含量与病变程度呈负相关(r=-0.819,P0.01)。同一冠脉病变程度组在不同采血时间EPCs培养的CD133含量比较,入院48 h采血测的CD133数量比入院当天采血的CD133含量均有所增加,并且差异均具有统计学意义(P0.05);但冠脉造影阴性组在不同采血时间CD133含量差异无统计学意义。相同采血时间(入院当天和入院48 h)EPCs培养的CD133含量在三组间比较,冠脉病变组(单支组和≥2支组)与冠脉造影阴性组相比较差异均具有统计学意义(P0.05);单支病变组和多支病变组的差异无统计学意义,但随着病变支数的增多CD133的数量下降。结论:循环内皮细胞的损伤与冠心病病情的轻重程度有关,循环内皮组细胞水平表达变化可能预测冠心病病情变化,为临床上冠心病合理治疗提供一定的依据。     

重楼皂苷Ⅰ抗肝癌细胞作用的初步研究

目的:本文旨在研究重楼皂苷Ⅰ对肝癌细胞株Huh7和HepG2的抑制作用并探讨其是否具有抑制肝癌干细胞的功效.方法:不同剂量的重楼皂苷Ⅰ处理肝癌细胞后,MTT方法检测细胞存活情况;流式细胞术检测细胞凋亡情况;Transwell实验检测细胞迁移能力;磁珠分选出CD133+-Huh7和CD133--HepG2细胞后,成球实验...     

白细胞介素8参与CD133^+肝癌细胞的侵袭与转移北大核心

背景:白细胞介素8是一种重要的炎性趋化因子,其在调节肿瘤细胞增殖、血管新生方面发挥了重要作用。目的:探讨白细胞介素8对CD133^+肝癌细胞侵袭转移能力的影响。方法:分离培养人肝癌MHCC97-H细胞株,免疫磁珠分选出CD133^+/CD133-MHCC97-H细胞,流式细胞仪检测CD133表达,ELISA检测上清液白细胞介素8水平。克隆形成实验、裸鼠成瘤实验、Transwell小室分别检测CD133^+/CD133-MHCC97-H细胞克隆形成率、小鼠成瘤能力、细胞迁移侵袭能力。另外,采用白细胞介素8中和抗体处理细胞,比较CD133^+/CD133-MHCC97-H细胞CD133表达、上清液中白细胞介素8水平及细胞克隆形成能力和迁移、侵袭能力。结果与结论:(1)CD133^+MHCC97-H细胞的CD133表达率、上清液白细胞介素8水平及克隆形成率均显著大于CD133-MHCC97-H细胞(P<0.05);(2)细胞浓度为1×10~6 L^(-1)和1×10~7 L^(-1)的CD133^+MHCC97-H细胞接种后有部分小鼠出现皮下移植瘤,但CD133-MHCC97-H细胞在同样细胞浓度下,均未出现皮下移植瘤;(3)CD133^+MHCC97-H细胞的透膜细胞数量均显著大于CD133-MHCC97-H细胞(P<0.05);(4)经白细胞介素8中和抗体处理之后,CD133^+/CD133-MHCC97-H细胞的CD133表达率、上清液中白细胞介素8水平、克隆形成率均显著下降(P<0.05),且CD133^+MHCC97-H细胞下降程度更大(P<0.01);CD133^+MHCC97-H细胞的迁移、侵袭透膜细胞数显著减少(P<0.05),CD133-MHCC97-H细胞则未出现明显的改变(P>0.05);(5)结果表明,白细胞介素8可以特异性参与调节CD133^+MHCC97-H细胞侵袭、转移能力。     

