Research On Paracrine Of Bone Marrow Mesenchymal Stem Cells By Click Chemistry

Cardiovascular disease is one of several diseases that seriously threaten human health.The treatment of bone marrow mesenchymal stem cells(BMSCs)after lower extremity ischemia and myocardial infarction has shown amazing results in angiogenesis and functional recovery,and may lead to revolutionary treatments method.At present,it is believed that mesenchymal stem cell therapy works mainly through its paracrine pathway,and the microenvironment of BMSCs in the organism makes it exhibit completely different biological effects.Therefore,if a certain technology can be used to screen the new protein status of BMSCs in the body,it will surely become a key to promote stem cell therapy and open the door to a new world for us.Click chemistry technology is based on the covalent bonding reaction of azide and alkyne groups.It has a very small molecular weight,mild reaction conditions,and rapid and efficient.The mutant methionyl-tRNA synthetase(MetRS)of specific cells produced by genetic engineering can incorporate the non-classical amino acid azidoleucine(ANL)instead of methionine(Met)in the translation process.Because methionine is the protein translated by most eukaryotic start codons,this allows ANL to be widely and comprehensively present in all newly synthesized proteins.In the next step,the alkyne group modification enrichment based on the click reaction can be used for mass spectrometry detection to accurately analyze the synthesis of new proteins in specific cell populations.Based on the previous research of the our team,we constructed the MetRS-MSCs cell line successfully.The ability of clcik technology to label MSCs nascent protein was verified,and it was found to be highly sensitive and specific.In this study,we explored the application of click technology on extracellular vesicles(EVs),and verified the feasibility of click technology on EVs through a series of optimized experimental designs.We also constructed a mouse lower extremity ischemia model.Through protein sorting and mass spectrometry analysis based on click technology,we observed the changes in the protein expression profile of BMSCs in the lower extremity ischemic tissue,and found that the first 7 days after the mouse lower extremity ischemia is the decision The key stage of the therapeutic effect of BMSCs,because during this period,the expression of apoptotic proteins represented by Atp5a1,Mdh2,and Sdhb dominates.In the following 7-14 days,energy metabolism-related proteins(such as Gm6096,Rpl34,Rpl4,Eef2,etc.)gradually increased,indicating that BMSCs mainly help their functional recovery and revascularization by changing the energy metabolism of themselves and ischemic tissues.In addition,BMSCs secrete a large amount of tissue remodeling proteins Mybpc2,Myl2,Myh4,and Mylpf during the repair process of ischemic injury.The increased expression of these structural proteins may be the key to their cytoprotective effect.Subsequently,EVs labeled with ANL were injected into a mouse animal model of myocardial infarction through the tail vein,and the distribution and uptake of MSC-EVs in the body under the state of myocardial infarction were observed.We found that exogenous MSC-EVs chemoattracted to the heart of mice with myocardial infarction.Further analysis revealed that MSC-EVs-related proteins appeared on the cell membranes of heart cells in the sham operation group,while in the myocardial infarction group they appeared in the cytoplasm.In part,it may be caused by the increase in membrane permeability.EVs-mediated transfer of DSG-1 to cardiomyocytes may be involved in the cardiac repair of MSC-EVs.The whole research,from the establishment of click technology to the test,from the observation of protein level to the exploration of EVsomal level,three-dimensionally explored the application value of click chemistry technology in the treatment of ischemic cardiovascular diseases with stem cells,which is a future stem cell therapy.The study of the mechanism provides a powerful weapon.