乳腺干细胞培养基的建立及有效性验证

背景：干细胞培养基,尤其是乳腺干细胞培养基现阶段尚无有效的制备方法。目的：利用Sca-1＋乳腺细胞验证自制乳腺干细胞培养基的有效性。方法：用BM培养基培养乳腺器官样小体,6 d后以免疫荧光方法监测成纤维细胞Sca-1和vimentin的表达。筛选出MaECM培养基,培养乳腺细胞6 d后,免疫磁珠筛选Sca-1＋和Sca-1-细胞群,流式细胞术分析Sca-1阳性细胞的纯度,1×10^4 Sca-1＋或Sca-1-细胞种植在4只鼠双侧乳腺脂肪垫上,6-8周后取出乳腺脂肪垫,以卡红整体染色和苏木精-伊红染色分析长出乳腺的结构数量。结果与结论：乳腺器官样小体用BM培养基培养6 d后,检测到大量Sca-1和vimentin阳性的成纤维细胞,说明BM培养基不适合分离Sca-1＋乳腺干细胞。筛选出的MaECM培养基能够抑制成纤维的生长。磁珠筛选后,流式细胞术检测Sca-1＋细胞在Sca-1＋和Sca-1-细胞群的纯度分别是92%和5%。移植实验显示在8个种植Sca-1＋细胞的脂肪垫生长出6个乳腺结构;而在8个种植Sca-1-细胞的脂肪垫中除1只鼠死亡,其余脂肪垫中均未长出乳腺结构。提示MaECM培养基适用于培养鼠乳腺干细胞。     

应用时间分辨荧光免疫分析技术检测SARS病毒抗原的初步研究

目的 建立定量检测SARS冠状病毒N蛋白的时间分辨荧光免疫分析(TRFIA)。方法 采用经SARS-CoVN蛋白和配对实验筛选的两株抗SARSN蛋白单克隆抗体,以双抗体夹心法为基础建立检测SARS-CoVN抗原的时间分辨荧光免疫分析技术,进行方法学的评价,并与相应ELISA试剂盒进行比较。结果 该法的测量范围为(0.02~150)ng/ml,灵敏度为0.02ng/ml;批内、批间CV分别为(3.3~6.2)%和(5.3~9.6)%,与采用ELISA试剂盒检测灭活SARS-CoVN蛋白情况比较的结果一致。结论 本研究建立的检测SARSN蛋白的时间分辨荧光免疫分析技术灵敏度高,特异性好,具有潜在的临床应用价值。     

Prognostic significance of urokinase plasminogen activator receptor and its cleaved forms in blood from patients with non‐small cell lung cancer

Urokinase plasminogen activator (uPA) cleaves its three‐domain cell surface receptor, uPAR, liberating domain I [uPAR(I)] and leaving the cleaved uPAR(II‐III) on the cell surface. Both intact and cleaved uPAR can be shed from the cell surface. uPAR(I) was previously shown to be a prognostic factor in lung tumour extracts. Here we analyse uPAR forms in blood from patients with non‐small cell lung cancer (NSCLC). Preoperatively sampled plasma/serum from 32 patients with NSCLC was analysed. Three time‐resolved fluoroimmunoassays (TR‐FIAs) measuring intact uPAR(I‐III) (TR‐FIA 1), uPAR(I‐III) + uPAR(II‐III) (TR‐FIA 2) and uPAR(I) (TR‐FIA 3) were applied. The Spearman rank correlations between plasma and serum levels of uPAR(I‐III), uPAR(I‐III) + uPAR(II‐III), and uPAR(I) were 0.89, 0.94 and 0.68 respectively. Survival analysis demonstrated that high levels of all uPAR forms were associated with shorter survival. Adjusted for histological subtype high plasma uPAR(I‐III) and uPAR(I) levels as well as serum uPAR(I) levels were significantly associated with shorter OS (hazards ratios = 4.3, 2.8 and 3.8 respectively). High blood levels of intact uPAR and its cleaved forms are associated with poor prognosis in NSCLC.     

免疫荧光法在研究人卵母细胞、受精卵及胚胎结构和功能中的应用

目的寻找适合研究人类生殖的免疫荧光方法。方法对蛋白激酶C(PKC)在人卵母细胞、受精卵、种植前胚胎及皮质颗粒进行免疫荧光染色。结果激光共聚集显微镜可以显示PKC在人GV、MI和MⅡ期卵母细胞及受精卵中的位置,并随着细胞的不同时期分布不同。在多精受精的胚胎中,免疫荧光显微镜可以清楚地显示PKC及细胞核在卵裂球的位置。使用A23187处理卵母细胞后,激光共聚集显微镜从表面可以观察到呈新月型点状分布的皮质颗粒。结论通过改进的免疫荧光法可以获得理想的实验结果。     

急性心肌梗塞患者肺炎衣原体感染的血清学研究

目的了解急性心肌梗塞病人肺炎衣原体感染程度。方法采用间接显微免疫荧光法检测３６例急性心肌梗塞病人和７３例健康献血员血清肺炎衣原体ＩｇＧ和ＩｇＭ滴度。结果急性心肌梗塞组肺炎衣原体既往感染率（８３．３％）明显高于对照组（３１．５％），两组急性感染率差别无显著性。结论肺炎衣原体既往感染在冠状动脉粥样硬化性心脏病和急性心肌梗塞发病中具有一定的影响，值得临床重视     