原发性肝癌组织中B7-H3表达与浸润的CD8~+ T细胞数负相关

目的探讨原发性肝癌组织中B7-H3的表达与CD8+T细胞浸润之间的关系。方法采用免疫组织化学技术分析原发性肝癌组织和正常肝组织中B7-H3的表达、CD8+T细胞的浸润数量,对B7-H3的表达与CD8+T细胞浸润数量的相关性进行统计学分析。结果 63例肝癌患者中B7-H3高表达26例(41.3%),低表达37例(58.7%),总阳性率为90.5%,其中正常肝组织基本不表达B7-H3。B7-H3高表达组CD8+T细胞浸润数量明显低于B7-H3低表达组,CD8+T细胞浸润数量与肝癌组织中B7-H3的表达呈负相关。肝癌组织中B7-H3的表达和CD8+T细胞数量在肝癌患者的临床病理分期、是否有局部淋巴结转移以及远隔器官转移等方面具有显著性差异。结论原发性肝癌组织中高表达B7-H3,与肿瘤组织中CD8+T细胞浸润呈负相关。     

沙培利乳剂栓塞治疗原发性肝癌患者后免疫变化的初步临床观察

评价原发性肝癌患者免疫栓塞治疗前后的免疫指标变化。经肝动脉灌注沙培利碘化油乳剂治疗肝癌患者6例,治疗前后用流式细胞仪检测外向血淋巴细胞亚群的比例,并与单纯栓塞组（19例）比较。结果,治疗前患者的CD3^ 、CD4^ 细胞比例、CD4^ /CD8^ 比值低于正常,而CD8^ 细胞比例则高于正常。治疗后CD3^ 、CD4^ 、NK细胞比例、CD4^／CD8^ 比值均较治疗前升高,并且较单纯栓塞组更为显著。在原发性肝癌患者的细胞免疫状态低下,免疫栓塞治疗可显著改善肝癌患者的细胞免疫状态。     

ZBP-89通过HIF-1α/Notch1通路对肝癌干细胞干性的调控作用

目的 探究ZBP-89(Zinc-binding protein-89)通过HIF-1α/Notch1通路对肝癌干细胞干性的调控作用.方法 构建ZBP-89过表达慢病毒载体(Len-ZBP-89)并进行慢病毒包装,微球体培养法富集肝癌干细胞,流式细胞术检测细胞中EpCAM、CD133的表达,CoCl2(HIF-1α激活剂)处理肝癌干细胞,RT-PCR检测细胞中ZBP-89、HIF-1α和Notch1mRNA表达,Western blot检测细胞中ZBP-89、HIF-1α、Notch1、CD44、CD133和EpCAM蛋白表达,Transwell检测细胞迁移和侵袭能力,光学显微镜观察特征性肿瘤球体的形成.结果 Len-ZBP-89慢病毒包装成功,病毒滴度为1.32×109 TU/mL;肝癌细胞PLC/PRF/5中ZBP-89 mRNA和蛋白表达水平下调,HIF-1α和Notch1 mRNA和蛋白表达水平上调;PLC/PRF/5细胞成功富集肝癌干细胞PLC/PRF/5.C,PLC/PRF/5.C中EpCAM、CD133的表达水平均上调;Len-ZBP-89可下调PLC/PRF/5.C细胞中HIF-1α、Notch1、CD133、CD44和EpCAM的蛋白表达,下调细胞迁移、侵袭能力和特征性肿瘤球体克隆数量;CoCl2(HIF-1α激活剂)可上调PLC/PRF/5.C细胞中HIF-1α、Notch1、CD133、CD44和EpCAM的蛋白表达,上调细胞迁移、侵袭能力和特征性肿瘤球体克隆数量(P＜0.05);CoCl2处理可逆转Len-ZBP-89对PLC/PRF/5.C干性的抑制作用.结论 过表达ZBP-89通过抑制HIF-1α/Notch1通路抑制肝癌干细胞干性.     

