ABSTRACT
Objective To explore the effects of ultrasound-exposed microbubbles (UM) on the expression of CXC chemokine receptor 4 (CXCR4) in bone marrow mesenchymal stem cells (BMSCs) and the mechanisms involved.Methods Mesenchymal stem cells were isolated from bone marrow taken from male Sprague-Dawley rats.They were divided into a control group,an ultrasound (US) group,an ultrasound-exposed microbubbles (UM) group,a UM plus catalase (UMC) group,a UM plus AMD3100 (UMA) group,and a UM plus anti-CXCR4 antibody (UMCX) group.The control group was not given any treatment.The US group was treated with 1 MHz ultrasound at 1 Watt per square centimetre for 30 seconds.The UM group was treated with ultrasound plus microbubbles.The UMC group was treated with catalase,microbubbles and ultrasound.The UMA group was treated with AMD3100,microbubbles and ultrasound.The UMCX group was treated with anti-CXCR4 antibody,microbubbles and ultrasound.Quantitative polymerase chain reaction (qPCR) and Western blotting were performed to determine the levels of CXCR4 mRNA transcription and the expression of BMSCs in the control,US,UM and UMC groups.Immediately,5 minutes and 15 minutes after the intervention,fluorescence intensities were observed in the cells labeled with Fluo-4/AM of the control group,US group and UM group under a fluorescence microscope.Migration assays were conducted to determine the chemotactic ability of the BMSCs with respect to stromal-derived factor-1α (SDF-1α) in all six groups.Results No significant differences were found in the levels of CXCR4 mRNA transcription and protein expression between the US and control groups(P＞0.05),but the levels in those groups and the UMC group were lower than those observed in the UM group.Fluorescence intensity in the cells of the US group was not significantly different from that in the control group (P＞0.05),but those levels were both significantly lower than that in the UM group (P＜0.05).There was no significant difference in the number of cells migrating to the SDF-1α between the US (22.4±2.2) and control group (20.5±2.3).However,the number of cells migrating to SDF-1α in the UM group (53.1±3.8) was significantly larger than that in the US group,the control group,the UMC group (35.2+3.1),the UMA group (32.5±2.8) and the UMCX group (30.7+2.9) (P＜ 0.05).Conclusion UM can increase mRNA transcription and the expression of CXCR4 protein in BMSCs,and promote BMSCs migration to SDF-lα.This may in part be mediated by an increase in calcium influx.
ABSTRACT
Objective To explore the effects of pretreatment of bone marrow mesenchymal stem cells (BMSCs) by ultrasound-exposed microbubbles (UM) on both homing to ischemic myocardium and cardiac function after acute myocardial infarction (AMI).Methods Rats of AMI model established by ligation of left anterior descending coronary artery were divided into four groups randomized:phospho-buffered saline (PBS) group,stem cells treatment (SCT) group,ultrasound and stem cells treatment (USCT) group,and UM stem cells treatment (UMSCT) group,and each group was injected with PBS,stem cells,US-pretreated stem cells and UM-pretreated stem cells through the caudal veins after AMI respectively.Homing of BMSCs to the ischemic myocardium was examined by confocal microscopy at 48 h after implantation,and cardiac function was examined by ultrasonic cardiogram (UCG) after 4 weeks.Masson staining was used to examine the changes of local ischemic cardiac tissues,and immunohistochemistry was used to detect the density of local neo-capillaries (CD31).Results 1) The numbers of CM-Dil-positive cells counted under confocal microscopy in the ischemic myocardial tissues of each groups 48 hours after implantation were not the same:there was no significant difference of the numbers of positive cells between USCT group (19.67 ±2.08) and SCT group (18.67 ± 2.08).However,the number of positive cells in the UMSCT group (39.33 ±3.06) was larger than that in USCT group and SCT group (P ＜0.05).2) UCG examinations showed that there was no significant difference of left ventricular systole function between the USCT group [LVEF (44.92 ± 2.77)％,LVFS (22.83 ± 1.79)％] and SCT group [LVEF (42.28 ± 2.82)％,LVFS (21.52 ±1.88) ％,P ＞0.05],but both were better than that in PBS group [LVEF (20.52 ± 1.88)％,LVFS (9.55 ±0.85) ％,P ＜0.05].The left ventricular systolic function in UMSCT group [LVEF (61.85 ± 3.15)％,LVFS (32.74± 2.45)％] was significantly higher than that in USCT group and SCT group (P ＜0.05),while which was still significantly lower than that in pseudo-surgery group [LVEF (75.88± 4.52)％,LVFS (42.76 ± 2.88)％,P ＜0.05].3) Pathological examinations showed the percentages of AMI areas in the USCT group (35.9 ± 1.1％) were not different compared with that in SCT group [(36.5 ± 1.3)％,P ＞0.05],while both were significantly smaller than that in PBS group [(45.2± 1.4)％,P ＜0.05].The percentages of AMI areas in the UMSCT group [(25.8 ± 1.0)％] were significantly smaller than that in USCT group and SCT group (P ＜0.05).The density of neo-capillaries (25.9 ± 1.3) in USCT groups had no difference compared with that in SCT group (25.2 ± 1.3),while both were significantly higher than that in PBS group (17.6 ± 1.1,P ＜0.05);the density of neo-capillaries (33.2 ± 1.6) was significantly higher in UMSCT group than that in both USCT group and SCT group (P ＜0.05),which were examined by immunohistochemistry.Conclusions Homing to ischemic myocardium of BMSCs transplanted intravenously could be promoted by UM pretreatment,which stimulates development of capillaries,reduces AMI areas,and improves the cardiac function after AMI.