小鼠精原细胞分选方法改良的研究

目的:通过特异抗体结合的磁珠分选,改进小鼠未分化和分化中两类精原细胞分选方法。方法:利用Thy1和c-Kit两个特异抗体结合的磁珠分选技术,分别将出生后7 d雄性小鼠睾丸中的Thy1~+细胞和c-Kit~+细胞分别做为未分化和分化中的精原细胞分选出来,利用免疫荧光技术和流式细胞术,检测两种精原细胞的纯度,利用实时荧光定量PCR检测两种精原细胞中Gfrα1、Plzf、c-kit和sohlh2 mRNA的表达差异来鉴定两种精原细胞,并将Thy1~+细胞进行原代细胞培养。结果:Thy1~+细胞和c-Kit~+细胞的分选纯度分别达到(85.6±8.35)%和(89.40±2.77)%(P0.01)。Thy1~+细胞中Gfrα1和Plzf基因的相对表达量分别可达到c-Kit~+细胞的(9.47±1.29)、(4.40±0.59)倍,而在c-Kit~+细胞中kit和sohlh2基因的相对表达量分别可达到Thy1~+细胞的(7.38±1.07)、(3.88±0.28)倍(P0.01)。原代培养后,可见细胞状态良好,能够顺利的自我增殖,并具有精原干细胞增殖特征。结论:利用Thy1和c-Kit两个特异抗体结合的磁珠分选技术可以有效地将未分化和分化中的精原细胞分选出来,并将未分化的精原细胞进行体外培养。     

免疫磁珠分离技术纯化和检测泰泽氏病原体的研究

目的建立泰泽氏病原体(Ty)纯化方法,获得纯的菌体供抗原研究,为ELISA诊断抗原的制备提供依据;并尝试建立Ty隐性感染血清学抗原检查方法。方法选择特异性抗体包被磁珠,从感染大鼠肝脏中富集和纯化Ty;用SDS-PAGE和Western blot技术考察纯化Ty的蛋白和抗原图谱;同时用免疫磁珠分离技术(IMS)直接检查隐性感染大鼠肠道上皮细胞内的Ty。结果用辛酸-硫酸铵纯化的Ty单克隆抗体M5以0·5μg/107beads以上浓度包被抗IgG抗体预结合的磁珠4h,可最大效率地富集Ty;分离反应进行1h,敏感性达到103菌体/mL;吖啶黄染色镜检法可以直接、快速地观察到结合于磁珠上的细菌;抗原分析表明,IMS较好地去除了肝组织和真核细胞成分,纯化的Ty RJ株具有3个免疫优势的抗原成分,相对分子质量(Mr)分别为160×103、116×103、55×103;此外,IMS法可直接从隐性感染大鼠盲肠上皮细胞中检测到少量寄生的Ty。结论用IMS技术可有效地富集和纯化Ty,并可以作为Ty隐性感染血清学抗原检查的候选方法。     

使用抗体-生物纳米磁珠复合体的免疫凝集检测法

使用从磁性细菌体内提取的生物纳米磁珠，在磁珠表面联上特定的抗体，以玻片免疫凝集检测法检测癌胚抗原（carcinoembryonic AntigenCEA）。结果表明使用5μg的CEA抗体-磁珠复合体，可以检测出100pg／ml浓度的CEA，在操作性、经济性上优于免疫荧光检测法。     

脐血CD34^＋细胞免疫表型的分析

应用免疫磁珠法分离脐血ＣＤ３４＾＋细胞，以比较ＣＤ３４＾＋细胞亚群及各系相关抗原的变化。经过分离，ＣＤ３４＾＋细胞由１．４７±０．７７％升至５７．３９±１６．１７％，分离后ＣＤ３４＾＋ＣＤ３８＾－细胞占ＣＤ３４＾＋细胞的比率为７．８２±４．７９％ＣＤ３４＾＋。细胞纯化同时可减少ＣＤ３＾＋细胞达１４．１２±９．１８倍，ＣＤ１５＾＋细胞减少１６．４２±２１．４７倍；Ｂ系（ＣＤ１９）、巨核系（ＣＤ４１）     

人外周血CD68^＋单核细胞体外的分选及诱导分化为破骨细胞

OBJECTIVE: To establish a method for obtaining highly purified primary human osteoclast precursors for the biochemical and molecular biological research. METHODS: CD68(+) mono/macrophages were separated from peripheral blood mononuclear cells (PBMCs) of healthy donors by means of immunomagnetic cell sorting for subsequent analysis with flow cytometry. The isolated cells were incubated on coverslips or bone slices in the presence of dexamethasone(10(-8) mol/L), macrophage colony-stimulating factor (25 microg/L ) and soluble receptor activator of nuclear factor (NF)-kappaB ligand (s-RANKL, 16 microg/L). Calcitonin receptor (CR) immunocytochemistry and tartrate-resistant acid phosphatase (TRAP) histochemistry were employed. The bone slices were also studied by scanning electron microscopy (SEM). RESULTS: Fluorescence-activated cytometric analysis showed that 93.06%+/-0.61% n=4 of the selected cells were CD68(+) cells. After 7 days of culture of the CD68(+) cells, VR+, TRAP+ multinucleated giant cells appeared, and resorption lacunae could be observed by SEM. CONCLUSION: Highly purified CD68(+) cells can be obtained from human PBMCs as the osteoclast precursors, and mature osteoclasts can be induced from CD68(+) mono/macrophages by RANKL.     

