Objective To specifically extract and analyze nascent proteins synthesized by bone marrow mesenchymal stem cells(BMSCs)after transplantation into ischemic hearts using a technique employing mutant methionyl-tRNA synthetase(MetRSL247G)for nascent protein labeling,in order to explore the potential mechanisms of action in BMSCs post-transplantation.Methods Point mutation at position 274 of the MetRS gene in BMSCs was induced via lentiviral infection to enable azidonorleucine(ANL)-mediated labeling of nascent proteins in BMSCs.The labeling efficiency was verified by means of fluorescent non-canonical amino-acid tagging(FUNCAT).Thirty healthy female C57BL/6J mice(8-10 weeks old)were divided into control and experimental groups,with 15 mice in each group.The acute myocardial infarction model was constructed by ligating the left anterior descending coronary artery in experimental group,while control mice underwent only thoracotomy without coronary ligation.After modeling,both groups received intramyocardial injections of MetRSL247G-modified BMSCs(MetRSL247G-BMSCs)at 3 different sites in the peri-infarct ischemic region.Mice were intraperitoneally injected with ANL every 6 hours for 4 times on postoperative days 0,2,and 6(n=5 for each time point)respectively,euthanized 24 h after the last injection,and cardiac tissues were isolated.The newly synthesized and labeled proteins produced by BMSCs after transplantation into the myocardium of experimental and control groups were collected,using an enrichment technique for ANL-tagged proteins and liquid chromatography-tandem mass spectrometry(LC-MS)analysis.Gene ontology(GO)analysis,Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis,protein-protein interaction(PPI)analysis,and heatmap visualization analysis were performed to identify differentially expressed proteins at the 3 time points and screen key pathways and genes.Results Under fluorescence microscopy,the MetRSL247G lentivirus-infected BMSCs were observed to be labelled with mCherry signals,confirming the successful construction of the MetRSL247G-BMSCs cell line.Green fluorescent signals were detected only in nascent proteins in culture medium containing both MetRSL247G-BMSCs and ANL,validating the sensitivity and specificity of the labeling method.GO analysis revealed that differentially expressed proteins were primarily involved in basic cellular biological processes such as extracellular exosome formation,extracellular matrix organization,and focal adhesion.KEGG and PPI analyses indicated that the differential proteins were mainly involved in complement and coagulation cascade pathway,actin cytoskeleton regulation pathway,and apoptosis pathway.Heatmap analysis showed significantly upregulated expression of anti-apoptosis and cell adhesion-related factors in experimental group on day 1(P＜0.05),upregulated anti-apoptotic factors,pro-apoptotic factors,and cell adhesion-related factors on day 3(P＜0.05),and upregulated anti-apoptotic factors,cell differentiation-related factors,and cell adhesion-related factors on day 7(P＜0.05)compared with control group.Expression of apoptosis-inducing factor 1 was significantly downregulated on days 1 and 7(P＜0.05).On day 3,most differentially expressed proteins,including anti-apoptosis factors(Protein S100-A11,Clusterin,Gelsolin),pro-apoptosis factor(Cathepsin B),cell differentiation-related factor(Transgelin-2),and cell adhesion-related factors(Cofilin-1,Periostin,Fibronectin)were significantly upregulated(P＜0.05).Conclusions The MetRSL247G mutation enables BMSCs to incorporate ANL and synthesize labeled proteins,confirming the feasibility of this nascent protein labeling technique.Nascent proteins of BMSCs in ischemic myocardium primarily contribute to extracellular exosome secretion and extracellular matrix organization.BMSCs may adapt to and respond to ischemic and hypoxic environments by influencing complement and coagulation cascades,activating inflammatory factors,regulating actin cytoskeleton structure,and modulating apoptosis,thereby maintaining the survival of BMSCs.

BACKGROUND AND OBJECTIVECT-guided microwave coagulation is a minimally invasive surgery for patients with liver cancer. Total intravenous anesthesia with propofol and fentanyl is commonly used. The depth of anesthesia during microwave coagulation for liver cancer is still monitored by clinical signs. There are few subjective and effective indicators. This study explored the application of Narcotrend-assisted "depth of anesthesia" monitoring on microwave coagulation for patients with liver cancer during total intravenous anesthesia with propofol and fentanyl.

