Rensning og Transplantation af Myogenic stamfader celle afledte Exosomes for at forbedre hjertefunktion i Duchenne muskuløs dystrofe mus
Summary
Vi præsenterer her, en protokol for at forbedre forbigående hjertefunktion i Duchenne muskeldystrofi mus ved omplantning exosomes stammer fra normal myogenic stamceller.
Abstract
Duchene muskeldystrofi (DMD) er en X-linked recessive genetisk sygdom forårsaget af en mangel på funktionelle dystrofin protein. Sygdommen kan ikke helbredes, og som sygdommen skrider frem patienten udvikler symptomer på forstørrede kardiomyopati, arytmi og kongestiv hjerteinsufficiens. DMDMDX mutant-mus udtrykke ikke dystrofin, og er almindeligt anvendt som en musemodel af DMD. I vores undersøgelse for nylig, konstaterede vi, at intramyocardial injektion af bred type (WT)-myogenic Stamform celler-afledte exosomes (MPC-Exo) forbigående restaureret udtryk af dystrofin i myokardiet af DMDMDX mutant-mus, der var forbundet med en forbigående forbedring i hjertefunktion tyder på, at kan WT-MPC-Exo give en mulighed for at lindre de kardiale symptomer af DMD. Denne artikel beskriver teknik af MPC-Exo rensning og transplantation i hjerter af DMDMDX mutant mus.
Introduction
Duchenne muskeldystrofi (DMD) er en X-linked recessive, progressive neuromuskulær sygdom forårsaget af en mutation i DMD-genet og tabet af funktionelle dystrofin1. Dystrofin udtrykkes primært i skeletmuskulatur og myokardiet og er mindre udtrykt i glatte muskelceller, endokrine kirtler og neuroner2,3. DMD er den mest almindelige form for muskeldystrofi med en forekomst af en pr. 3.500 til 5.000 nyfødte drenge verden over4,5. Personer typisk udvikle progressiv muskel nekrose, tab af uafhængige walking af tidlig pubertet, og død i de anden til tredje årtier af deres liv på grund af hjertesvigt og respirationssvigt6.
Forstørrede kardiomyopati, arytmier og kongestiv hjerteinsufficiens er fælles hjerte-kar-manifestationer af DMD7,8. Sygdommen kan ikke helbredes, støttende behandling kan forbedre symptomer eller forsinke progression af hjertesvigt, men det er meget vanskeligt at forbedre hjerte funktion9,10.
Svarende til DMD patienter, X-linked muskeldystrofi (MDX) mus er mangelfuld i dystrophin protein og nuværende symptomer på kardiomyopati11, og er derfor meget udbredt i DMD forbundet kardiomyopati forskning. For at gendanne dystrofin i sygdomsramte muskler, allogen stamcelleterapi har vist sig for at være en effektiv behandling af DMD12,13,14. Exosomes, 30-150 nm membran vesikler udskilles af forskellige celletyper, spiller en central rolle i celle-til-celle kommunikation gennem genetisk materiale transport, såsom messenger RNA (mRNA) og ikke-kodende RNA’er15,16,17 ,18,19,20,21.
Vores tidligere undersøgelser har vist, at exosomes stammer fra myogenic stamceller (MPC), såsom C2C12 cellelinie, kan overføre dystrofin mRNA til værten cardiomyocytes efter direkte hjertets indsprøjtning22, der angiver at allogene levering af MPC-afledte exosomes (MPC-Exo) kan forbigående gendanne DMD genekspression i MDX-mus. Denne artikel fokuserer på MPC-Exo rensning og transplantation teknikker.
Protocol
Representative Results
Discussion
Metode til at isolere ren exosomes er afgørende for at studere funktionen af exosomes. En af de almindelige teknikker til exosome isolation er polyethylen glykoler (pinde) medieret nedbør17,18,25. Exosomes kan være i en pløkkerne, og pelleted ved lav hastighed centrifugering. PIND-medieret rensning er meget praktisk, billigt, behøver det ikke nogen avanceret udstyr, men der er bekymring om renheden af exosomes, da andre lip…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Tang blev delvist støttet af American Heart Association: GRNT31430008, NIH-AR070029, NIH-HL086555, NIH-HL134354.
