Origins of circulating endothelial cells and endothelial outgrowth from blood

Normal adults have a small number of circulating endothelial cells (CEC) in peripheral blood, and endothelial outgrowth has been observed from cultures of blood. In this study we seek insight into the origins of CEC and endothelial outgrowth from cultures of blood. Fluorescence in situ hybridization analysis of blood samples from bone marrow transplant recipients who had received gender-mismatched transplants 5-20 months earlier showed that most CEC in fresh blood had recipient genotype. Endothelial outgrowth from the same blood samples after 9 days in culture (5-fold expansion) was still predominantly of the recipient genotype. In contrast, endothelial outgrowth after approximately 1 month (102-fold expansion) was mostly of donor genotype. Thus, recipient-genotype endothelial cells expanded only approximately 20-fold over this period, whereas donor-genotype endothelial cells expanded approximately 1000-fold. These data suggest that most CEC in fresh blood originate from vessel walls and have limited growth capability. Conversely, the data indicate that outgrowth of endothelial cells from cultures of blood is mostly derived from transplantable marrow-derived cells. Because these cells have more delayed outgrowth but a greater proliferative rate, our data suggest that they are derived from circulating angioblasts.     

外周血红系祖细胞来源诱导多能干细胞的建立

目的建立和鉴定外周血中红系祖细胞来源的诱导多能干细胞(induced pluripotent stem cells,i PSCs)。方法从健康人外周血标本中分选红系祖细胞,将表达Oct4、Sox2、Lin28、L-Myc和Klf4转录因子的ori P/EBNAl附着体电转染红系祖细胞使其重编程获得i PSCs,并通过核型鉴定、碱性磷酸酶染色、RT-PCR反应、畸胎瘤形成实验及类胚体形成实验检测其干细胞多能性特性。结果获得的i PSCs核型正常,碱性磷酸酶染色呈阳性,表达干细胞多能性基因Sox2、Oct4、Nanog和Klf4和Lin28,体内畸胎瘤形成实验可分化为内、中、外三胚层细胞,体外可形成类胚体。结论成功建立外周血红系祖细胞来源具有多向分化潜能的i PSCs。     

Analysis of circulating mesenchymal progenitor cells in arterial and venous blood after fracture

Mesenchymal progenitor cells (MPCs) play a critical role in fracture healing. Increasing evidence suggests that circulating MPCs in peripheral blood are mobilized during fracture healing and may contribute to fracture repair. However, to date, there have been no reports comparing the number of circulating MPCs in arterial blood (AB) with that in venous blood (VB) after fracture. In this study, we investigated the numbers of MPCs in AB, VB and bone marrow (BM) after fracture in rabbits via the colony‐forming unit–fibroblasts (CFU‐Fs) assay, and clarified the time‐course change. After femoral fracture in one side, the number of BM‐MPCs in the contralateral femur increased from day 1 to day 7. Correspondingly, the number of circulating MPCs in AB and VB increased. The number of circulating MPCs in AB was highest at post‐fracture day 4, whereas that in VB was highest at post‐fracture day 1, with significant difference compared to the control. Circulating MPCs in AB and VB after fracture may serve as new cell sources for bone tissue engineering. As the peaks of the number of circulating MPCs in AB and VB after fracture were different, our findings may provide new insights about when to collect circulating MPCs after fracture and from which blood to obtain them. Copyright © 2012 John Wiley & Sons, Ltd.     

Normal circulating adult human red blood cells contain inactive NOS proteins

Human red blood cells (RBCs) are considered to play a significant role in both blood pressure (BP) regulation and tissue oxygenation, because they can bind as well as release previously bound nitric oxide (NO) from hemoglobin (Hb) and other intracellular components. Two reports indicate that the human RBC possesses nitric oxide synthase (NOS) activity-by the accumulation of nitrite across a membraned chamber in one and by the hydrolysis of labeled L-arginine, presumably to labeled L-citrulline, in the other. Furthermore, NOS proteins have been identified by immunoblot in RBCs. If true, the presence of NOS activity would convert the human RBC to a donor with limited ability to bind exogenously generated NO. In considering the importance of the question of the presence or not of NO synthetic capacity of this cell in BP regulation and tissue perfusion, whether human RBCs are, indeed, able to hydrolyze L-arginine to L-citrulline, the coproduct of NO was explored. RBC samples collected from control subjects were assayed for NOS activity by incubation of homogenized cellular fractions with labeled tritiated L-arginine in the presence of 0.5 mmol/L NADPH. By this method, the amino acid coproduct of NO, tritiated L-citrulline, would be recovered in the supernatant after removal of unused substrate by cationic resin treatment. At first, activity appeared to be present in the RBC supernatant but not in the pellet. However, activity was not suppressed by known inhibitors of NOS, whereas activity was suppressed by norvaline, an inhibitor of arginase activity with no known effect on NOS. By contrast, RBC arginase activity was not inhibited by N(omega)-nitro-L-arginine, NG-methyl-L-arginine, or aminoguanidine, known inhibitors of NOS, but was inhibited by norvaline. The label recovered by thin-layer chromatography was determined not to be tritiated L-citrulline but was instead tritiated L-ornithine, the product of arginase activity. Thus the enzymatic hydrolysis of L-arginine was not caused by NOS but was a result of the action of the enzyme arginase, which abounds in this cell. However, proteins interacting with antibodies to the endothelial and inducible isoforms of NOS were detected in human RBCs by immunoblot. Together, these findings indicate that human RBCs collected from normal adult individuals possess proteins that react with monoclonal antibodies to the Inducible and endothelial isoforms of NOS, but the proteins are without catalytic activity.     