原发性肝癌组织中B7-H3表达与浸润的CD8^+ T细胞数负相关北大核心CSCD

目的探讨原发性肝癌组织中B7-H3的表达与CD8+T细胞浸润之间的关系。方法采用免疫组织化学技术分析原发性肝癌组织和正常肝组织中B7-H3的表达、CD8+T细胞的浸润数量,对B7-H3的表达与CD8+T细胞浸润数量的相关性进行统计学分析。结果 63例肝癌患者中B7-H3高表达26例(41.3%),低表达37例(58.7%),总阳性率为90.5%,其中正常肝组织基本不表达B7-H3。B7-H3高表达组CD8+T细胞浸润数量明显低于B7-H3低表达组,CD8+T细胞浸润数量与肝癌组织中B7-H3的表达呈负相关。肝癌组织中B7-H3的表达和CD8+T细胞数量在肝癌患者的临床病理分期、是否有局部淋巴结转移以及远隔器官转移等方面具有显著性差异。结论原发性肝癌组织中高表达B7-H3,与肿瘤组织中CD8+T细胞浸润呈负相关。     

急性早幼粒细胞患者治疗前后循环内皮细胞的变化及其影响因素的分析

目的:探讨急性早幼粒细胞白血病(acute promyelocytic leukemia,APL)患者治疗前后循环内皮细胞(circulating endothelial cells,CECs)数量、表面抗原及生物学特性的变化,并分析其与临床指标的关系。方法:免疫磁珠结合三色流式细胞仪分选计数APL患者初诊及治疗后CECs的数量;免疫荧光染色检测APL患者初诊及治疗后CECs表面CD146、CD31、CD144、VEGFR-2、CD45及CD133的表达水平。体外培养CECs进行血管生成实验及细胞集落形成率的测定。结果:APL患者CECs在外周血白细胞10×10~9/L及CD34阳性患者中数量明显增多(P0.05),高危、低危及中危3组患者的CECs数量也呈现明显的差异(P0.05)。32例APL患者诱导化疗后均获得完全缓解,治疗后CECs数量及其表面CD133表达水平均明显下降(P0.05)。同时治疗后APL患者CECs体外形成血管数目及细胞的集落形成率均明显减少(P0.05)。APL患者CECs数量治疗后/治疗前的比值与As_2O_3治疗1周后的尿砷浓度呈现明显负相关性(P0.05)。结论:精确计数并深入了解CECs生物学特性可能有助于评价APL的预后及设计治疗策略。     

血小板、红细胞对T细胞亚群的调控作用

目的: 在体外实验中, 探讨血小板和红细胞对T细胞亚群的调控作用. 方法: 流式细胞术测定血小板CD59、红细胞CD59、 T细胞CD3 CD4 、 CD3 CD8 的表达水平.按不同的实验条件进行分组, 全血细胞组、有PL无红细胞(RBC)组、无血小板(PL)组、无RBC无PL组.同时观测各组抗原刺激前后T细胞变化.结果: 各组T细胞CD3 CD4 、 CD3 CD4 /CD3 比较: 全血细胞组＞有PL无RBC组, 抗原刺激前后(P＜0.001); 有PL无RBC组＞无RBC无PL组刺激前(P＞0.05)刺激后(P＜0.05).各组T细胞CD3 CD8 表达无明显变化.各组CD3 CD8 /CD3 比较: 全血细胞组＜有PL无RBC组抗原刺激前后(P＜0.01); 有PL无RBC组＜无RBC无PL组刺激前(P＞0.05)刺激后(P＜0.05).全血细胞组和无PL组比较各项指标均无统计学意义(P＞0.05).抗原刺激前RBCCD59与CD3 CD4 在全血细胞组中负相关(P＜0.05); 而在无PL组中则无相关性; PLCD59与CD3 CD8 CD3 全血细胞组中的正相关无统计学意义(P＞0.05); 而在无红细胞组中则呈负相关(P＜0.05).结论: 活化血小板、红细胞均可正向调节CD3 CD4 /CD3 , 负向调节CD3 CD8 /CD3 .     

