微小磁珠法分离胚胎大鼠神经干细胞的实验研究

目的　旨在以微小免疫磁珠 (平均直径≤ 5 0 nm)分离提纯 SD大鼠胚胎大脑皮层组织中的神经干细胞并进行培养 ,观察细胞的增殖分化情况并对其分化产物进行鉴定 ,评价该分离方法的可行性及其在神经干细胞研究中的应用价值。方法　取 SD大鼠的胚胎大脑皮层组织制单细胞悬液 ,用微小磁珠分选出神经巢蛋白(nestin)阳性的细胞群 ,光镜下检测其纯度及活力后进行体外培养 ,特定条件下诱导分化并以免疫细胞方法检测产物的细胞类型。结果　分离的神经干细胞纯度为 (89.2± 4.8) % ,细胞存活率 (97.0± 1 .6) % ,诱导分化产物中出现 NSE阳性、GFAP阳性、巢蛋白 (nestin)反应阳性细胞群落。结论　微小磁珠法分离提纯神经干细胞纯度高 ,获得细胞数多 ,并对细胞的生物活性无明显影响 ,提纯的细胞可用于后续实验     

CD146-based immunomagnetic enrichment followed by multiparameter flow cytometry: a new approach to counting circulating endothelial cells

Summary. Background: Circulating endothelial cells (CECs) have emerged as non‐invasive biomarkers of vascular dysfunction. The most widely used method for their detection is CD146‐based immunomagnetic separation (IMS). Although this approach has provided consensus values in both normal and pathologic situations, it remains tedious and requires a trained operator. Objectives: Our objective was to evaluate a new hybrid assay for CEC measurement using a combination of pre‐enrichment of CD146+ circulating cells and multiparametric flow cytometry measurement (FCM). Patients and methods: CECs were determined in peripheral blood from 20 healthy volunteers, 12 patients undergoing coronary angioplasty, and 30 renal transplant recipients, and blood spiked with cultured endothelial cells. CD146+ cells were isolated using CD146‐coated magnetic nanoparticles and labeled using CD45–fluorescein isothiocyanate and CD146–PE or isotype control antibody and propidium iodide before FCM. The same samples were also processed using CD146‐based immunomagnetic separation as the reference method. Results: The hybrid assay detected CECs, identified as CD45^dim/CD146^bright/propidium iodide⁺, with high size‐related scatter characteristics, and clearly discriminated these from CD45^bright/CD146^dim activated T lymphocytes. The method demonstrated both high recovery efficiency and good reproducibility. Both IMS and the hybrid assay similarly identified increased CEC levels in patients undergoing coronary angioplasty and renal transplantation, when compared to healthy controls. In patients, CEC values from these two methods were of the same order of magnitude and highly correlated. Bland–Altman analysis revealed poor statistical agreement between methods, flerrofluid–FCM providing higher values than IMS. Conclusion: This new hybrid FCM assay constitutes an accurate alternative to visual counting of CECs following CD146‐based IMS.     

Comparison of current methods used to detect Cryptosporidium oocysts in stools

In this review all of the methods that are currently in use for the investigation of Cryptosporidium in stool material are highlighted and critically discussed. It appears that more qualifications and background knowledge in this field regarding the diagnosis of the Cryptosporidium parasite is required. Furthermore, there is no standardization for the protocols that are commonly used to either detect oocysts in faeces or to diagnose the Cryptosporidium infection. It is therefore necessary to initiate further education and research that will assist in improving the accuracy of the diagnosis of Cryptosporidium oocysts in the faecal micro-cosmos. Where ambient concentrations of oocysts are low in stool material, detection becomes a formidable task. Procedures for ring tests and the standardization of multi-laboratory testing are recommended. It is also necessary to enhance the routine surveillance capacity of cryptosporidiosis and to improve the safety against it, considering the fact that this disease is under diagnosed and under reported. This review is intended to stimulate research that could lead to future improvements and further developments in monitoring the diagnostic methodologies that will assist in harmonizing Cryptosporidium oocysts in stool diagnosis.     