脓毒症患者外周血祖细胞和内皮祖细胞数量的变化

目的 检测脓毒症患者外周血单个核细胞(peripheral blood mononuelear cell,PBMC)中祖细胞和血管内皮祖细胞(endothelial progenitor cells,EPC)相对数量的变化,探讨感染性休克和非休克患者外周血EPC变化的特点.方法 收集2007年8月至2008年2月复大学附属中山医院急诊科收治的脓毒症患者27例进行前瞻性研究,其中感染性休克患者12例、非休克患者15例,另选10例健康成年人作为正常对照,ICU非脓毒症患者10例作为ICU对照.Ficoll梯度离心法分离外周血PBMC,通过流式细胞仪检测外周血PBMC标记的CDl33,CIY34和血管内皮牛长因子受体-2(vascular endothelialgrowth factor receptor-2.VEGFR-2)的表达情况,计算祖细胞以及内皮祖细胞的相对数量.组间比较采用单因素方差分析.结果 健康成年人外周血祖细胞、EPC数量较少,分别占PBMC的0.25%.4-0.14%和0.09%.4-0.02%;ICU非脓毒症患者祖细胞和EPC数量分别占PBMC的0.38%.4-0.29%和0.12%.4-O.02%,与正常对照组相比无明显的变化(P>0.05);脓毒症非休克组患者外周血祖细胞、EPC的数量明显增加,分别占PBMC的0.57%±0.12%和0.22%±0.10%,与正常对照组相比差异具有统计学意义(P<0.05);感染性休克患者外周血祖细胞和EPE的数量明显减少,分别占PBMC的0.20%.4-0.12%和0.04%±O.01%,与非休克组、ICU对照组和正常对照组相比差异均具有统计学意义(P相似文献   

大肠癌外周血循环肿瘤细胞数的检测及临床意义

目的 检测大肠癌（CRC）患者外周血循环肿瘤细胞（CTCs）数,探讨其临床意义.方法 58例CRC患者术前静脉采血,使用多重聚合酶链式反应（PCR）结合荧光定量PCR（RTFQ PCR）方法检测外周血细胞角蛋白20（CK20）及癌胚抗原（CEA） mRNA表达,比较CTCs细胞阳性率与临床病理参数的关系.结果 58例CRC患者外周血中CK20及CEAmRNA表达的阳性率为93.1％.CTCs的阳性检出率与Ducke＇s分期及淋巴结转移相关（P＜0.05）.结论 多重RTFQ PCR方法可应用于CRC患者外周血中CTCs检测,对预后判断有着重要的临床意义.     

外周血液中循环肿瘤细胞和循环游离DNA检测在乳腺癌患者中的应用

目的 探究外周血液中循环肿瘤细胞(CTCs)和循环游离DNA(cfDNA)检测在乳腺癌患者中的应用效果.方法 收集本院2018-02-2019-02收治的94例乳腺癌患者、48例乳腺良性疾病患者及56例健康体检者外周血液标本,采用基于尺寸的高通量微流控芯片捕获CTCs、基于Alu序列的实时荧光定量PCR检测游离DNA长...     

外周血叶酸受体阳性循环肿瘤细胞检测在非小细胞肺癌筛查中的应用价值

目的观察非小细胞肺癌患者外周血叶酸受体阳性循环肿瘤细胞(circulating tumor cells,CTCs)水平变化,探讨外周血叶酸受体阳性CTCs在非小细胞肺癌筛查中的应用价值。方法非小细胞肺癌患者136例为肺癌组,肺部良性病变患者10例为良性病变组,健康志愿者54例为对照组。3组均采用以叶酸受体为靶点的免疫磁珠阴性富集+实时荧光定量PCR法检测外周血叶酸受体阳性CTCs水平;采用化学发光免疫分析法检测血清癌胚抗原(carcino-embryonic antigen,CEA)、糖链抗原(carbohydrate antigen,CA)125、CA724、细胞角蛋白19片段(cytokeratin 19fragment,CYFRA21-1)、神经元特异性烯醇化酶(neuron-specific enolase,NSE)水平。比较3组CTCs水平;比较不同临床特征非小细胞肺癌患者CTCs水平;绘制ROC曲线,评价CTCs及血清CEA、CA125、CA724、CYFRA21-1、NSE 5项指标联合诊断非小细胞肺癌的价值。结果肺癌组CTCs水平[11.21(8.58,15.30)FU/3mL]高于良性病变组[7.55(5.23,10.25)FU/3mL]和对照组[4.95(3.55,7.62)FU/3mL](P<0.05),良性病变组高于对照组(P<0.05)。TNM分期Ⅰ、Ⅱ、Ⅲ、Ⅳ期非小细胞肺癌患者CTCs水平[11.00(8.58,13.30)、13.25(10.48,16.88)、14.77(11.47,16.55)、17.89(17.07,19.22)FU/3mL]两两比较差异均有统计学意义(H=16.443,P<0.05);不同年龄、性别、肿瘤最大径、T分期、分化等级、病理类型非小细胞肺癌患者CTCs水平比较差异均无统计学意义(P>0.05)。ROC曲线分析结果显示,CTCs以8.70FU/3mL为最佳截断值,诊断非小细胞肺癌的AUC为0.953(95%CI:0.926~0.979,P<0.05),灵敏度为79.40%,特异度为98.10%,诊断效能优于CEA、CA125、CA724、CYFRA21-1、NSE联合检测。结论非小细胞肺癌患者外周血叶酸受体阳性CTCs水平升高,且增高程度与TNM分期有关;外周血叶酸受体阳性CTCs可用于非小细胞肺癌的早期筛查。     

