RESUMEN
Extracellular vesicles isolation from urine was severely interfered by polymeric Tamm-Harsefall protein due to its ability to entrap exosome. Studies had been reported to optimize the extraction of urine extracellular vesicles by using reducing agents, surfactants, salt precipitation or ultrafiltration, but rarely based on highly specific purification methods. We optimized the density gradient centrifugation method for the isolation of urinary small extracellular vesicles (sEV) and compared seven differential centrifugation protocols to obtain the high-yield and high-purity sEV isolation procedures. Our study showed Tris sucrose gradient centrifugation at 25°C had more concentrated distribution of exosomal marker in the gradient compared to Tris sucrose gradient centrifugation at 4°C and PBS sucrose gradient centrifugation. Dissolving the 16000 g pellet using Tris, Nonidet™ P 40 or Dithiothreitol then pooling the supernatants did not increase the exosomal markers and number of nanoparticles in sEV preparation compared to the control and PBS groups. Differential centrifugation at room temperature without ultrafiltration recovered more exosome-like vesicles, exosomal markers and nanoparticles than that at 4°C or combining ultrafiltration. Differential centrifugation at RT without ultrafiltration and salt precipitation recovered the highest number of nanoparticles than other protocols. However, differential centrifugation at RT combining 100 kd ultrafiltration obtained the highest purity of sEV calculated by Nanoparticle number/Total protein. In conclusion, we had established two urinary sEV isolation procedures that can recovered higher yield of sEV and more pure preparation of sEV. It is not recommended to treating 16000 g pellet with reducing agents or surfactants to increase the yield of sEV.
RESUMEN
A failure of bone marrow mesenchymal stem cells (BM-MSCs) to adhere to hematopoietic cells is an essential cause of the progression of chronic myelogenous leukemia and is also a cause of failure of bone marrow (BM) transplantation, but the exact mechanisms of this have not been fully elucidated. Recent studies have indicated that microRNAs (miRNAs) are contained in leukemia-derived exosomes and are involved in modulating the BM microenvironment. In this study, we found that K562 cell-derived exosomes transfer miR-711 to BM-MSCs and suppress the adhesive function of BM-MSCs. Using qRT-PCR, we also confirmed a significantly higher level of miR-711 in exosomes derived from K562â¯cells than in exosomes derived from parental cells. The BM-MSCs co-cultured with exosomes derived from K562â¯cells showed a lower adhesion rate than did controls. We further demonstrated that exosomal transfer of miR-711 induced decreased adhesive abilities by inhibiting expression of adhesion molecule CD44 in BM-MSCs. In conclusion, our study reveals that K562 cell-derived exosomal miR-711 can be transferred to BM-MSCs and weaken adhesive abilities by silencing the expression of the adhesion molecule CD44.
Asunto(s)
Adhesión Celular , Exosomas/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Células Madre Mesenquimatosas/metabolismo , MicroARNs/metabolismo , Células Cultivadas , Regulación hacia Abajo , Exosomas/genética , Exosomas/patología , Humanos , Receptores de Hialuranos/genética , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/patología , MicroARNs/genéticaRESUMEN
Association of chronic inflammation, primary tumor sidedness, adjuvant therapy and survival of metastatic colorectal cancer (mCRC) remains unclear. Circulating inflammatory cell, fibrinogen (Fib), albumin (Alb), pre-albumin (pAlb), Alb/Fib (AFR) and Fib/pAlb (FPR) were detected, and clinical outcome was obtained to determine the predictive, prognostic and monitoring roles of them in discovery and validation cohort. We found that elevated FPR, low AFR and poor survival was observed in right-sided mCRC comparing to the left-sided disease, elevated FPR harbored the highest areas under curve to independently predict poor progression-free survival and overall survival in overall and left-sided mCRC case in two cohorts. No survival difference was examined between the two-sided patients in subgroups stratified by FPR. Radiochemoresistance was observed in high FPR case. However, the patient could benefit from bevacizumab plus radiochemotherapy. Low FPR patient showed the best survival with treatment of palliative resection plus radiochemotherapy. Moreover, circulating FPR was significantly increased ahead imaging confirmed progression and it reached up to the highest value within three months before death. Additionally, c-indexes of the prognostic nomograms including FPR were significantly higher than those without it. These findings indicated that FPR was an effective and independent factor to predict progression, prognosis and to precisely identify the patient to receive optimal therapeutic regimen.