利用生物纳米技术检测外周血肺癌细胞的实验研究

目的建立磁性纳米颗粒从肿瘤患者外周血中富集与纯化肿瘤细胞的方法,应用荧光纳米晶生物探针检测方法,实现对肺癌早期微转移的诊断。方法体外培养人肝癌细胞和肺癌细胞,采集健康人外周血单个核细胞（PBMC）,将PBMC与肝癌细胞和肺癌细胞按比例混合,通过磁粒抗体复合物富集癌细胞,用荧光纳米晶探针进行肺癌特异性鉴定。结果光镜下可见较多的人肝癌细胞和人肺癌细胞与磁性纳米颗粒紧密结合,而PBMC与磁性纳米颗粒不结合。荧光显微镜下可见荧光纳米晶表面蛋白A探针特异性地与肺癌细胞结合产生荧光信号,但与肝癌细胞不结合,无荧光信号出现。结论本方法可成功地从外周血中富集到肺癌细胞,经荧光纳米晶探针成功地鉴定为肺癌细胞,达到诊断肺癌早期微转移的目的。     

RT-PCR/Southern杂交方法检测肝癌患者外周血AFP mRNA

目的寻找一种敏感的方法以检外外周血中的肝癌细胞.方法分离原发性肝癌患者以及肝硬化、急慢性肝炎、肝转移癌、肝脏良性肿瘤患者和健康志愿者的外周血单个核细胞,提取单个核细胞的总RAN,进行RT-PCR/Southern杂交检测AFPmRNA.结果原发性肝癌患者外周血AFP mRAN的检出率为41.4%,其检出率与肿瘤大小以及TNM分期有关,对照组均为阴性.结论肝癌患者外周血中的AFPmRNA可以作为肝癌细胞的血液微小转移的标志物.     

胃癌患者外周血微转移的细胞学检测

目的：肿瘤的浸润和转移是影响患者预后的重要因素，而肿瘤转移大多通过淋巴及血循环播散。为此该文研究建立胃癌患者外周血微转移癌细胞的细胞学检测方法并探讨其临床意义。方法：取进展期胃癌患者外周静脉血5ml，经Ficoll液密度梯度离心及免疫磁珠法（磁珠采用细胞角蛋白7和细胞角蛋白8包被）分离出单核细胞，然后对细胞涂片进行苏木精－伊红染色观察；并采用免疫荧光染色和免疫细胞化学法检测癌胚抗原、人端粒酶逆转录酶、CD34和CD45，对其中的胃癌细胞进行生物学特性分析。结果：在24例胃癌患者的外周血中有10例找到了癌细胞，阳性率为41．7％；癌细胞的检出与肿瘤原发灶的病理分级差异有显著性（P＜0．05）。结论：应用Ficoll密度梯度离心和免疫磁珠分选技术在胃癌患者的外周血中可分离检测到癌细胞,并可据此拟定治疗方案并对患者的预后作出判断。     

免疫磁珠技术应用于肺癌中的研究进展

免疫磁珠技术利用结合有特定单克隆抗体的微小磁珠在磁力场中的力学移动来分离相应的抗原。近年来该技术在肺癌研究中的应用越来越广泛,譬如肺癌肿瘤指标的磁珠检测,肺癌患者循环癌细胞的磁珠检测和分离,以及肺癌免疫净化治疗和磁导向治疗等。     

柚皮素对人自然杀伤细胞杀伤肝癌细胞的影响及其机制

目的 将人外周血单个核细胞体外扩增为自然杀伤(NK)细胞,与肝癌细胞1∶1共培养。观察柚皮素对NK细胞杀伤率的影响并初步研究相关机制。方法 淋巴细胞分离液分离人外周血单个核细胞,在体外经重组人白细胞介素2诱导培养NK细胞;0、3.125、6.25、12.5、25、50μmol/L浓度柚皮素作用于人肝癌细胞0、24、48 h后CCK8检测肝癌细胞增殖情况;不同浓度柚皮素作用于人NK细胞24 h后,CCK8检测NK细胞增殖情况;柚皮素作用于NK-肝癌细胞1∶1共培养体系24 h后,CellTiter-LumiTM检测NK细胞杀伤率;实时荧光定量PCR法检测NK细胞中激活型受体NKG2D的基因表达,以及肝癌细胞中NKG2D配体的基因表达。计量资料多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。结果 经重组人白细胞介素2诱导培养后,NK细胞可扩增至人外周血单个核细胞的82.33%±0.70%;柚皮素作用24 h后,所有质量浓度组CLC5肝癌细胞的增殖率无明显差异(P值均>0.05),25及50μmol/L质量浓度组柚皮素对NK细胞的增殖有较明显的促进作用(P值均<0...     