Development of a time-resolved fluoroimmunoassay for insulins and its application to monitoring of insulin secretion induced by feeding in the barfin flounder, Verasper moseri

A time-resolved fluoroimmunoassay (TR-FIA) system was developed to quantify insulin levels in the barfin flounder. This TR-FIA system is a solid-phase assay based on competition of unlabeled insulins and biotinylated barfin flounder insulin-II against an anti-barfin flounder insulin-II antibody. The minimum detectable level of barfin flounder insulin-I and -II in this TR-FIA was 10 pg/well which corresponded to 1.0 ng/ml, and insulin-II showed slightly higher crossreactivity than insulin-I. The accuracy of this TR-FIA was assured by specificity test, validation test, and recovery test using plasma added insulin-II. The results indicated the high specificity and sufficient accuracy of this assay system for insulin level measurement. This system was applied to the measurement of plasma insulin levels of fed and fasted barfin flounders. Plasma insulin levels (average +/- SEM) in fed flounders reached a maximum 2 h (9.3 +/- 1.7 ng/ml) and decreased gradually thereafter, while those in fasted flounders remained at low levels (1.1 +/- 0.1-2.0 +/- 0.2 ng/ml) during the experiment. After removing proteins by acidification and subsequent gel filtration, plasma samples taken from fed and fasted flounders at 2 h after feeding were fractionated separately by reversed-phase HPLC. In fed flounders, insulin immunoreactivity was detected in fractions corresponding to those of insulin-I or -II. The ratio of integrated insulin immunoreactivities of each peak was 0.378 +/- 0.044 (average +/- SD). This value was in good agreement with those (0.355 +/- 0.019) of absorbance areas of each insulin from Brockmann body extracts of the barfin flounder on reversed-phase HPLC. In fasted flounders, very weak insulin immunoreactivities were observed at retention times corresponding to those of insulin-I and -II. These results indicated that both insulin-I and -II were secreted into the blood being induced by feeding stimulation with approximately the same ratio as that of the quantities harbored in the Brockmann body.     

Prestin蛋白的稳定高表达卵巢细胞株的建立

目的:建立体外稳定高表达耳蜗外毛细胞马达蛋白(prestin)的多克隆中国仓鼠卵巢细胞株(CHO)。方法:采用prestin质粒转染CHO细胞进行表达,高浓度博来霉素400~800mg/L筛选出多克隆细胞株,传代培养,同时长时间维持低浓度抗生素(200~400mg/L)。收集所筛选的多克隆细胞株,提取总RNA,采用针对prestin蛋白不同片段设计的3对引物,进行逆转录-聚合酶链反应(RT-PCR),对扩增产物进行凝胶电泳分析。采用免疫荧光染色方法鉴定所筛选多克隆细胞株的prestin蛋白表达。结果:采用3对不同的prestin引物的RT-PCR结果均为阳性,而对照组则为阴性;利用prestin蛋白的特异性抗体,对所筛选的多克隆细胞株进行免疫荧光染色鉴定结果显示阳性率>90%。结论:成功获得稳定高表达prestin蛋白的多克隆CHO细胞株,转染表达率高。     

Development of highly specific fluorescence immunoassay and enzyme-linked immunosorbent assay for detection of dimethyl phthalate in water samples

A direct competitive fluorescence immunoassay (dc-FIA) and a direct competitive enzyme-linked immunosorbent assay (dc-ELISA) for the screening of dimethyl phthalate (DMP) in water samples were developed. The immunoassays utilise polyclonal antibodies against DMP raised in rabbits. The anti-DMP antibodies were linked to horseradish peroxidase (HRP) and fluorescein isothiocyanate (FITC). Under the optimal experimental conditions, the dc-ELISA has a linear working range of 0.1–2000 ng/ml (R ²=0.993) with a limit of detection of 0.09 ng/ml. In the dc-FIA, the linear working range was 0.05–30 ng/ml (R ²=0.996), and the limit of detection was 0.02 ng/ml, which is approximately four-fold more sensitive than the dc-ELISA using the same antibody and coating antigen. The results show low cross-reactivity with other structurally related compounds. The proposed methods are successfully applied to determine the DMP contaminants with a simple extraction procedure, and good recoveries were obtained.     

NSE时间分辨荧光免疫分析法的建立

采用夹心法建立神经元特异烯醇化酶（NSE）时间分辨荧光免疫分析技术（TRFIA）来测定血清中NSE的含量。以NSE单克隆抗体E1包被板,双功能螯合剂异氰酸苄基二乙烯三胺四乙酸络合Eu3＋及标记NSE单克隆抗体E7,发光增强系统为以β-二酮体为主的增强液。采用平衡饱和法建立NSE-TRFIA,数据采用双对数函数数据处理程序处理。本方法的连续批内和批间CV分别为2.4%和4.8%,平均回收率为102.6%,灵敏度为0.31ng/mL,可测范围为0.31～320ng/mL,ED20、ED50和ED80分别为20.72ng/mL、57.23ng/mL和157.25ng/mL。本方法与AFP、CEA均无交叉反应。Eu3＋标记抗体-20℃保存8个月免疫反应基本无损失,同批试剂连续8个月应用分析结果稳定。临床应用检测结果与E170所测值高度相关。实验结果表明,本文所建立的NSE-TRFIA的灵敏度、特异性、准确度等均符合临床应用要求。