Materials
| 0.22-μm Filter | Fisherbrand | 09-720-004 | |
| 15-cm Cell Culture Dish | Thermo Fisher Scientific | 157150 | |
| 24-gauge catheter | TERUMO | SR-OX2419CA | |
| 31-gauge insulin needle | BD | 328291 | |
| 4% paraformaldehyde | Affymetrix | AAJ19943K2 | |
| 50 mL Centrifuge Tubes | Thermo Fisher Scientific | 339652 | |
| 6-0 suture | Pro Advantage by NDC | P420697 | |
| Alexa Fluor 488 goat anti-rabbit IgG | Thermo Fisher Scientific | A-11008 | |
| Antibiotic Antimycotic Solution | Corning | 30-004-CI | |
| Anti-Dystrophin antibody | Abcam | ab15277 | |
| Antigen retriever | Aptum Biologics | R2100-US | Antigen recovery |
| Autofluorescence Quenching Kit | Vector Laboratories | SP-8400 | |
| C2C12 cell line | ATCC | CRL-1772 | |
| Centrifuge | Unico | C8606 | |
| Change-A-Tip High Temp Cauteries | Bovie Medical Corporation | HIT | |
| Confocal microscopy | Zeiss | Zeiss 780 Upright Confocal | |
| DBA/2J-mdx mice | The Jackson Laboratory | 013141 | |
| DMEM | Corning | 10-013-CM | |
| Fetal Bovine Serum (FBS) | Corning | 35-011-CV | |
| Goat serum | MP Biomedicals, LLC | 191356 | |
| Isoflurane | Patterson Veterinary | 07-893-1389 | |
| Ketamine | Henry Schein | 056344 | |
| Mounting Medium with DAPI | Vector Laboratories | H-1500 | |
| Mouse Retractor Set | Kent Scientific | SURGI-5001 | |
| Polyethylene glycol tert-octylphenyl ether | Fisher Scientific | BP151-100 | |
| Rodent ventilator | Harvard Apparatus | 55-7066 | |
| SW-28 Ti rotor | Beckman | 342207 | |
| The Vevo 2100 Imaging Platform | FUJIFILM VisualSonics | Vevo 2100 | Ultrasound System |
| Ultracentrifuge | Beckman | 365672 | |
| Ultra-Clear Tubes | Beckman | 344058 | |
| Xylazine (XylaMed) | Bimeda-MTC Animal Health Inc. | 1XYL003 8XYL006 |
References
- Yiu, E. M., Kornberg, A. J. Duchenne muscular dystrophy. Journal of Paediatrics and Child Health. 51 (8), 759-764 (2015).
- Nudel, U., et al. Duchenne muscular dystrophy gene product is not identical in muscle and brain. Nature. 337 (6202), 76-78 (1989).
- Rae, M. G., O’Malley, D. Cognitive dysfunction in Duchenne muscular dystrophy: a possible role for neuromodulatory immune molecules. Journal of Neurophysiology. 116 (3), 1304-1315 (2016).
- Mah, J. K., et al. A systematic review and meta-analysis on the epidemiology of Duchenne and Becker muscular dystrophy. Neuromuscular Disorders. 24 (6), 482-491 (2014).
- D’Amario, D., et al. A current approach to heart failure in Duchenne muscular dystrophy. Heart. 103 (22), 1770-1779 (2017).
- Koeks, Z., et al. Clinical Outcomes in Duchenne Muscular Dystrophy: A Study of 5345 Patients from the TREAT-NMD DMD Global Database. Journal of Neuromuscular Diseases. 4 (4), 293-306 (2017).
- Kamdar, F., Garry, D. J. Dystrophin-Deficient Cardiomyopathy. Journal of the American College of Cardiology. 67 (21), 2533-2546 (2016).
- Wang, Z., et al. Regenerative Therapy for Cardiomyopathies. Journal of Cardiovascular Translational Research. , (2018).
- Fayssoil, A., Nardi, O., Orlikowski, D., Annane, D. Cardiomyopathy in Duchenne muscular dystrophy: pathogenesis and therapeutics. Heart Failure Reviews. 15 (1), 103-107 (2010).
- Hagan, M., Ashraf, M., Kim, I. M., Weintraub, N. L., Tang, Y. Effective regeneration of dystrophic muscle using autologous iPSC-derived progenitors with CRISPR-Cas9 mediated precise correction. Medical Hypotheses. 110, 97-100 (2018).
- Quinlan, J. G., et al. Evolution of the mdx mouse cardiomyopathy: physiological and morphological findings. Neuromuscular Disorders. 14 (8-9), 491-496 (2004).