非小细胞肺癌循环肿瘤细胞的定量检测

目的探讨非小细胞肺癌循环肿瘤细胞（CTCs）定量检测方法。方法CD45免疫磁珠阴性分选组20侧，CD326免疫磁珠阳性分选组25例，均为明确诊断的非小细胞肺癌患者。磁性分离富集后肺静脉与外周静脉血标本应用多参数流式细胞仪对CTCs进行定量检测。结果阴性分选由于只能去除CD45阳性细胞，回收目的细胞纯度低。阳性分选组中25例术中肺静脉血CTCs定量检测阳性率为64％（16／25），外周静脉血CTCs阳性率40％（10／25，P〈0．05）。结论免疫磁珠富集联合流式细胞分析检测CTCs的敏感性和特异性较高。     

多抗原肽免疫血清体外阻断丙型肝炎病毒感染外周血单个核细胞的研究

目的探讨多抗原肽（MAP）免疫兔血清体外阻断丙型肝炎病毒（HCV）感染人外周血单个核细胞。方法用兔抗MAP血清按一定比例混合后接种于HCV感染的人外周血单个核细胞,采用逆转录聚合酶链反应、免疫组化和原位杂交技术分别检测细胞内或上清中HCV RNA、细胞内HCV NS3和HCV NS5抗原的表达。结果兔抗MAP血清按1：1混合后,外周血单个核细胞仍可检出HCV RNA,细胞有HCV NS3抗原表达;而按5：1和10：1混合后,单个核细胞内未检测出HCV正负链RNA,未见特异性抗原表达,原位杂交也未检测出HCV RNA存在。结论兔抗MAP血清在体外能部分阻断HCV感染。     

人骨髓多能成体祖细胞的分离培养及向肝细胞样细胞分化的研究

目的 培养人骨髓多能成体祖细胞(multipotent adult progenitor cells,MAPCs)并研究其向肝细胞样细胞的分化。方法 应用体外细胞培养技术将人骨髓中的单个核细胞从无血液疾病患者的髂嵴骨髓中分离出来后，用磁式细胞分离仪分离富集骨髓单个核细胞中的CD45^-HIA—DR^-细胞，把其接种于DMEM培养基中进行培养，到达对数生长期时，加入成纤维细胞生长因子4(fibroblast growth factor-4，FGF—4)并改用维持液来诱导其向肝细胞样细胞分化，最后用免疫细胞化学方法检测其分化情况。结果 成功地进行了人骨髓MAPCs的原代和传代培养，并在体外成功地把其诱导分化为具有肝细胞表型特征的细胞。结论 FGF-4可诱导人MAPCs向具有肝细胞表型特征的细胞发生分化。     

原发性乳腺癌患者外周血循环肿瘤细胞及肿瘤干细胞的检测及临床意义

目的探究原发性乳腺癌患者外周血中循环肿瘤细胞(CTCs)及肿瘤干细胞(TSCs)与乳腺癌临床、病理特征的关系。方法采集44例初治原发性乳腺癌患者和15例正常女性志愿者外周血,制备外周血单个核细胞悬液,以CK19单克隆抗体标记,免疫磁性激活细胞分选获得的CTCs,将提取的CK19+细胞标记单克隆抗体CD44、CD24进行免疫荧光抗体双染色,免疫荧光显微镜检测表达CD44+/CD24-/low的TSCs含量。结果 15例正常女性志愿者PBMC中未检测到CK19+细胞。44例原发性乳腺癌患者中33例检测到CK19+细胞,按临床分期和淋巴结转移分组:每组患者均能检测到CK19+细胞,差异具有明显统计学意义。33例CK19阳性样本中21例(63.63%)检测到CD44+/CD24-/low细胞,按临床分期、病理学指标及分子亚型分组均无明显统计学差异。结论原发性乳腺癌患者外周血中可检测到CTCs及TSCs,CTCs的阳性率与临床分期、淋巴结转移密切相关。临床分期越晚,TSCs阳性率越高。     

对过继免疫治疗中外周血单个核细胞采集术的探讨

目的:探讨在恶性肿瘤患者过继免疫治疗中外周血单个核细胞的采集技术.方法:根据患者外周血单个核细胞计数值,应用CS-3000PLUS血细胞分离机,采集外周血单个核细胞,运用Ficoll法分离纯化,再用贴壁法分离出贴壁细胞进行诱导分化为树突状细胞(DC),未贴壁细胞(主要为淋巴细胞)诱导分化为肿瘤杀伤细胞(CIK)或体外扩增为记忆性淋巴细胞(ALT),最后进行细胞回输.结果:在8例13人次的采集中,处理总循环血量,获得单个核细胞总数,单个核细胞纯度,细胞采集效率,血小板丢失率分别为(6.99±1.74)L,(3.47±1.44)×109/L,(90±6)%、(5L 3±13)%、(43.5±13.1)%.DC加CIK治疗1例,CK治疗3例,化疗后ALT免疫重建治疗4例,均取得了一定的临床疗效.结论:外周血单个核细胞采集术,不良反应发生率低、单个核细胞的采集效率高,用于恶性肿瘤过继免疫治疗的临床效果较好.但采集后血小板的丢失严重,对于恶性肿瘤化疗后血小板减低的患者应引起关注.     

