Using the microfluidic disk system to isolate urinary podocytes as template to analyze proteinuric glomerular diseases

• Main Authors: Wai-San Wong, 黃蔚珊
• Other Authors: Tai-Ping Lee
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/84vf59

碩士 === 國立陽明大學 === 醫學生物技術暨檢驗學系 === 102 === Taiwan ranked first in the world in the prevalence of dialysis and the incidence ranked 4th. Nephritis, nephrotic syndrome and nephropathy ranked tenth in the top ten causes of death of Ministry of health and welfare in 2013. Two-thirds of chronic kidney diseases belong to glomerular diseases and often become into the end-stage renal diseases (ESRD). The symptoms contain proteinuria, edema, high cholesterol, low albumin. Diagnostic methods are based on urine and blood analysis, and confirmed with the tissue sections. Recent research indicates that impaired glomerular basement membrane causes the loss of specialized epithelial cells on glomerular, podocyte, in the urine. In addition, we could use the podocalyxin (PCX) to detect the podocyte. Compared to proteinuria, the number of podocytes in the urine could be as an early indicator of glomerular diseases. In this study, we used a microfluidic disk system to separate urinary podocytes. In addition, using the positive selection mainly capture podocalyxin positive cells. First, we presented a means of detecting urinary podocytes using microfluidic disk with a model cell system [Human pluripotent embryonal carcinoma cells (NCCIT cells)]. The spiked cells were stained Hoechst 33342 and anti-podocalyin-PE and finally confirmed by fluorescence microscopy. Under the volume of inlet reservoir 500 l, the detection rate reached 77.73% and recovery rate reached 54.97%. In order to prove the advantages of microfluidic disk for separating cells, the detection and recovery rate from the microfluidic disk with that from the BD™ IMag Cell Separation System were required to demonstrate. When spiking 25 to 1000 of NCCIT cells in a buffer solution, the detection rate of microfluidic disk was 83.84%. In addition, the recovery rate was at 46.08%. The system can compete with BD IMag Cell Separation System. Overall, the microfluidic disk (detection rate up to 94.67%) was more suitable than BD™ IMag Cell Separation System for separation in rare cells system. In addition, the microfluidic disk was used to determine the number of urinary podocytes in the patients with glomerular diseases and healthy people. About 3 ~ 2667 PCX+ cells/mL had been found in the patients. It was statistically significant (p = 0.01) between patients and healthy controls (about 0~172 cells/mL). We analyzed the urinary podocalyxin by ELISA kit. There was no significant difference between patients and healthy controls. According to the urine protein/creatinine ratio for patients’ clustering, podocyturia was more increased in the group of urine protein/creatinine ratio ≧1 than that of group of urine protein/creatinine ratio ≦1 (p = 0.02). These results suggested urinary podocyte may be an indicator of diagnosis criteria for glomerular diseases. Moreover, the urine protein/creatinine ratio was moderately strong correlated and statistically significant with the urinary podocyte (/mg.Cr) (r = 0.43, p = 0.02). Moreover, the negative correlation had been found between podocyturia (/mg.Cr) with urine total creatinine and CKD-EPI (r = -0.5, p = 0.005；r = -0.32, p = 0.08). At the same time, we analyzed the urinary podocyte sorted by the microfluidic system with the podocalyxin gene by direct PCR. Until now, no mutation has been found. We will try to find the single nucleotide polymorphism (SNP) or the somatic mutation with podocalyxin gene in glomerular diseases. From above data, we believe the novel microfluidic method and information from the study will bring the brand new dimension for non-invasive diagnosis and predict the progression of the glomerular diseases in the future.