| Objectives: 1.Research on exosome extraction methods: comprehensively compare the purity of exosomes extracted by ultracentrifugation and dialysis ultrafiltration,and screen out the extraction methods suitable for human umbilical cord mesenchymal stem cell-derived exosomes and use them for subsequent experiments.2.Preparation and characterization of exosomal liposomes: explore the characterization changes and fusion degrees of exosomal liposomes prepared by ultrasound method and thin film dispersion method with different amplitudes,and screen the best ultrasound amplitude.3.Study on the stability of exosomal liposomes prepared by different methods: explore the changes of physical stability and biological stability of exosomal liposomes prepared by ultrasound and film dispersion after short-term storage at room temperature(RT)/4°C,and screen out the preparation methods with better stability improvement effect.Methods: 1.Research on exosome extraction methods:(1)Exosomes in the supernatant of human umbilical cord mesenchymal stem cells were extracted by ultracentrifugation and dialysis ultrafiltration,respectively.(2)Nanoparticle size and zeta potential analyzer to detect particle size;BCA detection of total protein concentration;Western blot detected exosome-specific marker TSG101 and CD9.2.Preparation and characterization of exosomal liposomes:(1)Exosomal liposomes were prepared by ultrasound method(amplitude 20%,23%,25%,30%)and film dispersion method.(2)Nanoparticle size and zeta potential analyzer detect exosome liposome particle size and potential.(3)Western Blot detected the expression of exosomal liposomal TSG101 and CD9;Fluorescence resonance energy transfer(FRET)investigates the effects of exosomal liposome fusion.3.Study on the stability of exosome liposomes prepared by different methods: exosomes(EXO group),ultrasound preparation of exosomal liposomes(HC group),and membrane dispersion method of exosomal liposomes(HB group)were stored at room temperature and 4 °C for 1-7 days,and the particle size,polymer dispersity index(PDI)of the three groups of samples were detected by nanoparticle size and Zeta potential analyzer on the 1st,3rd,5th and7 th days.Zeta potential;CCK-8 was detected in the EXO,HC and HB groups that promoted the proliferation of human skin fibroblasts in the EXO,HC and HB groups when stored at room temperature for 1 day and at 4°C for 1 day,3 days and 5 days.Western Blot detected the expression of exosome-specific markers TSG101 and CD9 in the EXO,HC and HB groups stored at 4°C for 1,3 and 5 days.Results: 1.Research on exosome extraction methods:(1)The particle size of exosomes extracted by ultra-high-speed centrifugation was 169.03 nm,with only a single main peak;The particle size of exosomes extracted by dialysis ultrafiltration was35.13 nm,and there were heterogeneous peaks.(2)The concentration of total exosome protein extracted by dialysis ultrafiltration was significantly higher than that extracted by ultracentrifugation(p<0.05).(3)The exosomes extracted by both methods expressed CD9,and the expression level of exosomes extracted by ultracentrifugation was significantly higher than that of dialysis ultrafiltration(p<0.05);exosomes extracted by ultracentrifugation expressed TSG101,but TSG101 was not detected in exosomes extracted by dialysis ultrafiltration.2.Preparation and characterization of exosomal liposomes:(1)The particle size of exosomal liposomes prepared by film dispersion method increased,and the particle size of exosomal liposomes prepared by ultrasound method(amplitude 20%,23%,25%,30%)was similar to that of exosomes;The exosomal liposome potential prepared by ultrasound method(amplitude 23%,25%,30%)and thin film dispersion method is between exosomes and liposomes.(2)The exosomal liposomes prepared by ultrasound method(amplitude 20%,23%,25%,30%)and thin film dispersion method expressed TSG101 and CD9,and when the amplitude of ultrasound method was 30%,the expression level of CD9 was low.(3)FRET effect occurred in exosomal liposomes prepared by ultrasound method(amplitude 23%)and thin film dispersion method,while FRET effect occurred in the 20% amplitude group.3.Study on the stability of exosomal liposomes prepared by different methods:(1)Physical stability changes:(1)Particle size: The particle size change of the HC group was not obvious when stored at room temperature and 4 °C for 1-7 days,and the particle size change of the EXO group and HB group was more obvious than that of the HC group.(2)PDI: The PDI values of the EXO group were stored at room temperature for3 days and 4°C for 5 days were higher than 0.3,the PDI values of the HC group were always lower than 0.3 at room temperature and 4°C for 1-7 days,and the PDI values of the HB group were always higher than 0.3.(3)Zeta potential: The Zeta potential value of HC group and HB group stored for 1-7 days at room temperature and 4 °C is always higher than that of EXO group.(2)Changes in biological stability:(1)CCK-8 test results: On day 0,the EXO group,HC group and HB group all had the effect of promoting the proliferation of human skin fibroblasts(BJ).Left at room temperature for 1 day,none of the three groups of samples had a proliferative effect on BJ cells(p>0.05).Stored at 4 °C for 1,3,and 5 days,only the HC group had a proliferative effect on BJ cells(p<0.05).Compared with day 0,the proproliferation effect of BJ cells was weakened in the HC group stored at 4°C for 1,3 and 5 days(p<0.05),but there was no significant change in the pro-BJ cell proliferation effect between 1,3 and 5 days of storage at 4°C(p>0.05).(2)WB test results: the expression level of TSG101 in the EXO group was gradually reduced(p<0.05)when stored at 4°C for 3-5 days;the expression level of TSG101 in the HC group was stored at 4°C for 1-5 days without significant change(p>0.05);the expression level of TSG101 decreased(p<0.05)in the HB group when stored at 4°C for 5 days.CD9 expression levels decreased(p<0.05)in the EXO group when stored at 4°C for 1 and 5 days,CD9 expression levels decreased in the HC group when stored at 4°C for 5 days,and CD9 expression levels decreased in the HB group when stored at 4°C for 1-5 days(p<0.05).Conclusion(s): 1.Research on exosome extraction method: The purity of exosomes derived from cell supernatant extracted by ultracentrifugation method is better than that of dialysis ultrafiltration.2.Preparation and characterization of exosomal liposomes: both ultrasound(amplitude 23%)and film dispersion can fuse liposomes and exosomes,and both inherit exosome-specific marker TSG101 and CD9.3.Study on the stability of exosome liposomes prepared by different methods:(1)The physical stability and biological stability of exosomal liposomes prepared by ultrasound method were improved.(2)Ultrasound fusion treatment can improve the biological stability of exosomes stored at 4 °C for a short time. |
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