免疫磁性捕获ELISA法检测甘草中甘草蛋白的研究

目的 制备甘草蛋白免疫磁性微球，并建立快速、精确的免疫磁性捕获ELISA法检测甘草蛋白。方法 采用种子聚合法合成聚苯乙烯磁性微球，并以兔抗甘草蛋白IgG抗体致敏，制备特异性捕获甘草特征蛋白的免疫磁性微球。以生物素标记抗体为示踪抗体，结合辣根过氧化物酶标亲和素建立ELISA检测系统，用于甘草药材和含甘草中成药中甘草蛋白的分析。结果 利用该方法对甘草药材和中成药中甘草蛋白抗原检测，检测灵敏度达到10ng／mL。结论 免疫磁性捕获ELISA检测技术方便、快速、准确，为生药的品种鉴定及中成药的质量控制提供一种新方法。     

肿瘤微环境对树突细胞功能状态的影响

目的：探讨肿瘤细胞分泌的可溶性细胞因子营造的微环境对树突细胞(DCs，dendritic cells)的分化发育的影响，以进一步揭示肿瘤的免疫逃逸机制。方法：用免疫磁珠从人外周血分离CD14^ 单核细胞，加入粒细胞巨噬细胞集落刺激因子(GM-CSF)、白细胞介素4(IL-4)和人白血病细胞Jurkat培养上清液体外培养DCs，以正常培养诱导的DCs作为对照，采用傅立叶变换红外光谱(FTIR)技术分析人白血病细胞培养上清液对DCs分化发育的影响。结果：正常培养DCs与肿瘤上清液培养的DCs相比，在细胞内蛋白质和核酸的相对含量和细胞内消耗葡萄糖重新合成磷脂的量方面差异无显著性，但是，在细胞的转录状态方面差异有显著性。结论：肿瘤细胞上清液培养液所营造的微环境细胞转录水平上对DCs的功能状态有明显的抑制作用。     

Immunomagnetic enrichment and detection of isolated tumor cells in bone marrow of patients with epithelial malignancies

The detection of isolated tumor cells (ITC) in the bone marrow of patients with epithelial malignancies is an independant prognostic factor for several entities as breast cancer, colorectal cancer or non-small lung cancer. However, with conventional immunocytology using Ficoll density gradient and APAAP staining, only a small proportion of the bone marrow samples can be scanned for cytokeratin-positive (CK+) cells. To improve detection rates, we evaluated the enrichment of ITC by magnetic activated cell sorting (MACS) compared to regularly stained cytospins. Recovery experiments with a CK+ breast cancer cell line (SKBR3) were performed to calculate the MACS enrichment rate. Bone marrow was obtained by aspiration from 20 patients with carcinomas of epithelial origin and from 17 controls. ITC were enriched and stained with magnetically labeled CAM 5.2 antibodies directed to cytokeratin 7 and 8. MACS of SKBR3 seeded in peripheral blood revealed average recovery rates of 62% and 48% and average enrichment factors of 104-fold and 8139-fold of the CK+ cells after one and after two separations, respectively. After immunomagnetic enrichment, CK+ cells were detected in 16 of 20 (80%) cancer patients, whereas only 7 (35%) patients showed CK+ cells without magnetic enrichment (P=0.002). Ten of twelve (83%) patients with metastatic disease (stage M₁) and six of eight (75%) patients without any overt metastases (M₀) had CK+ cells in their bone marrow. None of the negative controls showed any CK+ cells. Enrichment with magnetically labeled anti cytokeratin antibodies increases the detection rate of epithelial cells in bone marrow of cancer patients compared to immunocytology. This revised version was published online in July 2006 with corrections to the Cover Date.     