人鼻咽癌细胞株中肿瘤干细胞分离鉴定方法及肿瘤多药耐药机制

目的:研究人鼻咽癌细胞株中肿瘤干细胞分离鉴定方法及对肿瘤多药耐药机制。 方法:取鼻咽癌细胞株（SUNE）,进行细胞的培养、传代及鉴定,采用免疫荧光细胞化学技术联合流式细胞仪检测SUNE中CD133-及CD133+细胞体外分化能力。利用免疫磁珠分选技术完成CD133+肿瘤细胞纯化,测定CD133+细胞体外增殖能力,将其与未分选及CD133-细胞进行比较。取顺铂、紫杉醇化疗药物,采用CCK-8法测定CD133+细胞耐药性。取10只4周龄ICR小鼠,皮下注射人鼻咽癌细胞株和普通细胞株,分析耐药性。 结果:分离培养的SUNE在含有血清的培养基中呈贴壁生长,并且生长活跃,培养2~3 d后倒置显微镜下显示细胞呈梭形、扁平状,光泽度良好,培养2 周左右细胞融合瓶底80.00%;流式细胞仪检测结果显示:约0.35%细胞表面膜存在抗原CD133。免疫细胞化学显示:SUNE细胞贴壁爬行,部分细胞能与SUNE中的CD133-相互结合,荧光显微镜下显示呈现橙红色,球状;向经免疫磁珠分选后的细胞中加入培养基,细胞呈单细胞,球形,悬浮生长。随着培养时间的不断延长,细胞开始出现团状生长,体积增加,细胞数量增多,培养24 h后细胞开始贴壁生长,培养7 d后呈串状生长;CD133+肿瘤细胞、CD133-肿瘤细胞及未分选细胞随着时间的延长细胞均出现增长,且CD133+肿瘤细胞增殖能力显著高于CD133-肿瘤细胞及未分选细胞（P<0.05）。10只小鼠均可见瘤体形成,种植人鼻咽癌细胞株组的瘤体体积显著大于普通细胞株组（P<0.05）。结论:鼻咽癌干细胞中CD133+对化疗药物的耐药性强。     

Flow Cytometric Analysis of CD133- and EpCAM-Positive Cells in the Peripheral Blood of Patients with Lung Cancer

Lung tumors are characterized by their high metastatic potential, which is the main cause of therapeutic failure. However, the exact cellular origin of metastasis remains unknown. Since the introduction of the cancer stem cell theory, lung cancer stem cells (LCSCs) have been thought to represent metastasis-founding cells. The current study aimed to evaluate whether LCSCs could be found in the circulation. Expression of the stem cell markers CD133 and EpCAM was confirmed in tumor and normal lung tissue by flow cytometry. Then, this technique was further used to investigate the expression of CD133 and EpCAM in the peripheral blood of 41 patients with primary lung cancer. Putative LCSCs (CD133⁺EpCAM⁺) were present in 6/7 tumor samples, and CD133⁺EpCAM⁺ cells were identified in the blood samples of 15 patients at a median level of 40/ml of blood. EpCAM⁺ cells were detected in 60 % of the patients, and the number of these cells was higher in patients with adenocarcinoma than patients with squamous cell carcinoma and was also higher in patients with less advanced disease. Moreover, the frequency of this subpopulation significantly correlated with the circulating level of SSEA-4⁺ cells. Additionally, CD133⁺EpCAM^? cells were found in 87 % of the patients, and the numbers of these cells were significantly higher in patients with distant metastases and correlated with disease stage. This study confirmed the presence of an LCSC subpopulation with a CD133⁺EpCAM⁺ phenotype in the tumors and blood of patients with lung cancer, and these results suggest an important role for CD133 and EpCAM in lung cancer progression and their potential application as novel biomarkers of the disease.     

