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Objective:To explore the regulatory role of exosomes in calcification of rat vascular smooth muscle cells (VSMC) induced by high phosphorus.Methods:VSMC (A7r5 cells) were cultured in vitro and randomly divided into three groups: normal phosphorus group (0.9 mmol/L), high phosphorus group (2.6 mmol/L) and high phosphorus exosomes induction group (i.e. the exosomes extracted from high phosphorus group were added to VSMC in normal culture). Until the 7th day of culture, the culture medium of normal phosphorus group and high phosphorus group obtained during the change of cell culture medium was collected, and the precipitate was obtained by ultracentrifugation and suspended by phosphate buffered saline. The protein content of the precipitate was determined by BCA protein quantitative method. The precipitates were identified. The structure and size of exosomes were observed by transmission electron microscope. The exosomes marker proteins tumor susceptibility gene 101 protein (TSG101) and CD9 were detected by Western blotting. The miRNA in exosomes was extracted, and the expression of related miRNA (miR-30b, miR-204, miR-211) were observed by real-time quantitative PCR. After 7 days of cultivation, the exosomes uptake process of VSMC in high phosphorus exosomes induction group was observed. The calcium deposition was detected by Alizarin stain, and the calcium content was detected by O-cresol complex copper. The content of alkaline phosphatase was detected by colorimetry. The protein expression of Runx2 was quantified by Western blotting. Results:(1) The precipitate obtained by ultracentrifugation of the cell culture fluid was identified as exosomes by electron microscopy morphology. Western blotting confirmed that the expression of the exosomal marker proteins TSG101 and CD9 were positive. (2) The exosomes were rich in miRNAs. The expression of miR-30b, miR-204, miR-211, which negatively regulated the transcription of Runx2, was significantly down-regulated in the high-phosphorus group compared with the normal group ( P<0.05). (3) After culturing rat VSMC with high phosphorus for 7 days, calcium salt deposition was obvious. Compared with the normal phosphorus group, calcium content and alkaline phosphatase activity were significantly increased (both P<0.05), and Runx2 expression was also significantly increased ( P<0.05). (4) Added the obtained high-phosphorus exosomes to the normal cultured VSMC, the exosomes could be taken up by VSMC and successfully induced VSMC calcification. The levels of cell calcification indicators and Runx2 expression were significantly increased. Conclusions:High phosphorus induces calcification of VSMC and promotes the increase of Runx2 expression. The mechanism may be realized by releasing exosomes from VSMC to transmit cell signals.
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Objective To evaluate the therapeutic effect and safety of bilirubin absorption(BA) combined with low volume plasma exchange(PE) in the treatment of severe hepatitis.Methods Forty-five inpatients with severe hepatitis in this hospital from January 1,2015 to December 31,2016 were performed the prospective study.All cases were given the therapy of BA combined with low volume PE.The indicators of liver function (ALT,AST,TBIL,CHE,ALB),coagulation function (PTA,INR),blood routine (WBC,Hb,PLT),electrolytes(K+,Na+,Cl-,Ca2+) and renal function(BUN,Cr) were collected before and after treatment.The changes of clinical symptoms and signs(weak,anorexia,abdominal distension,etc.) before and after treatment were recorded in all cases.The complications during the treatment process were also observed and recorded.The t-test was used for the inter-group comparison of the measurement data and the abnormal distribution adopted the Wilcoxon rank sum test.Results After the treatment of BA and low volume PE,the clinical symptoms of the patients were improved in different levels.The levels of ALT,AST and TBIL were decreased(P＜0.01),the CHE level was increased(P＜0.01),ALB level was decreased(P＜0.01);PTA was increased(P＜0.05),INR was decreased(P＜0.01);WBC,HGB and PLT were decreased(P＜0.05).Nineteen cases(31.1％) developed adverse reactions,which were recovered to normal after general symptomatic treatment.The treatment compliance was good without influence on artificial liver therapy.Conclusion BA combined low volume PE for treating severe hepatitis can significantly improve the liver function with safety and effectiveness.