小鼠肺纤维化模型中基质金属蛋白酶-9及其抑制剂表达水平的变化

目的 :研究小鼠博来霉素在肺纤维化模型中肺泡巨噬细胞(AM)分泌的基质金属蛋白酶 9(MMP 9)和基质金属蛋白酶抑制剂 1(TIMP 1)的表达随着时间的变化 ,观察肺纤维化中MMP 9和TIMP 1的表达是否存在失衡。方法 :以博来霉素诱导建立小鼠肺纤维化模型 ,采用HE染色观察肺部胶原沉积状况。选用抗CD6 8mAb ,采用微小免疫磁珠法分离纯化肺泡灌洗液中的AM ,用Hoechst 332 5 8染色AM ,在下光镜高倍视野计数法评估AM的纯度和活性 ,用EIA法检测AM上清液中MMP 9和TIMP 1的表达。结果 :肺组织切片HE染色显示小鼠肺部胶原沉积在 1、3、7、14、2 8d呈逐渐加重趋势。粗分离的AM纯度为 (82 .5± 2 .5 ) %。采用微小免疫磁珠法分离肺泡灌洗液中的AM纯度可达到 (99.3± 0 .7) % (P <0 .0 5 )。纯化后的细胞存活率为 (92 .5± 1.8) % ,与纯化前的 (92 .7± 2 .0 ) % ,相差不大 (P >0 .0 5 )。随着小鼠肺间质纤维化的进展 ,MMP 9的分泌量逐渐减少 ,而TIMP 1的表达量逐渐增加。结论 :在小鼠肺纤维化中AM分泌的MMP 9和TIMP 1之间存在失衡 ,表明基质降解系统受到抑制     

人外周血CD4~+树突状细胞的纯化及鉴定

目的　建立人外周血树突状细胞 (dendriticcell,DC)的分离方法 ,观察其形态学和免疫组织化学特点 ,为下一步细胞融合提供DC来源。方法　以免疫磁珠分选法从人外周血单个核细胞中分离CD4 + DC ,流式细胞仪检测所得细胞的纯度 ,光镜、电镜和激光共聚焦扫描显微镜观察其形态 ,SP免疫细胞化学方法检测DC的分子表达。结果　此纯化方法所得细胞纯度可达到 80 %以上 ,形态学观察可见纯化细胞具有典型的DC特征 ,该细胞能高表达HLA DR和S 10 0分子。结论　免疫磁法可获得较高纯度典型DC ,为进一步进行DC与肿瘤的融合实验及临床应用提供了可能     

肺癌肿瘤干细胞的分选及生物学特性

目的 探讨肺癌肿瘤干细胞分选及生物学特性的研究方法。方法 采用免疫磁珠分选经盐酸阿霉素(ADM)诱导的人肺腺癌SPC-A-1(SPC-A-1/ADM)细胞系中的ABCG2 ⁺和ABCG2 ^-细胞,流式细胞术和裸鼠体内移植针对ABCG2 ⁺和ABCG2 ^-细胞体内的成瘤率进行测定分析。结果 SPC-A-1细胞中的荧光强度平均为(1.001±0.014)×10 ²,明显低于SPC-A-1/ADM细胞的(10.257±0.023)×10 ²(t=17.320,P=0.001)。SPC-A-1细胞ABCG2/BCRP-FITC处理组与SPC-A-1细胞对照组(t=5.269,P=0.021)和SPC-A-1细胞同型对照组(t=6.869,P=0.012)间差异有统计学意义;SPC-A-1/ADM细胞对照组与SPC-A-1/ADM细胞同型对照组间差异有统计学意义(t=8.112,P=0.015)。SPC-A-1/ADM 细胞 ABCG2/BCRP-FITC 处理组阳性率为9.8%,是SPC-A-1细胞组的39.84倍。SPC-A-1/ADM 细胞 ABCG2/BCRP-FITC 处理组与SPC-A-1/ADM细胞组(t=9.120,P=0.005)和SPC-A-1/ADM细胞同型组(t=8.257,P=0.006)间差异有统计学意义。B 群细胞阳性率是 A 群细胞的 684 倍,差异有统计学意义(t=11.235,P=0.001),且 B 群细胞的荧光强度较强。接种SPC-A-1 细胞、A群细胞和B 群细胞的裸鼠平均成瘤体积分别为(6.96±1.82)、(6.70±2.55)和(9.17±2.41)mm ³,以接种B群细胞组最高,但3组间差异无统计学意义(F=2.362,P=0.086)。接种B群细胞组的成瘤率为75.00%,明显高于SPC-A-1 细胞组和A 群细胞组(F =19.780,P=0.002)。结论 ABCG2抗体与免疫磁珠分选法结合可以从人肺腺癌 SPC-A-1/ADM 细胞系内分离ABCG2 ⁺细胞,其在裸鼠体内成瘤情况良好,可用于肺癌肿瘤干细胞的分选及生物学特性研究。     