大肠癌患者外周血细胞CD133的表达

背景：近期有报道称在肿瘤患者外周血中检测出了高于正常值的CD133阳性细胞。 目的：观察CD133 mRNA和蛋白在大肠癌患者外周血中的表达并探讨其临床意义。 方法：应用流式细胞技术与RT-PCR反应检测29例大肠癌患者和20例正常对照外周血中CD133 抗原与mRNA的表达。 结果与结论：发生转移的大肠癌患者外周血中CD133阳性细胞比例显著高于正常对照及无转移的大肠癌患者(P < 0.01)。正常健康人群和无转移大肠癌患者外周血中CD133mRNA表达为阴性,发生转移的部分大肠癌患者外周血中CD133mRNA表达阳性,阳性率为35.7%(5/14),在超过38个以上循环时表达为弱阳性。结果表明,无转移大肠癌患者外周血中CD133 mRNA和蛋白表达与正常对照无区别,发生转移的大肠癌患者外周血中CD133 mRNA和蛋白高于正常对照和未发生转移者。     

Elevated serum levels of vascular endothelial growth factor are associated with tumor-associated macrophages in primary breast cancer

We analyzed serum and tumor samples from 133 patients with operable primary breast cancer to determine the possible relationship between presurgery and postsurgery circulating serum vascular endothelial growth factor (VEGF) levels and tumor-associated macrophage (TAM) numbers, tumor VEGF expression, and other immunohistochemical parameters. A significant positive correlation was observed between the number of TAM and postsurgery circulating VEGF values (P < .05). Moreover, patients with a p53+ tumor had higher postsurgery serum VEGF levels than those with a p53- tumor (P < .05), and tumor p53 overexpression correlated significantly with TAM number (P = .007). We observed no significant association between serum values and tumor VEGF expression. Although the macrophage index was higher in VEGF+ than in VEGF- tumors, the differences were not statistically significant. Our data show a positive interrelation between high circulating VEGF levels, the number of TAM, and p53 overexpression, a relationship that might have an important role in the enhanced angiogenesis processes in breast cancer.     

Major Hepatectomy Induces Phenotypic Changes in Circulating Dendritic Cells and Monocytes

Introduction  Patients undergoing major hepatectomy are at increased risk for post-operative morbidity and mortality, and changes in the phenotype of effector cells may predispose these patients to infectious sequelae. Methods  To better understand post-hepatectomy immune responses, peripheral blood from 15 hepatectomy patients was drawn immediately before and after liver resection and on post-operative days 1, 3, and 5. Circulating monocytes and dendritic cells were analyzed by flow cytometry for quantity, phenotype, activation status, human leukocyte antigen DR (HLA-DR) expression, and toll-like receptor-2 and -4 expression. Results  Major hepatectomy increased the numbers of activated CD16^bright blood monocytes and the percentage of activated dendritic cells, although monocyte HLA-DR expression was reduced. These results may represent both dysfunctional antigen presentation and pending anergy, as well as cellular priming of immune effector cells. Better understanding of the alterations in innate immunity induced by hepatectomy may identify strategies to reduce infectious outcomes.     

Four-color flow cytometric analysis of peripheral blood donor cell chimerism

Passenger leukocytes have been demonstrated to play significant roles in initiating and also regulating immune reactions after organ transplantation. Reliable techniques to detect donor leukocytes in recipients after organ transplantation are essential to analyze the role, function, and behavior of these leukocytes. In this report we describe a simple, reliable method to detect donor cells with low frequencies using peripheral blood samples. Detection of small numbers of major histocompatibility complex (MHC) mismatched cells was first studied using four-color flow cytometry in artificially created cell mixtures. By selecting the CD45(+) population and simultaneous staining with several leukocyte lineage markers (CD3, CD4, CD8, CD56, and CD19), MHC-mismatched leukocytes were consistently detected in cell suspensions prepared from directly stained whole blood samples with a threshold sensitivity as low as 0.1%-0.2%. When the fresh peripheral blood mononuclear cells were separated by conventional Ficoll gradient purification, similar, but slightly lower levels of donor cells were detected. Blood samples obtained 1-5 months after liver, kidney, and intestine transplants revealed that the kind of organ allograft influenced levels and lineage pattern of the circulating donor cells. This procedure provided a simple and reliable method in determining early chimerism in transplant recipients. However, the detection of MHC-mismatched leukocytes of all lineages was much lower when frozen peripheral blood mononuclear cells were used.     

