Establishment Of Immortalized Umbilical Cord Mesenchymal Stem Cell Line Expressing SV40 LT And Extraction Of Its Secretions

Objective: In this study,SV40 LT plasmid was designed and constructed to transform human umbilical cord mesenchymal stem cells,and the immortalization function and atomization effect of exosomes on emphysema were studied and evaluated.Methods: The SV40 LT gene was encoded into the lentivirus vector by molecular cloning technology,and the recombinant vector was obtained.Then,the recombinant vector was packed with lentivirus,and the human umbilical cord mesenchymal stem cells infected by virus were obtained.The stable SV40 LT expression cell lines were obtained by hygromycin screening and monocloning.The surface markers CD29,CD73 and CD105 were detected by flow cytometry,and the changes of hematopoietic stem cell markers CD34 and CD45 were also detected.The stem cell characteristics were determined by osteogenic and lipogenic differentiation.The exosomes of primary and SV40-h UCMSCs were extracted by PEG centrifugation.The extracted exosomes were compared and analyzed by automatic exosome fluorescence detection and analysis system.To establish a beagle model of emphysema and observe the therapeutic effect of atomizing exosome administration.Results: 1.The lentivirus vector of SV40 LT was constructed successfully;2.The mesenchymal stem cells of human umbilical cord were successfully infected,and the immortalization function of the modified cells was verified: SV40-hucmscs were passed to 60 generations and the morphology of the original cells was consistent with that of the original cells(the original cells were passed to about 10 generations and lost their activity).Some of the original cells expressed CD34 but did not express CD45,while none of the SV40 cells expressed two kinds of antibodies.MSC markers CD29,CD73 and CD105 were expressed in both cells.SV40-h UCMSCs were successfully induced and differentiated into bone and lipoblast cells at 28 days.3.There was no significant difference between the exosomes extracted from primary cells and the modified SV40 mesenchymal stem cells(subgroup CD63+,CD81+,CD9+,the mean particle size of the CD63-positive exosomes was 69 nm,the CD81-positive exosomes was 69 nm,and the CD9-positive exosomes was 85nm)4.In the human emphysema Beagle model,there was no significant difference in the therapeutic efficacy of exosomes from primary mesenchymal stem cells,and both achieved good therapeutic efficacy after the 15 th round of administration.After 2 weeks of drug withdrawal,the RL of primary cells was 0.409 SV40-h UCMSCs and 0.308 RL,which still fluctuated within the range of normal animal RL.Conclusion: In this study,the immortalization of SV40 LT into the mass production of mesenchymal stem cells is possible.The unlimited expansion of mesenchymal stem cells facilitates the large-scale extraction of exosomes.At the same time,it was proved that atomizing drug delivery method had better effect on exosome treatment.It lays a good experimental basis for the treatment of lung diseases by mesenchymal exosomes in the future.