- Siemionow, M., et al. Creation of Dystrophin Expressing Chimeric Cells of Myoblast Origin as a Novel Stem Cell Based Therapy for Duchenne Muscular Dystrophy. Stem Cell Reviews and Reports. 14 (2), 189-199 (2018).
- Sienkiewicz, D., Kulak, W., Okurowska-Zawada, B., Paszko-Patej, G., Kawnik, K. Duchenne muscular dystrophy: current cell therapies. Therapeutic Advances in Neurological Disorders. 8 (4), 166-177 (2015).
- Zhang, Y., et al. Long-term engraftment of myogenic progenitors from adipose-derived stem cells and muscle regeneration in dystrophic mice. Human Molecular Genetics. 24 (21), 6029-6040 (2015).
- Ju, C., et al. Transplantation of Cardiac Mesenchymal Stem Cell-Derived Exosomes for Angiogenesis. Journal of Cardiovascular Translational Research. 11 (5), 429-437 (2018).
- Ju, C., et al. Transplantation of Cardiac Mesenchymal Stem Cell-Derived Exosomes Promotes Repair in Ischemic Myocardium. Journal of Cardiovascular Translational Research. 11 (5), 420-428 (2018).
- Ruan, X. F., et al. Suxiao Jiuxin pill promotes exosome secretion from mouse cardiac mesenchymal stem cells in vitro. Acta Pharmacologica Sinica. 39 (4), 569-578 (2018).
- Ruan, X. F., et al. Exosomes from Suxiao Jiuxin pill-treated cardiac mesenchymal stem cells decrease H3K27 demethylase UTX expression in mouse cardiomyocytes in vitro. Acta Pharmacologica Sinica. 39 (4), 579-586 (2018).
- Chen, Y., Tang, Y., Fan, G. C., Duan, D. D. Extracellular vesicles as novel biomarkers and pharmaceutic targets of diseases. Acta Pharmacologica Sinica. 39 (4), 499-500 (2018).
- Chen, Y., Tang, Y., Long, W., Zhang, C. Stem Cell-Released Microvesicles and Exosomes as Novel Biomarkers and Treatments of Diseases. Stem Cells International. 2016, 2417268 (2016).
- Murphy, C., et al. Emerging role of extracellular vesicles in musculoskeletal diseases. Molecular Aspects of Medicine. 60, 123-128 (2018).
- Su, X., et al. Exosome-Derived Dystrophin from Allograft Myogenic Progenitors Improves Cardiac Function in Duchenne Muscular Dystrophic Mice. Journal of Cardiovascular Translational Research. , (2018).
- Hu, G., et al. Exosome-mediated shuttling of microRNA-29 regulates HIV Tat and morphine-mediated neuronal dysfunction. Cell Death & Disease. 3, 381 (2012).
- Bayoumi, A. S., et al. A carvedilol-responsive microRNA, miR-125b-5p protects the heart from acute myocardial infarction by repressing pro-apoptotic bak1 and klf13 in cardiomyocytes. Journal of Molecular and Cellular Cardiology. 114, 72-82 (2018).
- Wang, Y., et al. Exosomes/microvesicles from induced pluripotent stem cells deliver cardioprotective miRNAs and prevent cardiomyocyte apoptosis in the ischemic myocardium. International Journal of Cardiology. 192, 61-69 (2015).
- Cheruvanky, A., et al. Rapid isolation of urinary exosomal biomarkers using a nanomembrane ultrafiltration concentrator. American Journal of Physiology-Renal Physiology. 292 (5), 1657-1661 (2007).
- Oksvold, M. P., Neurauter, A., Pedersen, K. W. Magnetic bead-based isolation of exosomes. Methods in Molecular Biology. 1218, 465-481 (2015).
- Pedersen, K. W., Kierulf, B., Neurauter, A. Specific and Generic Isolation of Extracellular Vesicles with Magnetic Beads. Methods in Molecular Biology. 1660, 65-87 (2017).
- Teng, X., et al. Mesenchymal Stem Cell-Derived Exosomes Improve the Microenvironment of Infarcted Myocardium Contributing to Angiogenesis and Anti-Inflammation. Cellular Physiology and Biochemistry. 37 (6), 2415-2424 (2015).
- Aminzadeh, M. A., et al. Exosome-Mediated Benefits of Cell Therapy in Mouse and Human Models of Duchenne Muscular Dystrophy. Stem Cell Reports. 10 (3), 942-955 (2018).
