临近闪烁分析技术在生命科学研究中的新进展

起源于放射免疫分析的临近闪烁分析法(scintillation proximity assay,SPA)是一种均相、灵敏、快速和简便的基于闪烁载体的分析平台.放射性标记分子和亲和标签分子的多样化和商业化,以及液闪计数器和液相操作等技术的发展,使得SPA操作具有无需分离、被测目标分子易于固定和检测的优势.SPA已成为高通量检测的重要方法,广范应用于筛选药物靶点及先导化合物筛选、药物作用机理研究,以及病毒、肿瘤标志物等抗原的高通量检测.本文在简要介绍SPA技术的原理及系统分析SPA操作中环境、条件对测试结果影响的基础上,比较全面地介绍了SPA技术在生物学研究中的应用,尤其是在膜蛋白和细胞水平上的应用进展,介绍了应用实例,归纳了国内研究人员开展相关技术应用与技术创新的制约因素,给出了未来的发展趋势.基于SPA的生物学应用技术创新必然提升我们对细胞体系生物学的全面理解.

Upregulated CD133 expression in tumorigenesis of colon cancer cells

AIM: To analyze the upregulated CD133 expression in tumorigenesis of primary colon cancer cells. METHODS: Upregulated CD133 expression in tumorigenesis of colorectal cancer cell lines (Lovo, Colo205, Caco-2, HCT116 and SW620) was analyzed by flow cytometry. Human colon cancer tissue samples were stained with anti-human CD133. SW620 cells were sorted according to the CD133 expression level measured by fluorescence-activated cell sorting. Spheroids of colorectal cancer cells were cultured with the hanging drop. Expression of CD133 and Lgr5 in spheroids of colorectal cancer cells and monolayer culture was detected by RT-qPCR. Spheroids of colorectal cancer cells were analyzed using anti-human CD133 with immunohistochemical staining. RESULTS: CD133 antigen was expressed in colorectal cancer cell lines (Lovo, Colo205, Caco-2, HCT116 and SW620) as well as in primary and metastatic human colon cancer tissues. However, the CD133 was differently expressed in these cell lines and tissues. The expression levels of CD133 and Lgr5 were significantly higher in spheroids of parental, CD133hi and CD133-cells than in their monolayer culture at the mRNA level (P < 0.05). Immunohistochemical staining of spheroids of CD133-cells showed that CD133 was highly expressed in colorectal cancer cell lines. CONCLUSION: Upregulated CD133 expression plays a role in tumorigenesis colorectal cancer cells, which may promote the expression of other critical genes that can drive tumorigenesis.

Morphology and ontogeny of dendritic cells in rats at different development periods

AIM： To study the morphology and ontogeny of dendritic cells of Peyer＇s patches in rats at different development periods. METHODS： The morphometric and flow cytometric analyses were performed to detect all the parameters of villous-crypts axis and the number of OX62＋DC, OX62＋CD4＋SIRP＋DC, and OX62＋CD4-SIRP-DC in the small intestine in different groups of rats. The relationship between the parameters of villous-axis and the number of DC and DC subtype were analyzed. RESULTS： All morphometric parameters changed significantly with the development of pups in the different age groups （F = 10.751, 12.374, 16.527, 5.291, 3.486; P = 0.000, 0.000, 0.000, 0.001, 0.015）. Villous height levels were unstable and increased from 115.24μm to 140.43 μm as early as 3 wk postpartum. Villous area increased significantly between 5 and 7 wk postpartum, peeked up to 13817.60 tam2 at 7 wk postpartum. Villous height and crypt depth ratios were relatively stable and increased significantly from 2.80 ＋ 1.01 to 4.54 =1= 1.56, 9-11 wk postpartum. The expression of OX62＋DC increased from 33.30%±5.80% to 80%± 17.30%, 3-11 wk postpartum （F =5.536, P = 0.0013）. OX62＋CD4＋SIRP＋DC subset levels detected in single-cell suspensions of rat total Peyer＇s patch dendritic cells （PP-DCs） increased significantly from 30.73% ± 5.16% to 35.50% ± 4.08%, 5-7 wk postpartum and from 34.20% ±1.35% to 43.60% ± 2.07% 9-11 wk postpartum （F = 7.216, P = 0.005）. CONCLUSION： This study confirms the agerelated changes in villous-crypt axis differentiation in the small intestine. Simultaneously, there are also development and maturation in rat PP-DCs phenotypic expression. Furthermore, the morphological changes of intestinal mucosa and the development of immune cells （especially DC） peaked at 9-11 wk postpartum, indicating that the intestinal mucosae reached a relatively mature state at 11 wk postpartum.