CXCR4/CXCL12 expression profile is associated with tumor microenvironment and clinical outcome of liver metastases of colorectal cancer

Interaction between CXCR4 and CXCL12 plays a role in tumor progression. The present study examined CXCR4, CXCL12 and CD133 expression in liver metastases of colorectal cancer (CLM) and determined whether the expression profiles affect the tumor microenvironment and thus progression, and whether they could serve as a prognostic marker for survival. Liver metastases of colorectal cancer collected from 92 patients were evaluated by CXCR4, CXCL12 and CD133 immunohistochemistry and clinicopathological data were analyzed. The expression profile of CXCR4 was determined in the colorectal cancer cell line, SW48. The expression of cytoplasmic CXCR4 was higher in 36 (39%) patients than that indicated by CXCR4 staining intensity of hepatocytes. High levels of nuclear CXCR4 expression in 23 (25%) patients significantly correlated with CXCL12 expression in hepatocytes. Nuclear CXCR4 expression was increased in the cancer cells after exposure to CXCL12. Univariate and multivariate analyses demonstrated that the high levels of nuclear CXCR4 and CXCL12 expression in hepatocytes were significantly better prognostic factors for overall and hepatic disease-free survival in patients with CLM. The expression of CXCR4 and CXCL12 in CLM may have an interactive effect that could alter the tumor microenvironment. CXCR4 expression in metastatic liver tumors together with the upregulation of CXCL12 in hepatocytes may help to predict the clinical outcomes of patients with CLM after hepatectomy.     

Mitochondrial membrane potential is reduced in peripheral natural killer cells following partial hepatectomy

The mechanism underlying immunosuppression after partial hepatectomy remains unclear. Hepatectomy induces lymphopenia, which is related to immunomodulation. The aim of this study was to determine whether peripheral blood lymphocytes (PBL) are susceptible to mitochondria-mediated apoptosis after hepatic resection. We compared the changes in mitochondrial membrane potential in lymphocytes from hepatectomized patients with metastatic liver tumor with the corresponding changes in lymphocytes from cholechystectomized patients, because changes in mitochondrial membrane potential have been reported to frequently occur during the early stages of apoptosis. Mitochondrial membrane potential, subpopulation, and apoptosis of lymphocytes were estimated with flow cytometry. Hepatectomy significantly (P<0.001) reduced postoperative mitochondrial membrane potential, while cholecystectomy slightly decreased it. Apoptosis of lymphocytes was increased on post-hepatectomy day, and this increase was correlated with the extent of mitochondrial membrane potential reduction. The major subset of lymphocytes with low mitochondrial membrane potential consisted of CD56(+) natural killer (NK) cells, and NK cell activity and cell counts significantly decreased after hepatectomy. Mitochondrial membrane potential of PBL was reduced after hepatectomy, and some lymphocytes underwent apoptosis through the mitochondrial pathway, which was one of the causes for lymphopenia. NK cells were more responsible for the decrease of mitochondrial membrane potential after hepatectomy than other lymphocytes, and the reduction in mitochondrial membrane potential in NK cells appeared to reflect modulation of the innate immune system.     

Increased activation and oligoclonality of peripheral CD8(+) T cells in the chronic human helminth infection alveolar echinococcosis

Alveolar echinococcosis (AE) in humans is a chronic disease characterized by slowly expanding liver lesions. Cellular immunity restricts the spreading of the extracellular pathogen, but functional contributions of CD4(+) and CD8(+) T cells are not defined. Here we studied ex vivo the phenotype and function of circulating T-cell subsets in AE patients by means of flow cytometry, T-cell receptor spectratyping, and lymphocyte proliferation. AE patients with parasitic lesions displayed a significant increase of activation of predominantly CD8(+) T cells compared to healthy controls and AE patients without lesions. In vitro, proliferative T-cell responses to polyclonal stimulation with recall antigens and Echinococcus multilocularis vesicular fluid antigen were sustained during chronic persisting infection in all AE patients. Only in AE patients with parasitic lesions did T-cell receptor spectratyping reveal increased oligoclonality of CD8(+) but not CD4(+) T cells, suggesting a persistent antigenic drive for CD8(+) T cells with subsequent proliferation of selected clonotypes. Thus, our data provide strong evidence for an active role of CD8(+) T cells in AE.     

