Mechanism Of Ultrasound Microbubble-Enhanced Mesenchymal Stem Cells Homing To Treat Liver Fibrosis In Mice:A Pilot Study

Objective:To investigate the potential mechanism of UTMD technology to promote the homing of mesenchymal stem cells to fibrotic liver.Methods:Cryopreserved GFP-labeled mice bone marrow mesenchymal stem cells which provided by cellular central laboratory of 901 hospital of PLA were resuscitated and passaged into 25 cm~2.GFP-labeled mice bone marrow mesenchymal stem cells were divided into five groups with random assignment method:M group(Control group),M+U60s group(Ultrasound60s group),M+UTMD60s group,M+UTMD90s group,and M+UTMD180s group.The M group was left untreated.The ultrasound group was irradiated for 60 s at a frequency of 1 MHz and an output power of 0.6 W/cm~2.In the UTMD60s group,5μL of ultrasonic microbubbles were added to the same ultrasonic conditions for 60s.In the UTMD90s group,5μL of ultrasonic microbubbles were added to the same ultrasonic conditions for 90s.In the UTMD180s group,5μL of ultrasonic microbubbles were added to the same ultrasonic conditions for 180s.Flow cytometry,immunohistochemistry and western blot were used to detect the expression of CXCR4 on the surface of MSCs in each group.Trypan blue staining was used to detect the survival rates of cells in different treatment groups at 0,12,36 and 48 hours.Forty cirrhotic mouse models with successful modeling were randomly divided into group I,group II,group III and group IV,with 10 in each group.In group I,1 ml of normal cultured MSCs(1×10~6cells/1 ml)were injected through the tail vein;In group II,1ml MSCs treated with UTMD60s via tail vein;In group III,1 ml MSCs treated with UTMD60s were input through the tail vein,and the ultrasound intensity of 1W/cm~2and the frequency of 1MHz was applied to the mouse liver for 2 min;In group IV,1 ml of microbubbles was first input through the tail vein,and then 1 ml of MSCs treated with UTMD60s was immediately input.At the same time,the same ultrasound effect as in group III was given to the mice above the liver for 2min.After 48 hours,the mice were sacrificed by cervical dislocation,and then liver tissue was removed for HE staining to observe the liver tissue structure.Finally,the number of GFP-positive cells in each group was observed under a fluorescence microscope.Results:(1)Flow cytometry(FCM)revealed that the GFP expression level in BMSCs was approximately 97.21±0.19%.Additionally,FCM analysis indicated that the positive rates of MSCs-specific antigens CD29 and CD44was 97.2 and 97.8%;the specific antigens CD34 and CD45 of other cell lineage were not expressed in the MSCs,and the positive rate of these genes was 2.06 and 2.74%.(2)Flow cytometry detection showed that the percentage of cells expressing surface CXCR4 in the M+UTMD180s group(8.44±1.00%)was 1.22-fold higher than the M+UTMD60s group(6.91±0.36%)and 4.53-fold higher than the M+U60s group(1.86±0.37%)and8.79-fold higher than the M group(0.96±0.18%).The number of CXCR4-positive cells in the M+UTMD60s group was significantly higher compared with the M group and the M+U60s group(P<0.01).There was no significant difference in the number of CXCR4-positive cells between the M+UTMD60s group,the M+UTMD90s group and the M+UTMD180s group(P>0.05).(3)Immunohistochemical staining showed that CXCR4 was predominantly localized on the cell membrane and cytoplasm.The number of CXCR4-positive cells was relatively smaller in the M group and the M+U60s group.the percentage of cells expressing surface CXCR4 in the M+UTMD180s group(8.33±0.79%)was 1.09-fold higher than the M+UTMD60s group(7.61±0.98%)and 7.30-fold higher than the M+U60s group(1.14±0.36%)and 16.02-fold higher than the M group(0.52±0.32%).The number of CXCR4-positive cells in the M+UTMD60s group was significantly higher compared with the M group and the M+U60s group(P<0.01).There was no significant difference in the number of CXCR4-positive cells between the M+UTMD60s group,the M+UTMD90s group and the M+UTMD180s group(P>0.05).(4)Western blot results showed that the level of CXCR4 was higher in the M+UTMD60s group(0.92±0.09)compared to the M group(0.06±0.04)and the M+U60s group(0.12±0.04)(P<0.01)and that the M+UTMD180s group(1.04±0.09)had the highest levels compared to all other groups.(5)Trypan blue staining results showed that ultrasound treatment for 60s and UTMD treatment for 60s had no significant effect on cell viability.With the further increase of UTMD treatment time from 60 to 180s,cell viability decreases from 86.33±1.89%to 38.00±1.63%after 48 h of treatment.(6)Fluorescence microscopy images revealed that GFP-labeled MSCs were mostly localized in the manifold area and around the central vein.The results indicated that the number of GFP-positive cells in the group IV(31.9±4.58)was significantly increased compared to the group II(5.7±1.26)and the group III(6.4±1.45),and the number of GFP-positive cells in the group II(5.7±1.26)was significantly increased compared to the group I(1.7±1.09).Conclusion:(1)This study explored the potential mechanism of ultrasound-targeted microvesicle destruction(UTMD)to promote MSCs homing to fibrotic liver.Our results showed that UTMD can promote the homing to fibrotic liver by increasing the expression of CXCR4 on the surface of MSCs.(2)In addition,reasonable ultrasound treatment has no significant effect on the survival rate of MSCs,and the application of UTMD technology on the liver surface can have better results.This study enriches the treatment of liver fibrosis and has broad prospects for stem cell treatment of liver fibrosis.