Objective To observe the impact of collagen patches using 1-ethyl-3- （3-dimethylaminopropyl) carbod-iimide hydrochloride chemistry （EDC） to conjugate vascular endothelial growth factor （VEGF） ＋ basic fibroblast growth factor （bFGF） or VEGF alone on the survival rate of transplanted human bone morrow mesenchymal stem cells （hBM-MSCs）in vitro and in vivo. Methods Collagen patches which were activated by EDC were used as the control group，and EDC activated collagen patches that were conjugated with VEGF or VEGF ＋ bFGF were used as the experiment groups（VEGF group and VEGF ＋ bFGF group）. hBM-MSCs （0.5×106/patch） were used as seeding cells to construct engineered heart tissue （EHT）. MTT assay was performed to assess in vitro proliferation of hBM-MSCs on 3 different collagen patches. Ventricular aneurysm model after myocardial infarction was created by left anterior descending artery （LAD） ligation in male SD rats，and EHT which were constructed with 3 different patches were used for ventricular plasty. Four weeks later，immunofluorescence staining was used to examine arteriole density （anti-α-SMA staining） and transplanted cell survival （anti-h-mitochondria staining）. Results （1） hMSCs proliferation in VEGF group and VEGF ＋ bFGF group was significantly better than that in the control group on the 2nd and 4th day after cell transplantation （P＜0.05）； （2） Four weeks afterEHT implantation，immunofluorescence staining for α-SMA revealed that arteriole density of VEGF group and VEGF ＋ bFGF group was significantly higher than that of the control group （P＜0.05）； （3） Immunofluorescence staining forh-mitochondria showed that survival rates of transplanted hBM-MSCs of VEGF group and VEGF ＋ bFGF group were significantly higher than that of the control group （P＜0.05）； （4） There was a significantly positive correlation between survival rate of hBM-MSCs and arteriole density （r 2=0.99，P=0.02）. Conclusion VEGF or VEGF ＋ bFGF conjugated collagen patch can significantly improve hBM-MSCs proliferation in vitro and enhance survival rate of transplanted hBM-MSCs by accelerating revascularization of EHT in vivo.
ObjectiveTo construct a cationic microbubble (CMB), and investigate the enhancement of gene transfection efficiency and therapeutic effect of ultrasound-targeted microbubble destruction (UTMD) in vivo with CMB compared to definity MB (DMB).Methods In vitro, the CMB was prepared by the method of thin film hydration. The morphology, size, zeta potential, and gene-carrying capacity of CMB were compared with the DMB. In vivo, the firefly luciferase gene which was used as a reporter gene was targeted transfected into myocardium of 16 rats with CMB and DMB, respectively. The gene transfection efficiency and targeting were observed dynamically. Then, ischemia-reperfusion (I/R) model was performed on 64 rats. The models of 60 rats were successfully confirmed by using ultrasonography at 5 days after I/R. The rats were divided into 3 groups (n=20) randomly. The control group received DMB carrying empty plasmid for transfection; DMB group received DMB carrying AKT plasmid for transfection; and CMB group received CMB carrying AKT plasmid for transfection. The cardiac perfusion, cardiac function, infarct size, and infarct thickness were measured by ultrasonography and histological observations after treatment. In addition, the capillary and arteriolar densities were measured with immunohistochemical staining. The myocyte apoptosis was measured with TUNEL staining. The protein expressions of AKT, phospho-AKT (P-AKT), Survivin, and phospho-BAD (P-BAD) were measured by Western blot.ResultsThe size of CMB was uniformly. The zeta potential of CMB was significantly higher than that of DMB (t=28.680, P=0.000). The CMB bound more plasmid DNA than the DMB (P<0.05). The luciferase activity of myocardium were higher in CMB group than in DMB group bothin vitro and in vivo measurements (P<0.05). There was no significant difference between groups in the ratio of signal intensity in anterior wall to posterior wall, ejection fraction (EF), and fractional shortening (FS) at 5 days after I/R (P>0.05), but the above indexes were significant higher in CMB and DMB groups than in control group at 21 days after I/R (P<0.05). Besides, the above indexes were significant higher in CMB group than in DMB group at 21 days after I/R (P<0.05). The infarct size was the smallest and infarct thickness was the thickest in the CMB group, followed by DMB group, control group at 21 days after I/R. The capillary and arteriolar densities of CMB and DMB groups were significant higher than those of control group at 21 days after I/R (P<0.05). Besides, the capillary and arteriolar densities of CMB group were significant higher than those of DMB group (P<0.05). The apoptotic cells were the most in the control group, followed by DMB group, CMB group at 3 days after gene transfection, showing significant differences between groups (P<0.05). The protein expressions of AKT, P-AKT, Survivin, and P-BAD were significant higher in CMB and DMB groups than those in control group at 3 days after gene transfection (P<0.05). Besides, these protein expressions were significant higher in CMB group than those in DMB group (P<0.05).ConclusionThe DNA-carrying capacity and gene transfection efficiency are elevated by CMB, although its physicochemical property is the same as DMB. When ultrasound-targeted AKT gene transfection is used to treat myocardial I/R injury in rats, delivery of AKT with the CMB can result in higher transfection efficiency and greater cardiac functional improvements compared to the DMB.