CD138/Syndecan-1在多发性骨髓瘤免疫表型中的意义

为了建立多发性骨髓瘤（MM）免疫分型的测定方法,分析各种抗原的表达特点,并分选高纯度原代骨髓瘤细胞,应用CD45/侧向角（SSC）设门的三色免疫荧光流式细胞术（FCM）测定18例MM患者、20例急性白血病（AL）患者和7例正常人骨髓免疫分型特点;应用抗CD138和免疫磁珠从MM患者的骨髓中分选骨髓瘤细胞.结果表明：骨髓瘤细胞CD45阴性或弱阳性,SSC介于有核红细胞和中性粒细胞之间,CD138和CD38均阳性,T、B细胞及髓系抗原多为阴性,CD56阳性率为83.3%,HLA-DR阳性率为44.4%.与MM患者相比,AL患者CD138均阴性,而CD38为100%阳性,CD56阳性率为25%.正常人CD138和CD56均为阴性.磁珠分选获得的原代骨髓瘤细胞纯度在73%-95%之间,平均为86%.结论：CD45/SSC设门的三色免疫荧光FCM是测定MM免疫分型的稳定可靠方法,CD138是骨髓瘤细胞表面较为特异的抗原标志,应用抗CD138和免疫磁珠可以获得高度富集的原代骨髓瘤细胞.     

Efficacy of a vaccine and a direct-fed microbial against fecal shedding of Escherichia coli O157:H7 in a randomized pen-level field trial of commercial feedlot cattle

Our primary objective was to determine the efficacy of a siderophore receptor and porin proteins-based vaccine (VAC) and a Lactobacillus acidophilus-based direct-fed microbial (DFM) against fecal shedding of Escherichia coli O157:H7 in commercial feedlot cattle fed a corn grain-based diet with 25% distiller's grains. Cattle projected to be on a finishing diet during the summer were randomly allocated into 40 study pens within ten blocks based on allocation dates. Blocks were complete; each of the four pens within a block was randomly assigned one treatment: control, VAC, DFM, or VAC+DFM. The DFM was fed (10(6)CFU/animal/day of Lactobacillus) throughout the study periods (84-88 days) and cattle were vaccinated at enrollment and again three weeks later. Fresh fecal samples (30/pen) from pen floors were collected weekly for four consecutive weeks (study days 52-77). Two concurrent culture procedures were used to enable estimates of E. coli O157:H7 shedding prevalence and prevalence of high shedders. From 4800 total samples, 1522 (31.7%) were positive for E. coli O157:H7 and 169 (3.5%) were considered high shedders. Pen-level linear mixed models were used for data analyses. There were no significant interactions among treatments and time of sampling. However, vaccinated pens had lower (P<0.01) overall prevalence of E. coli O157:H7 (model-adjusted mean ±SEM=17.4±3.95%) and lower (P<0.01) prevalence of high shedders (0.95±0.26%) than unvaccinated pens (37.0±6.32% and 4.19±0.81%, respectively). There was no evidence of a DFM effect on either measure of E. coli O157:H7 shedding. Results indicate that a two-dose regimen of the vaccine significantly reduces fecal prevalence of E. coli O157:H7 (vaccine efficacy of 53.0%) and prevalence of E. coli O157:H7 high shedders (vaccine efficacy of 77.3%) in commercial feedlot cattle reared in the summer on a finishing diet with 25% distiller's grains.     

Interlaboratory Pig-a gene mutation assay trial: Studies of 1,3-propane sultone with immunomagnetic enrichment of mutant erythrocytes

An international collaborative trial was established to systematically investigate the merits and limitations of a rat in vivo Pig-a gene mutation assay. The product of this gene is essential for anchoring CD59 to the plasma membrane, and mutations in this gene are identified by flow cytometric quantification of circulating erythrocytes without cell surface CD59 expression. Initial interlaboratory data from rats treated with several potent mutagens have been informative, but the time required for those flow cytometric analyses (～20 min per sample) limited the number of cells that could be interrogated for the mutant phenotype. Thus, it was desirable to establish a new higher throughput scoring approach before expanding the trial to include weak mutagens or nongenotoxicants. An immunomagnetic column separation method that dramatically increases analysis rates was therefore developed (Dertinger et al. [2011]: Mutat Res 721:163-170). To evaluate this new method for use in the international collaborative trial, studies were conducted to determine the mutagenic response of male Sprague Dawley rats treated for 3 or 28 consecutive days with several doses of 1,3-propane sultone (1,3-PS). Pig-a mutant frequencies were measured over a period of several weeks and were supplemented with another indicator of genetic toxicity, peripheral blood micronucleated reticulocyte (MN-RET) counts. 1,3-PS was found to increase Pig-a mutation and MN-RET frequencies in both 3- and 28-day study designs. While the greatest induction of MN-RETs was observed in the 3-day study, the highest Pig-a responses were found with 28-days of treatment. Pig-a measurements were acquired in approximately one-third the time required in the original method, while the number of erythrocyte and reticulocyte equivalents analyzed per sample were increased by factors of 100 and 10, respectively. The data strongly support the value of using the immunomagnetic separation technique for enumerating Pig-a mutation frequencies. These results also demonstrate that the ongoing international trial will benefit from the inclusion of studies that are based on both acute and protracted repeat dosing schedules in conjunction with the acquisition of longitudinal data, at least until more data have been accumulated.     

