ABSTRACT
Objective To investigate the effects of monocytes on phenotypic changes of human proximal tubular HK-2 cells and the mechanism.Methods Monocytes were co-cultured with HK-2 cells.Morphological changes of HK-2 cells were detected by inverted phase contrast microscope.Expressions of E-cadherin,α-SMA and fibronectin were assessed by RT-PCR,Western blotting and immunocytochemical staining.Flow cytometry techniques was applied to evaluate intercellular cell adhesion molecule-1 (ICAM-1) expression on HK-2 cells.The intracellular signal was investigated by gene microarr ay.Results The typical epithelial cell morphology of HK-2 cells disappeared after co-culture with monocytes,accompanied by decreased E-cadherin expression and increased α-SMA and fibronectin expression (all P＜0.05).The expression of ICAM-1 on HK-2 cells was increased by monocytes stimulation.Interestingly,administration of CD18 antibody directly inhibited the phenotypic change of HK-2 cells.Furthermore,NF-κB signaling might be critical in mediating this process,and blockade of this signaling pathway could inhibit 1CAM-1 expression and epithelial mesenchymal transition (EMT) formation.Conclusion Monocytes can directly induce EMT of HK-2 cells via up-regulating ICAM-1 through NF-κB signaling pathway.
ABSTRACT
Objective To build an antibody microarray based on direct labeling strategy for microalbnrninuria measurement, and evaluate it's technical potentiality for clinical application. Methods Urine samples of diabetic patients were collected. Antibody microarrays were constructed by preparation of array support, array fabrication, then protein assay and data analysis were performed. Procedure conditions for each step especially the labeling of samples were optimized. The set-ups were evaluated in terms of sensitivity, specificity and reproducibility. Urinary albumin excretion in the samples was detected by fabricated protein array, which was compared to that detected with immunoturbidimetry. Results The signal intensity was best when protein quality ratio of pure albumin or urine sample against NHS-biotin was 2:1. A calibration curve with a correlation coefficient of 0.9995 was established. The lower limit of detection was 0.0617 mg/L. Interehip and intrachip variation studies conducted on patient urine demonstrated CVs as 6.78%-9.22% and 3.35%-7.59%, respectively. Compared with the immunoturbidimetry, the antibody microarray was able to detect the extremely lower grade albumin in urine samples. The correlation coefficient of the results obtained by the two methods was 0.9199 (P <0.01). Conclusion An antibody microarray based on direct labeling strategy for microalbuminuria measurement is successfully established, which is comparable to immunoturbidimctry in its accuracy and will have great potential for clinical use with its high throughput, sensitivity, specifity and reproducibility.