重症肌无力患者造血干/祖细胞及其亚群富集纯化的研究

目的:获得高纯度造血干/祖细胞及其亚群,用于重症肌无力(MG)造血干细胞移植治疗和生物学特性的研究。方法:用免疫磁珠法(MiniMACS)从MG骨髓中富集CD34+细胞后再用荧光激活细胞分选(Fluorescent activatedcell sorter,FACS)纯化CD34+/CD38+和CD34+/CD38-细胞亚群。结果:MiniMACS分选的CD34+细胞群纯度达90％,FACS分选的CD34+/CD38-和CD34+/CD38+两细胞亚群纯度可达95％以上。结论:联合使用MiniMACS和FACS可迅速大量纯化HSC/HPC及其重要细胞亚群,可用于临床造血于细胞移植、基因治疗和研究HSC/HPC及其亚群生物学特性。     

双阴性选择法纯化培养人骨髓多能成体祖细胞

OBJECTIVE: To establish a method for purifying and culturing human multipotent adult progenitor cells (MAPCs) in vitro. METHODS: Mononuclear cells were separated from the bone marrow of healthy adult volunteers by density gradient centrifugation and cultivated in adherent culture. The plastic-adherent cultured bone marrow cells were isolated by magnetic-activated cell sorting with CD45 and GlyA magnetic microbeads, and the purity of CD45-/GlyA- cells evaluated by flow cytometry. Phase-contrast microscopy was used to detect morphological changes of the cells in different stages of culture. RESULTS: Approximately (5-10)x10(4)/ml MAPCs could be separated from every 1x10(6)/ml bone marrow mononuclear cells by magnetic- activated cell sorting. The viability of the cells before and after separation was (96.7+/-1.7)% and (96.0+/-2.4)%, respectively. The isolated MAPCs grew well in a self-prepared culture medium till the16th passage. The purity of CD45-/GlyA- separated from the bone marrow was more than 98% as examined by flow cytomety even till the 12th passage. CONCLUSIONS: MAPCs derived from adult human bone marrow can be purified by magnetic-activated cell sorting with CD45 and GlyA microbeads and retain the undifferentiated state for a long time. The self-prepared culture medium is appropriate for MAPCs cultures in vitro.     

免疫磁珠法分离白血病转基因小鼠骨髓造血干细胞

本文分离转基因小鼠骨髓造血干细胞。运用针对小鼠干细胞表面特异表达的干细胞抗原 1(stemcellantigen 1,Sca 1)的单克隆抗体和包被于磁颗粒表面的第二抗体 ,采用磁吸附细胞分选方法 (MACS )分离小鼠骨髓造血干细胞 ,用流式细胞术 (FACS )检测MACS分离后骨髓细胞中干细胞 (Sca 1阳性细胞 )比例 ,监测细胞分选效果。结果显示MACS分离后骨髓细胞中Sca 1阳性细胞所占比例达 85 %以上 ,涂片观察发现细胞组成和细胞形态学特征与FACS所得结果一致。MACS可以从转基因小鼠全骨髓细胞中分离出Sca 1阳性的骨髓造血干细胞 ,细胞纯度可达 85 %以上。     

磁分离酶联免疫荧光法(MEIF)检测人血清中胰岛素

目的:采用磁微粒分离酶联免疫荧光(MEIF)分析技术建立一种简便、高敏感性、定量检测人血清胰岛素(insulin)的新方法.方法:选用2株识别人胰岛素不同表位的单克隆抗体(mAb).一株mAb用异硫氰酸荧光素(FITC)标记, 另一株用碱性磷酸酶(AP)标记;偶联羊抗FITC抗体的磁珠用作固相分离载体, 4-甲基磷酸伞型酮用作荧光底物.结果:成功建立了定量检测人血清胰岛素的MEIF, 灵敏度2.0 μIu/mL, 线形范围0～188.52 μIu/mL, 批内变异系数(CV)为4.3%～5.2%, 批间CV为2.6%～9.5%, 稀释回收率为93%～117%, 加标回收率为94%～113%.实际样品的测定结果与倍爱康公司商品化人insulin磁分离发光系统检测试剂盒的检测结果比较, 具有良好的相关性.结论:MEIF定量测定人insulin方法成本低、灵敏度高、稳定性好, 在临床免疫检测领域具有广阔的应用前景.