Combined use of positive and negative immunomagnetic isolation followed by real-time RT-PCR for detection of the circulating tumor cells in patients with colorectal cancers

To establish a novel molecular diagnostic method of detecting circulating tumor cells (CTCs) LS174T colon cancer cells were serially diluted with normal blood. Additional peripheral blood samples were collected from 25 patients with colorectal carcinoma. Mononuclear cells (MNCs) were collected, equally divided into four parts, and then cancer cells were enriched by four methods: method A, nonimmunobead method; method B, negative immunobead method: CD45 immunomagnetic beads were used to deplete the leukocytes; method C, positive immunobead method: Ber-EP4 immunomagnetic beads were used to enrich cancer cells; method D, negative-and-positive immunobead method: CD45 immunomagnetic beads were first used to deplete the leukocytes from MNC and then Ber-EP4 immunomagnetic beads were used to enrich cancer cells. Finally, real-time quantitative RT-PCR was used to monitor mRNA expression of ₂-mircoglobulin (2M) and carcinoembryonic antigen (CEA). The relative CEA mRNA values were corrected with reference to 2M mRNA, to CEA mRNA/2M mRNA ratios according to a CEA mRNA external standards prepared with tenfold serial dilutions (1–10⁴ IS174T cells) of cDNA and 2M mRNA external standards prepared with tenfold serial dilutions (10²–10⁷ leukocytes) of cDNA. In recovery experiments a significant correlation between the number of cancer cells and CEA mRNA expression was found when CD45 or Ber-EP4 immunomagnetic beads were used alone. A highly significant correlation was found when CD45 and Ber-EP4 immunomagnetic beads were used successively. The sensitivity of method D was one cancer cell per milliliter of blood. Circulating cancer cells were detected in 19 of 25 patients with colorectal cancers. The relative CEA mRNA value obtained by method D was the smallest. The positive detection rate of circulating cancer cells in patients at Dukes B, C, and D stages were 25.0% (1/4), 83.3% (10/12), and 88.9% (8/9). Combinative use of immunomagnetic isolation followed by real-time RT-PCR is a useful technique to detect circulating tumor cells in patients with colorectal carcinomas. Applying negative and positive immunomagnetic beads successively yields the highest correlation with amount of tumor cells.     

肿瘤干细胞表面标志物CD133表达水平对Ⅱ期结直肠癌肝转移发生风险的预测价值分析

目的 分析CD133表达水平对Ⅱ期结直肠癌患者肝转移发生风险的预测价值.方法 本研究选取2012年至2016年间本院就诊的确诊Ⅱ期CRC患者98例进行研究,随访收集肝转移发生情况信息并讨论CD133表达水平对Ⅱ期CRC肝转移发生风险的预测价值.采用SPSS17.0软件分析数据.结果 基线CD133的表达阳性特征分析结果提示肿瘤组织和癌旁组织中CD133的表达阳性率未见性别差异.随访1年期间4例患者死亡,此4例患者均为基线测量中CD133阳性表达组患者,其中2例并见癌旁组织CD133表达阳性.其余94例患者中肝转移者检出率为22.34％.基线肿瘤组织CD133表达阳性的75例患者中随访检出肝转移患者发生率为26.67％,显著高于基线肿瘤组织CD133阴性者中的发生水平,P ＜0.05;此外基线癌旁组织CD133表达阳性者中随访肝转移的发生率为87.50％,高于阴性表达组水平,P＜0.05.结论 肿瘤组织中CD133表达增加会提高Ⅱ期结直肠癌肝转移的潜在风险,且癌旁组织中CD133表达增高也对肿瘤肝转移具有一定的预测功能.