Objective To investigate the impact of iron overload on apoptosis in primary mouse liver cells via a synchronous separation technology.Methods Hepatocyte(HC),liver sinusoidal endothelial cell(LSEC),and Kupffer cell(KC)were isolated and purified with collagenase,percoll density gradient centrifugation,and CD146 magnetic beads.Cell types were identified using flow cytometry and immunofluorescence staining.Cells of different types were cultured in vitro,and an iron overload model was established by treating the mice with 0,25,50 and 100 μmol/L ferric ammonium citrate(FAC)for 24 h.The iron content was quantified using Prussian blue staining,while cell viability and mitochondrial membrane potential were assessed by flow cytometry.Results The synchronous separation technology of primary liver cells exhibited stable efficiency.The yield of HC was(4.0±0.5)×107 cells per mouse,exhibiting an effective survival rate of(76.33±0.67)％.The yield of LSEC was(5.0±1.0)×106 cells per mouse,with a survival rate of(93.63±0.25)％and a purity level of(93.40±0.46)％.The yield of KC was(1.5±0.5)×106 cells per mouse while a high survival rate of(98.33±0.12)％and a purity level of(88.30±2.02)％were maintained.The obtained cells were large in number,with good vitality and high purity,which could meet the requirements of subsequent experiments.Treatment with FAC significantly elevated iron contents in different types of cells when compared with the control group.Upon stimulation of FAC,the survival rate of HC decreased from(73.97±5.54)％to(54.10±1.68)％,the mean fluorescence intensity of JC-1 aggregates decreased from 326.33±30.37 to 155.00±6.56,JC-1 monomer increased from 1700.00±1 44.04 to 3713.33±81.82.The survival rate of LSEC decreased from(90.60±1.74)％to(78.03±2.15)％,the mean fluorescence intensity of JC-1 aggregates decreased from 502.33±5.51 to 372.33±4.04,and JC-1 monomer increased from 750.00±67.51 to 1340.00±36.39.The survival rate of KC decreased from(94.23±1.44)％to(88.37±1.56)％,the mean fluorescence intensity of JC-1 aggregates decreased from 652.67±25.66 to 478.00±12.49,and JC-1 monomer increased from 1984.33±80.65 to 3062.33±245.20.Conclusion A robust and reliable simultaneous isolation technique of primary mouse HC,LSEC,and KC has been established.Moreover,our finding demonstrates that iron overload significantly enhances apoptosis levels in HC,LSEC and KC.

BACKGROUND:Due to the sudden release and the rapid removal by proteases,platelet-rich plasma hydrogel leads to shorter residence times of growth factors at the wound site.In recent years,researchers have focused on the use of hydrogels to encapsulate platelet-rich plasma in order to improve the deficiency of platelet-rich plasma hydrogels. OBJECTIVE:To prepare self-assembled polypeptide-platelet-rich plasma hydrogel and to explore its effects on the release of bioactive factors of platelet-rich plasma. METHODS:The self-assembled polypeptide was synthesized by the solid-phase synthesis method,and the solution was prepared by D-PBS.Hydrogels were prepared by mixing different volumes of polypeptide solutions with platelet-rich plasma and calcium chloride/thrombin solutions,so that the final mass fraction of polypeptides in the system was 0.1%,0.3%,and 0.5%,respectively.The hydrogel state was observed,and the release of growth factors in platelet-rich plasma was detected in vitro.The polypeptide self-assembly was stimulated by mixing 1%polypeptide solution with 1%human serum albumin solution,so that the final mass fraction of the polypeptide was 0.1%,0.3%,and 0.5%,respectively.The flow state of the liquid was observed,and the rheological mechanical properties of the self-assembled polypeptide were tested.The microstructure of polypeptide(mass fraction of 0.1%and 0.001%)-human serum albumin solution was observed by scanning electron microscope and transmission electron microscope. RESULTS AND CONCLUSION:(1)Hydrogels could be formed between different volumes of polypeptide solution and platelet-rich plasma.Compared with platelet-rich plasma hydrogels,0.1%and 0.3%polypeptide-platelet-rich plasma hydrogels could alleviate the sudden release of epidermal growth factor and vascular endothelial growth factor,and extend the release time to 48 hours.(2)After the addition of human serum albumin,the 0.1%polypeptide group still exhibited a flowing liquid,the 0.3%polypeptide group was semi-liquid,and the 0.5%polypeptide group stimulated self-assembly to form hydrogel.It was determined that human serum albumin in platelet-rich plasma could stimulate the self-assembly of polypeptides.With the increase of the mass fraction of the polypeptide,the higher the storage modulus of the self-assembled polypeptide,the easier it was to form glue.(3)Transmission electron microscopy exhibited that the polypeptide nanofibers were short and disordered before the addition of human serum albumin.After the addition of human serum albumin,the polypeptide nanofibers became significantly longer and cross-linked into bundles,forming a dense fiber network structure.Under a scanning electron microscope,the polypeptides displayed a disordered lamellar structure before adding human serum albumin.After the addition of human serum albumin,the polypeptides self-assembled into cross-linked and densely arranged porous structures.(4)In conclusion,the novel polypeptide can self-assemble triggered by platelet-rich plasma and the self-assembly effect can be accurately adjusted according to the ratio of human serum albumin to polypeptide.This polypeptide has a sustained release effect on the growth factors of platelet-rich plasma,which can be used as a new biomaterial for tissue repair.

Platelets transfusion is one of the most important methods to prevent and treat hemorrhagic diseases.At present, the vitro storage of platelets with gentle agitation at (22±2)℃ is short and susceptible to bacterial contamination, which greatly affects the availability and safety of clinical platelets transfusion.Studies at home and abroad have shown that storage at 4°C could prolong the preservation period of platelets.However, refrigeration may aggravate platelets storage lesion (PSL) and accelerate the clearance mechanism after transfusion, which seriously restricts the clinical application of refrigerated platelets.This paper reviews the research advances in PSL caused by 4℃ storage and the clearance mechanism after transfusion.

【Objective】 To explore the main development directions and research progress of blood transfusion in the U. S.military based on ClinicalTrials.gov. 【Methods】 The literature of clinical trials related to field transfusion medicine from 2002 to 2021 were retrieved based on www.clinicaltrials.gov with "United States Department of Defense" , "U.S.Army Medical Research and Development Command" and "United States Naval Medical Research Center" as the search terms, and the main characteristics were analyzed in terms of research progress, clinical stage, task allocation, regional distribution, research contents and results. 【Results】 A total of 931 clinical trials were retrieved, of which 16 were closely related to field blood transfusion medicine.Among them, pathogen reduction/inactivation of whole blood, platelet transfusion, plasma transfusion, whole blood transfusion and other types of researches accounted for 25% (4/16), 25% (4/16), 25% (4/16), 18.75 (3/16) and 6.25% (1/16), respectively.The U. S.military focused on the new technological breakthroughs in blood safety and blood storage, the evaluation of the countershock therapy, the application of whole blood early transfusion and the civil-military inosculation in research. 【Conclusion】 This study, based on ClinicalTrials.gov data, redounds to understand the research progress and current situation of the U. S.military field blood transfusion medicine.