内镜超声引导下细针穿刺胰腺癌门静脉血测定循环肿瘤细胞的临床研究

目的评估用内镜超声引导下细针抽吸术(EUS-FNA)于胰腺癌门静脉采血检测循环肿瘤细胞(CTCs)的安全性及有效性。方法该研究为一项前瞻性单中心临床研究,纳入5例胰腺癌患者,行EUS-FNA门静脉采血。以外周血CTCs为对照,采用阴性免疫磁珠联合FISH法和叶酸受体阳性CTCs检测试剂盒检测门静脉血和外周血CTCs的细胞含量。结果5例患者EUS-FNA下门静脉采血均成功。1例出现血液凝集,未能进行CTCs检测。4例患者进行检测,3例门静脉血和外周血检测到CTCs。其中门静脉血CTCs细胞含量为(10.5±4.0)FU/3.7 mL,外周血CTCs含量为(11.4±4.2)FU/3.7 mL,两组比较差异无统计学意义(P>0.05)。术中术后无感染、腹腔出血和休克等并发症。结论内镜超声引导下胰腺癌门静脉血CTCs测定是一项安全可行的方法,可有助于胰腺癌早期转移的预估和治疗方案的选择。     

Epidermal growth factor receptor-targeted immune magnetic liposomes capture circulating colorectal tumor cells efficiently

AIM To compare the capacity of newly developed epidermal growth factor receptor(EGFR)-targeted immune magnetic liposomes(EILs) vs epithelial cell adhesion molecule(Ep CAM) immunomagnetic beads to capture colorectal circulating tumor cells(CTCs).METHODS EILs were prepared using a two-step method, and the magnetic and surface characteristics were confirmed. The efficiency of capturing colorectal CTCs as well as the specificity were compared between EILs and Ep CAM magnetic beads. RESULTS The obtained EILs had a lipid nanoparticle structure similar to cell membrane. Improved binding with cancer cells was seen in EILs compared with the method of coupling nano/microspheres with antibody. The binding increased as the contact time extended. Compared with Ep CAM immunomagnetic beads, EILs captured more CTCs in peripheral blood from colorectal cancer patients. The captured cells showed consistency with clinical diagnosis and pathology. Mutation analysis showed same results between captured CTCs and cancer tissues. CONCLUSION EGFR antibody-coated magnetic liposomes show high efficiency and specificity in capturing colorectal CTCs.     

Purging of small cell lung cancer cells from bone marrow using immunomagnetic beads and a flow-through device.

Immunomagnetic beads can be used to remove subpopulations of cells from a mixed cell suspension in a flow-through system. One application of this process is the removal of tumor cells from bone marrow prior to its use in autologous bone marrow transplantation (ABMT). Based on preliminary data showing that three monoclonal antibodies (MoAb) (SCCL 175, HNK-1, and TFS-4) gave optimal separation in small-scale experiments, we have designed a large-scale separator suitable for clinical use. In our separator, the cell suspension flows through a 150 ml baffled transfer pack which is held over an array of permanent magnets. Direct (one MoAb only) and indirect (MoAb and anti-mouse antibody) methods of binding beads to cells were investigated as were the effects of temperature, bead to cell ratio, and medium additives on tumor cell removal and normal cell recovery. We determined the optimal separation conditions to be the indirect method of binding at 22 degrees C using a bead to tumor cell ratio of 25:1. Testing of the device on DMS-273 small cell lung cancer (SCCL) cells mixed with normal human bone marrow mononuclear cells resulted in a mean tumor cell removal of 3.64 logs (99.977%) with a concomitant mean normal granulocyte-monocyte colony forming unit (CFU-GM) recovery of 61.3%. These experiments form the basis to use the immunomagnetic beads to purify bone marrow from patients with SCCL for use in ABMT.     