Site‐specific cytomorphology of disseminated PC‐3 prostate cancer cells visualized in vivo with fluorescent proteins

Circulating tumor cells (CTC) may reach multiple organ sites. However, CTC seeding and growth in distant organs is not random. Each metastatic site may contain a specific subpopulation of the original metastatic tumor capable of growing at that site. The fluorescent orthotopic prostate cancer model (PC‐3‐GFP) model was used for immunomagnetic capture of CTC. The captured CTC were efficiently cultivated in vitro. PC‐3‐GFP cells were also isolated from various metastatic sites, grown in vitro and examined under fluorescence microscopy. The differential morphology was compared of primary tumor cells, CTC and disseminated (DTC) from multiple metastatic sites, from nude mice with orthotopic PC‐3‐GFP. The cultured captured CTC and DTC from various organs have distinctive morphologies. Distinct cancer cell morphologies were observed at different metastatic sites as well as among CTC. The distinct morphologies were maintained during in vitro culture. The results demonstrate extensive tumor heterogeneity that could account for the widely different behavior of cancer cells in a single tumor. Further hetereogeneity testing would be a big promise for personalizing the cancer treatment in the future. Diagn. Cytopathol. 2013. © 2012 Wiley Periodicals, Inc.     

免疫磁珠分选兔前软骨干细胞及细胞研究平台的建立

[目的]研究兔前软骨干细胞PSCs,(precartilagious stem cells)的分离、培养方法及增殖、表型特征,为细胞移植治疗椎间盘退变,提供合适的种子细胞.[方法]取胎兔骨骺周围软骨膜(LaCroix环)中的细胞进行原代培养,然后采用免疫磁珠分离系统分选纯化PSCs.体外培养,传代,冻存复苏,绘制生长曲线,观察细胞特性.分别采用免疫组化、免疫荧光、RT-PCR等方法鉴定纯化后的PSCs.[结果]免疫磁珠分离可获得纯度较高的兔PSCs,培养后成活率高,细胞状态良好.细胞冻存复苏后,细胞增殖速度、细胞形态及表面标志物无明显变化.免疫组化、免疫荧光、RT-PCR等方法鉴定后,细胞都有明显的PSCs表面特异性标记物的表达.[结论]PSCs存在于LaCroix环中,免疫磁珠分离法得到的PSCs,体外培养条件下,细胞增殖较快,生物学特性稳定.     

Practical blood dendritic cell vaccination for immunotherapy of multiple myeloma

Therapeutic vaccination combined with new drugs may cure multiple myeloma (MM). We have developed a bio-process to purify CMRF-56 monoclonal antibody (mAb) and a standard operating procedure to immunoselect blood dendritic cells (BDC). Leucopheresed mononuclear cells were cultured overnight, labelled with CMRF-56 mAb and BDC prepared using a clinical scale immunoselection system. The mean BDC yield from healthy donors was 48% (n = 6, purity 28%). Preparations from MM patients (n = 6, yield 47%, purity 35%) primed cytotoxic T lymphocytes (CTL) to clinically relevant MM antigens. This procedure can be performed readily by clinical cell manufacturing units to facilitate BDC vaccination studies.     

成年大鼠纹状体神经干细胞体外提纯及检测

目的建立成年大鼠纹状体区神经干细胞的体外提纯方法,并进行纯度检测。方法将成年大鼠的纹状体组织制成单细胞悬液,通过磁性分离系统从细胞悬液中分离出神经干细胞,并以流式细胞仪检测其纯度。结果提纯而来的细胞经干细胞特异性Nestin荧光染色后在荧光显微镜下可见细胞呈圆形且发出绿色荧光,经流式细胞仪检测Nestin荧光染色阳性率87.5 % ±2.5 %。结论利用磁性分离技术可成功将成年大鼠纹状体组织中的神经干细胞提纯。