METHODSForty liver cancer patients underwent CT-guided microwave coagulation were randomly assigned to receive Narcotrend index monitoring or standard clinical monitoring for depth of anesthesia with 20 patients in each group. All patients received total intravenous anesthesia with propofol and fentanyl. The depth of anesthesia for patients in the Narcotrend group was measured according to a Narcotrend index, which was maintained between D2 and E0. The depth of anesthesia for those in the standard clinical practice group was measured according to heart rate, mean arterial pressure, and patient movement. Changes of hemodynamics, the duration of the emergence from anesthesia, and the recovery of orientation were recorded. The doses of propofol and fentanyl, postoperative visual analogue scores (VAS), and the incidence of postoperative nausea and vomiting were also recorded.

RESULTSThere was no significant alteration in heart rate or mean arterial pressure between the two groups. Compared with other anesthetic stages, both heart rate and mean arterial pressure decreased during the induction of the anesthesia in the two groups(P<0.05). The doses of propofol were higher in the standard clinical practice group than in the Narcotrend group [(460+/-30) mg vs. (380+/-35) mg, P<0.01]. The duration of emergence and orientation were longer in the standard clinical practice group than in the Narcotrend group [(9.5+/-2.9) min vs. (4.9+/-2.2) min, P<0.01; (12.2+/-3.5) min vs. (6.6+/-3.2) min, P<0.01, respectively]. There was no difference in the dosage of fentanyl, VAS, or the incidence of postoperative nausea or vomiting between the two groups (P>0.05).

CONCLUSIONFor patients with liver cancer, monitoring the depth of anesthesia with Narcotrend on microwave coagulation can contribute to lower dosage of propofol and shorten duration of recovery during total intravenous anesthesia with propofol and fentanyl.

Adult ; Aged ; Anesthesia, Intravenous ; Anesthetics, Intravenous ; administration & dosage ; Electrocoagulation ; methods ; Fentanyl ; administration & dosage ; Hemodynamics ; Humans ; Liver Neoplasms ; surgery ; Male ; Microwaves ; Middle Aged ; Monitoring, Intraoperative ; instrumentation ; methods ; Propofol ; administration & dosage ; Tomography, X-Ray Computed

OBJECTIVETo investigate the anti-inflammatory effect of bone marrow stromal cells (MSCs) transfected with recombinant adenovirus-mediated ciliary neurotrophic factor (CNTF) gene in C57BL/6 mice with experimental allergic encephalomyelitis (EAE).

METHODSAn adenovirus vector containing CNTF gene Ad-CNTF-IRES-GFP was constructed and transfected in the MSCs (MSC-CNTF). After examination of CNTF expression, the transfected cells were transplanted in C57BL/6 mice with MOG 35-55-induced EAE, which were monitored for the changes in the symptoms scores. The levels of tumor necrosis factor-alpha (TNF-alpha), inteferon-gamma (IFN-gamma), interleukin-12P35 (IL-12P35), and IL-10 in the peripheral blood of the mice were detected, and the number of MSC-CNTF cells in the spleen and spinal cord was counted. CD3+ T cell infiltration and TNF-alpha and IFN-gamma expressions in the lesions were also observed after the cell transplantation.

RESULTSCNTF gene transfection resulted in significantly increased CNTF expression in the MSCs. The mice receiving MSC-CNTF transplantation exhibited significantly improved symptoms with shortened disease course and lessened disease severity. The cell transplantation also resulted in significantly decreased peripheral blood TNF-alpha levels, ameliorated CD3+T cell infiltrations and lowered TNF-alpha expression in the lesions, while the levels of IFN-gamma underwent no significant changes.

CONCLUSIONTransplantation of CNTF gene-transfected MSCs results in decreased peripheral blood TNF-alpha and IFN-gamma levels and reduced inflammatory cells, CD3-positive cells and TNF-alpha expression in the lesion of EAE, therefore providing better effect than MSCs in relieving the symptoms of EAE in mice.

Adenoviridae ; genetics ; metabolism ; Animals ; Bone Marrow Cells ; metabolism ; Ciliary Neurotrophic Factor ; biosynthesis ; genetics ; therapeutic use ; Encephalomyelitis, Autoimmune, Experimental ; therapy ; Female ; Genetic Therapy ; Interferon-gamma ; blood ; Mice ; Mice, Inbred C57BL ; Random Allocation ; Stromal Cells ; metabolism ; T-Lymphocytes ; immunology ; Transfection ; Tumor Necrosis Factor-alpha ; blood