4种方法在检测痰内结核分枝杆菌结果的比较分析

目的对结核病临床诊断方法的应用价值进行比较分析。方法对别采用涂片法、免疫磁珠技术分离涂片法（IMS-涂片）、痰培养法、荧光定量聚合酶链反应（FQ-PCR）对病例组112例和对照组53例痰标本进行检测。结果普通涂片法灵敏度最低为25.00%,其次是IMS-涂片和痰培养分别是39.29%和37.50%,FQ-PCR法的灵敏度为67.86%,明显高于其他方法。结论 FQ-PCR法和IMS-涂片可作为普通抗酸染色法和培养法的重要补充或是互补的方法,对提高检出率,早发现、早治疗极具临床意义。     

免疫磁珠对肺腺癌小鼠模型micro-CT的增强作用

  目的  研究偶联单抗NJ001的免疫磁珠对肺腺癌裸鼠模型micro-CT扫描的增强作用。  方法  经尾静脉注射SPC-A1-luc细胞建立肺腺癌裸鼠模型,生物发光成像定量监测肿瘤的大小。裸鼠分为:生理盐水组、裸磁珠组和免疫磁珠组,每周分别注射生理盐水、750 nm裸磁珠溶液和偶联NJ001的750 nm免疫磁珠溶液,于注射前和注射后4 h进行micro-CT扫描。利用免疫组织化学验证肿瘤组织中NJ001特异性抗原SP70的表达。  结果  免疫磁珠组在第4周即可检测到肿瘤,而生理盐水组和裸磁珠组均到第6周才能检测到。第6周生理盐水组、裸磁珠组和免疫磁珠组micro-CT扫描肿瘤的灰度值分别为注射前的59.05±0.66、60.69±0.55和58.25±0.32,注射后的60.30±1.83、61.05±0.68和67.41±3.82。与注射前相比,免疫磁珠组注射后的灰度值显著增加,差异具有统计学意义（P=0.007 9）。生理盐水组和裸磁珠组注射后的灰度值与注射前相比差异均无统计学意义（均P＞0.05）。  结论  偶联单抗NJ001的免疫磁珠对肺腺癌裸鼠模型micro-CT扫描有增强作用,并且有望用于肺癌的早期诊断。      

免疫磁珠分离涂片和PCR检测痰内结核分枝杆菌的诊断价值研究

目的：探讨免疫磁珠分离涂片染色镜检法和免疫磁珠分离-PCR法对痰标本中结核分枝杆菌的诊断价值。方法：利用纯化的兔抗H37Rv血清包被磁珠,对痰中结核分枝杆菌进行分离,再进行涂片和PCR检测。结果：免疫磁珠分离涂片法比直接涂片法（χ2=40．83,P=0．039）、免疫磁珠分离-PCR法比常规PCR（χ2=23．31,P〈0．01）的灵敏度高。结论：免疫磁珠分离涂片法和磁珠分离-PCR法因操作简单、阳性率高,适宜在临床上推广使用。     

IMB联合SEM检测乳腺癌患者骨髓微转移的临床意义

目的：探讨免疫磁珠（immunomagnetic beads,IMB）联合扫描电镜（scanning electron microscope,SEM）检测乳腺癌患者骨髓微转移（bone marrow micrometastasis,BMM）的临床意义。方法：采用IMB富集联合SEM鉴定的方法检测106例乳腺癌患者骨髓有核细胞中上皮来源细胞的阳性发生率,分析其与原发肿瘤大小、临床分期、腋淋巴结转移、病理组织学分级、雌孕激素受体蛋白（ER、PR）表达及术后1年远端转移发生情况等常用病理学观测指标的关系。结果：106例乳腺癌骨髓标本中,37例检测到肿瘤细胞,骨髓微转移阳性率为34．9％;乳腺癌BMM阳性率与原发肿瘤大小正相关（P〈0．05）;临床分期越晚,出现BMM的概率也越高（P〈0．01）;BMM不仅与腋淋巴结是否转移有关（P〈0．05）,且随淋巴结转移数目增加,BMM阳性率亦增高（P〈0．05）;此外,乳腺癌BMM阳性率还与病理组织学分级正相关（P〈0．05）;与ER、PR蛋白在肿瘤组织中的表达负相关（P〈0．05）;与术后1年内患者是否发生远端转移正相关（P〈0．05）。结论：IMB联合SEM检测骨髓微转移是具有潜在应用价值的乳腺癌早期诊断方法和预后指标。