ZM-66, a New Podophyllotoxin Derivative Inhibits Proliferation and Induces Apoptosis in K562/ADM Cells

Objective To investigate the anti-tumor effect of ZM-66 on multidrug-resistant leukemic cell line K562/ADM. Methods The K562/ADM cells were treated with varying concentrations （0, 1, 2, 4×10^-3 mmol/L） of ZM-66 or etoposide for 24 hours. The proliferation was detected by Sulforhodamine B Sodium Salt （SRB） assay and apoptosis was detected by flow cytometry analysis and fluorescent staining. In addition, the expression levels of p53 and bax genes in K562/ADM cells were detected by RT-PCR analysis. The level of P-glycoprotein （P-gp）, P53 and Bax protein in K562/ADM cells were detected by Western blot assay. Results SRB assay demonstrated that etoposide had little inhibitory effect on K562/ADM cells, whereas ZM-66 （1, 2, 4×10^-3 mmol/L） had significantly inhibitory effect on K562/ADM cells （all P〈0.01）. The acridine orange/propidium iodide dual staining showed that there were typical condensation of chromatin and nuclear fragmentation nuclei with red color in ZM-66 treated cells. Flow cytometric analysis showed that there was a significantly increase of apoptotic cells i~ K562/KDM cells after treated with ZM-66. RT-PCR showed that the p53 and bax mRNA expression levels in K562/ADM cells treated with ZM-66 at 1, 2, 4×10^-3 mmol/L were higher than those in the cell without treatment. Western blot showed that the P53 and Bax protein expression levels in K562/ADM cells treated with ZM-66 at 2, 4x 10 s mmol/L were higher than those in the cell without treatment. But the P-gp protein expression level in K562/ADM cells treated with ZM-66 at 2, 4×10^-3 mmol/L was gradually lower than those in the cell without treatment. Conclusion ZM-66 is able to induce cell death by apoptosis in vitro, as a result of the reverse of theapoptosis resistance in drug-resistant K562/ADM cells by modulating expression of key factors associated with apoptosis induction.

Red blood cell level is increased in obese but not in non-obese patients with coronary heart disease

Objective To examine the changes of red blood cell levels in the obese and non-obese patients with coronary heart disease （CHD） and its clinical significance. Methods 230 cases of coronary heart disease were selected and divided into the obese group and the non- obese group. Obesity and non-obesity were defined based on the body mass index （BMI if 28.0kg/m2）, or waist-hip ratio （men〉 0.9, women〉 0.85）. In addition, 130 healthy subjects were recruited as controls. The pathological status of coronary lesions was quantita- tively analyzed according to the Coronary Vascular Image Segmentation Evaluation Criteria （American Heart Association 1984） and the Gensini scoring system. Results of the changes of both the hemoglobin levels and the red blood cell count in the obese group, the non- obese group with CHD and the control group were compared. Besides, Multivariant Logistic Regression Analysis was applied to assess the correlation between the red blood cells and the coronary artery disease. Results The red blood cell count and the level of hemoglobin in the obese group with CHD was higher than that in the non-obese group with CHD [（4.35 ± 0,55） and （4.13 ± 0.56） 10^9/L; （136.71± 15.87） and （129.96 ±16.23） g/L, P 〈 0.05 in both]; the proportion of acute coronary syndrome in the obese group with CHD was higher in the obese group with CI-/D than that in the non-obese group with CHD （P〈0.05）; Multivariant logistic regression analysis also showed that the red blood cell count was positively correlated with obesity with CHD.Conclusion The red blood cell count and the level of hemoglobin in the obese group were higher than those in the non-obese group; the increase of red blood cell count and hemoglobin level is one of the independent risk factors for the obese patients with CHD.

Study on the extra X chromosome＇s effect on the hormone level of Klinefelter＇s syndrome

Klinefelter＇s syndrome is an inherited （genetic） disorder found only in men caused by at least one extra X chromosome in a cell. Does the extra X chromosome have any effect on the hormone level of Klinefelter＇s Syndrome？ In this paper, 25 subjects with Klinefelter＇s syndrome, 30 infertile subjects and 36 normal men without Klinefelter＇s syndrome were compared each other in endocrinology profile and cytogenetics. Subjects with Klinefelter＇s syndrome were identified by the karyotypes 47, XXY or 47, XXY/46XY, and positive of the X-chromatins （Barr bodies）. Hormone analysis of subjects with Klinefelter＇s syndrome showed that the testosterone （T） values were lower than those of the normal subjects, while the FSH and LH values were higher than those of the normal people; in the infertile experiment subjects without Klinefelter＇s Syndrome, the karyotypes are 46, XY, with negative of the X-chromatins. The testosterone （T） values of these subjects were also lower than those of the normal people, but the FSH and LH values were within the normal range. These results indicated that endocrinological test on infertile subjects can be used to determine whether a cytogenetic analysis is necessary, and hence exclude non- Klinefelter＇s syndrome. The mechanism of the occurrence of this difference, its clinical applications and the relationship among the karyotypes, the endocrinological test and the severity of the phenotype are discussed. Lyon＇s hypothesis stating that only one of the two X-chromosomes is genetically active in female cells, but our study concluded that the extra X chromosome do have effect on the hormone level of Klinefelter＇s Syndrome.