非小细胞肺癌患者外周血循环内皮干细胞的检测及其临床意义

目的探讨外周血循环内皮祖细胞（endothelial progenitors cells,EPCs）与非小细胞肺癌患者的关系及临床意义。方法选择非小细胞肺癌患者45例和对照组15例,用抗CD133和KDR的单克隆抗体标记外周血细胞,应用流式细胞术检测其外周血中循环内皮祖细胞。应用Real-time RT-PCR方法检测患者外周血中CD133和KDR的mRNA表达水平。密度梯度离心法从人外周血分离出单个核细胞,将其接种在人纤维粘连蛋白包被培养板,在加有EGM-2-MV-SingleQuots的培养液中培养,在荧光显微镜下进行鉴定。其中U EA-1和D iI-A c-LDL染色双阳性细胞为正在分化的内皮祖细胞。结果肿瘤患者外周血中EPCs含量高于正常对照组（P〈0.01）,外周血中CD34和KDR的表达水平明显高于正常对照组。结论非小细胞肺癌患者外周血EPCs明显增高,为今后将EPCs作为非小细胞肺癌患者辅助诊断和治疗效果检测的标志物奠定基础。     

应用套式RT—PCR方法检测大肠癌患者外周血中的肿瘤细胞

目的：探讨应用套式RT－PCR检测在肠癌患者外周血中的肿瘤细胞的可行性及其临床意义。方法：以CK－20mRNA为靶基因,应用套式RT－PCR方法检测10名健康人,17名非恶性消化系疾病患者,32名大肠癌患者手术前、后3天外周血中的肿瘤细胞,结果：CK－20mRNA在空白对照组中均呈阴性表达,大肠癌患者CK－20表达情况为：2名Duke'sA期患者外周血中未检出CK－20阳性细胞,15名Duke'sB期患者外周血中CK－20mRNA阳性表达4例,10名Duke'sC患者外周血CK－20mRNA阳性表达6例,5名Duke'sD期患者外周血中CK－20mRNA阳性率达4例。32名大肠癌患者术,术前CK－20mRNA阳性总检出率为43．8％;A、B期阳性率为23．5％;C、D期阳性率为66．7％。结论：以CK－20为靶基因,应用套式RT－PCR检测大肠癌患者外周血中的肿瘤细胞是一非损伤性且特异性、敏感性较高的检测方法。它将有助于肿瘤经血液微转移的早期诊断,并将有助于指导辅助化疗和监测其疗效。．     

Nucleated cells circulating in the peripheral blood contribute to the repair of osteochondral defects only in the early phase of healing

The role of cells circulating in the peripheral blood to participate in the natural repair process of osteochondral defects was evaluated in a green fluorescent protein (GFP) transgenic and wild rat parabiosis model. Two weeks after the parabiosis operation, vascular communication between the conjoined rats was confirmed by flow‐cytometry analysis. A 1.5 mm diameter and 1.0 mm depth osteochondral defect was made in the patellar groove of each rat femoral bone. Histological examination was performed at 1, 2, 4 and 24 weeks following surgery. In the early postoperative phase (1–4 weeks) there were GFP‐negative and ‐positive cells in the defects of both parabiotic rats. GFP‐positive chondrocytes were confirmed partly in the repair tissue of the wild parabiotic rat. In the late postoperative phase (24 weeks), the repaired defects were occupied by cells originating from the adjacent tissue and not from the peripheral blood. The ratio of cells originating from the peripheral blood was approximately 30–40% in the repair tissue at 1 week after surgery, reduced to 0–7% at 24 weeks. From these results it is confirmed that cells circulating in the peripheral blood contributed to the repair of the osteochondral defects, particularly in the early phase of healing. Thus, peripheral blood not only supplies the factors needed for repair but also provides a cell population involved in the wound‐healing process. Copyright © 2012 John Wiley & Sons, Ltd.