Detection and viability of tumor cells in peripheral blood stem cell collections from breast cancer patients using immunocytochemical and clonogenic assay techniques [see comments]

Although peripheral blood stem cell collections (PBSC) are thought to have less tumor involvement than bone marrow (BM), the incidence of circulating tumor cells in patients with breast cancer has not been widely investigated. We prospectively investigated the incidence and viability of tumor cell involvement in PBSC and BM collections from breast cancer patients undergoing high-dose chemotherapy/hematopoietic stem cell transplantation. Paired samples of PBSC and BM from 48 patients were analyzed using an immunocytochemical technique that detects one epithelial-derived tumor cell per 5 x 10(5) mononuclear cells. Immunostained tumor cells were detected in 9.8% (13/133) PBSC specimens from 9/48 (18.7%) patients and in 62.3% (38/61) BM specimens from 32/48 (66.7%) patients, a significantly higher rate than in PBSC (P < .005). The geometric mean concentration of tumor cells in contaminated PBSC specimens was 0.8/10(5) mononuclear cells (range 0.33 to 2.0/10(5)) compared with 22.9/10(5) mononuclear cells in BM (range 1 to 3,000/10(5), P < .0001). In culture experiments, clonogenic tumor colonies grew in 21/26 immunocytochemically positive specimens. No tumor colony growth was detected in 30/32 immunocytochemically negative specimens. Immunocytochemical detection of tumor involvement in BM and PBSC correlated significantly with in vitro clonogenic growth (P < .0001). We conclude that PBSC contain fewer tumor cells than paired BM specimens from patients with advanced breast cancer and that these tumor cells appear to be capable of clonogenic growth in vitro.     

Isolating highly enriched populations of circulating epithelial cells and other rare cells from blood using a magnetic sweeper device

The enumeration of rare circulating epithelial cells (CEpCs) in the peripheral blood of metastatic cancer patients has shown promise for improved cancer prognosis. Moving beyond enumeration, molecular analysis of CEpCs may provide candidate surrogate endpoints to diagnose, treat, and monitor malignancy directly from the blood samples. Thorough molecular analysis of CEpCs requires the development of new sample preparation methods that yield easily accessible and purified CEpCs for downstream biochemical assays. Here, we describe a new immunomagnetic cell separator, the MagSweeper, which gently enriches target cells and eliminates cells that are not bound to magnetic particles. The isolated cells are easily accessible and can be extracted individually based on their physical characteristics to deplete any cells nonspecifically bound to beads. We have shown that our device can process 9 mL of blood per hour and captures >50% of CEpCs as measured in spiking experiments. We have shown that the separation process does not perturb the gene expression of rare cells. To determine the efficiency of our platform in isolating CEpCs from patients, we have isolated CEpCs from all 47 tubes of 9-mL blood samples collected from 17 women with metastatic breast cancer. In contrast, we could not find any circulating epithelial cells in samples from 5 healthy donors. The isolated CEpCs are all stored individually for further molecular analysis.     

磁激活细胞分选术联合混合抗体提高模拟恶性腹水中游离癌细胞检出效率的分析

背景磁激活细胞分选术(magnetic activated cell sorting,MACS)是一种新的免疫磁性分离技术.其原理是基于抗体对抗原的特异性识别,将50 nm磁性微珠直接或者间接耦联在抗体上,与有相应抗原表达的细胞相连,在高强度、高梯度磁场中达到细胞磁性分离的目的.该法具有分离纯度和回收率均较高的优势,也能分离出体液中存在的少量肿瘤细胞.目的评价MACS联合一组肿瘤细胞标志物的方法对提高模拟恶性腹水中游离癌细胞检出效率的作用.方法 选择5种与恶性腹水病因有关的肿瘤细胞株作为研究对象,采用免疫荧光反应和流式细胞仪(FCM)检测上皮相关抗原(epithlial-related antigen,EpCAM)、CA125、癌胚抗原和TAG-72共4种单克隆抗体及其混合抗体在各肿瘤细胞的表达.将肿瘤细胞以不同比例掺入单个核细胞中模拟恶性腹水细胞成分,与联合单个抗体进行分选对比,观察MACS术联合混合抗体分选肿瘤细胞的效率.结果 FCM结果表明,混合抗体在5种肿瘤细胞的阳性表达率均高于4种抗体单独反应的阳性率.MACS术联合混合抗体检出模拟恶性腹水中肿瘤细胞的得率比联合单个抗体的得率要高,其中以2种胃癌细胞和结肠癌细胞的平均得率提高较多(分别为69.18%±20.84%比45.23%±11.54%、78.75%±15.42%比59.73%±16.64%和85.63%±12.30%比76.88%±8.65%),卵巢癌细胞次之(32.49%±3.58%比31.79%±4.82%),肝癌细胞最少(11.78%±0.43%比7.16%±0.46%).结论 与联合单个抗体进行分选相比,应用MACS术联合一组混合抗体的方法可有效提高恶性腹水中游离癌细胞的检出效率,尤其对胃肠道肿瘤所致的恶性腹水有潜在的临床应用价值.