Establishment of DCM rat model Male Sprague-Dawley rats (SYXK lu 20150003) aged 6 weeks were purchased from Jinan Pengyue Experimental Animal Breeding Co. (Jinan, Shandong, China), kept in the SPF animal feeding center of the Affiliated Hospital of Qingdao University the and obtain the approval of the Affiliated Hospital of Qingdao University Medical Ethics Committee. The rats in the DCM group were given intraperitoneal injection of Adriamycin (batch no. H33021980, 9 g/L saline diluted to 1g/L) of 1 mg/kg twice a week for 8 weeks, while the rats in the normal group were intraperitoneally injected with the same volume of saline 9 g/L twice a week for 8 weeks.Echocardiography was used to assess the success of the model. The rats were kept in comfortable environments, including comfortable cages, with appropriate temperature and light and free access to water and food.
During the modeling process, the rats were continuously observed.In the normal group, all the rats showed normal daily activities and normal growth without death incidents, while in the DCM group, 34 out of 40 rats (85%) survived for 8 weeks after modeling.In addition, the surviving rats showed loss of appetite, decreased exercise, slow reaction, slow growth, severe depilation and ascites.
Preparation of hucMSCs The umbilical cord was obtained from a healthy newborn baby immediately after birth with the full informed consent of its legal guardian and the approval of the Affiliated Hospital of Qingdao University Medical Ethics Committee.Screening was done for human immunodeficiency virus (HIV), hepatitis C virus (HCV), cytomegalovirus (CMV), hepatitis B virus (HBV) and treponema pallidum. Huatong glue from the umbilical cord was extracted and the organization block adherent method was used to cultivate hucMSCs, and subculture, in appropriate algebra cells cryopreserved in liquid nitrogen.
Extraction of hucMSC-Ex The 3rd generation hucMSCs with good growth status were resuscitated and screened for aerobic bacteria, mycoplasma, HIV, HBV, HCV, CMV and endotoxin. The morphology and immunophenoype of the cells were examined (CD34, CD44, CD45, CD146, CD105 and HLA-DR). The supernatants were collected after 48h of starvation, and the purified exosomes were obtained using ultra-high-speed centrifugation. Finally, the exosomes were resuspended by PBS.
Identification of hucMSC-Ex The morphology of the exosomes was observed using the transmission electron microscopy.Western blot was used to detect the exosomes’ specific markers CD63, CD81 and CD9 and the particle size of the exosomes was determined by NTA.
HucMSC-Ex treatment Two weeks after the establishment of the DCM rat model, the surviving DCM rats were randomly divided into two groups (17 in each group): the DCM control group that was injected with 2.0 mL PBS once a week, and the hucMSC-Ex group that was injected with 250ug/kg of hucMSC-Ex through the tail vein once a week.The general condition of the rats was observed 24 hours after the injection to observe the adverse effects. This initial treatment was followed for 4 weeks.
Echocardiography Before initiating the hucMSC-Ex injection and 2 weeks after the final treatment, the heart was explored by M-mode echocardiography using the Philips iE33 xMATRIX echocardiography system (Philips Healthcare, Amsterdam, Netherlands) that is equipped with 8-12 MHz ultrasound probe. The parameters related to the cardiac function were measured and calculated,including left ventricular inner systolic diameter (LVIDs), left ventricular inner diastolic diameter (LVIDd), left ventricular ejection function (LVEF) and left ventricular fractional shortening (LVFS).
Immunohistochemistry Two weeks after the final treatment, the rats were euthanized by intraperitoneal injection of a lethal dose of chloral hydrate solution (200mg/kg). The ventricular muscle tissues of the rats were then taken to be fixed with paraformaldehyde, embedded in paraffin, sliced, incubated with antibodies, stained and observed under an optical microscope (Olympus BX51, Tokyo, Japan).
Western blot After the rats were euthanized,we collect their ventricular muscle tissue.Western blot was used to detect their ventricular muscle tissueCOLI, Smad3 and a-SMA protein expression and calculate the relative expression quantity.
Real-time fluorescent quantitative PCR After collecting the ventricular muscle tissue of the euthanized rats,total RNA was extracted and then resuspended in DEPC-H2O.The PrimeScript RT reagent Kit with gDNA Eraser(Takara, Japan) was used to reverse transcribe the total extracted RNA to produce cDNA. Using the TB GreenTMPremix Ex TaqTMII(TliRNaseH Plus) (Takara, Roche cobasz480) for real-time quantitative cDNA, the quantitative process involved 95°C 30s for one cycle, 95°C 5s and 30s60°C for a total of 30 cycle.The GAPDH gene was used as the normalization control. The cycle number at the fluorescence threshold (CT) was used as an indicator of the expression level of the target gene. The expression levels in the normal controls were used as the comparative reference, and relative quantification of the expression in the study groups was determined by the 2-ΔΔCT values.The primers that were used in the process were designed and synthesized by the Huada gene company.
Electron microscope Two weeks after the final treatment, the rat myocardial tissue was fixed, embedded, sectioned and stained;the ultrastructure of the myocardium was then observed by the JEOL jem-1200ex transmission electron microscopy.
Statistical analysis Statistical analyses were performed using the SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). Data were expressed in the form of mean ± SEM. We performed one-way ANOVA in conjunction with the least significant difference test to determine the statistical significance (P<0.05).