ABSTRACT
Objective@#To explore the inhibitory effect of exosomes secreted by human umbilical cord mesenchymal stem cells(HUCMSC) on apoptosis of human umbilical vein endothelial cells(HUVEC) after model group(oxygen-glucose deprivation reoxygenation), and to clarify its possible mechanism.@*Methods@#Human umbilical cord mesenchymal stem cells were cultured. The collected cell supernatant was stored in a centrifugal tube. The exosomes secreted by human umbilical cord mesenchymal stem cells were extracted by ultracentrifugation and identified. Human umbilical vein endothelial cells were randomly divided into control group, model group and different concentrations of HUCMSC-EXO(20 μg/ml, 40 μg/ml, 60 μg/ml) treatment groups(adding HUCMSC-EXO into the model group) . The morphological changes of HUVEC cells in each group were observed by inverted phase contrast microscope, and the proliferation inhibition rate of HUVEC in each group was measured by CCK-8 reagent. Western blot was used to detect the expression of apoptosis-related proteins Caspase-3, Bax, Bcl-2 and hypoxia-associated protein hypoxia inducible factor 1α(HIF-1α). Inhibitor(HIF-1α inhibitor) + model group and HUCMSC-EXO + inhibitor + model group were added on the basis of the above experiments. Western blot analysis was performed to observe the effects of HUCMSC-EXO, inhibitor and both of them on HIF-1α and Bax expressions in HUVEC.@*Results@#HUCMSC-EXO was successfully extracted and identified. Compared with the control group, the volume of HUVEC in the model group and the HUCMSC-EXO group with different concentrations decreased, became round, connected and evacuated, and the growth state was poor under the inverted phase contrast microscope.CCK-8 detection showed that the cell viability in the HUCMSC-EXO group was significantly higher than that in the model group, the difference was statistically significant (t=9.23, P<0.05). Western blot analysis showed that compared with the control group, the expression levels of Caspase-3 ((0.296±0.038), (0.879±0.088); t=14.92, P<0.05), Bax((0.234±0.034), (0.762±0.084); t=14.36, P<0.05) of HUVEC in the model group were up-regulated, and the expression level of Bcl-2 was down-regulated ((0.863±0.103), (0.387±0.059); t=9.85, P<0.05), with statistically significant differences. Compared with the model group, the expression levels of Caspase-3( (0.586±0.075); t=6.24, P<0.05), Bax((0.311±0.055); t=11.01, P<0.05) and Bcl-2((0.665±0.071); t=7.45, P<0.05) of HUVEC in the HUCMSC-EXO treatment group were down-regulated and the differences were statistically significant. Inhibitor intervention experiments showed that there were no significant differences between the inhibitor+ model group and HUCMSC-EXO+ inhibitor+ model group in the expression of HIF-1α protein ((0.348±0.055), (0.388±0.077); t=1.04, P>0.05)and Bax protein ((0.363±0.069), (0.370±0.064); t=0.18, P>0.05). But both of them were down-regulated compared with the model group (HIF-1α protein (0.919±0.064), Bax protein (0.902±0.071)), the differences were significant( t=13.56, t=13.03, both P<0.05).@*Conclusion@#HUCMSC-EXO has a protective effect on OGD/R model of HUVEC, and its mechanism may be related to the down-regulation of HIF-1α expression.
ABSTRACT
Objective To observe the expression of neuropilin-1 (NRP-1) in glioma cells of different grades,and evaluate the application value of a novel molecular probe(USPIO-PEG-tLyP-1)in the grading diagnosis of heterotopic glioma in nude mice by magnetic resonance imaging (MRI).Methods Expression levels of NRP-1 in glioma cell lines of different grades were detected by Western-Blot.USPIO-PEG-tLyP-1 was synthesized by carbon diimine method.The U87-MG tumor-bearing mice model (U87-MG group) and CHG-5 tumor-bearing mice model(CHG-5 group) were established with 10 mice in each group.Six tumorbearing mice with a tumor volume about 0.6 cm3 were selected from each group,and they were given with 2mg/kg molecular probes via tail vein respectively and was detected by MRI at 0 h,6 h,12 h and 24 h,then R2 values were calculated.After the imaging,tumor-bearing mice were sacrificed,and tumor tissue sections were made.The iron particles in the sections was detected by Prussian blue staining.The binding ability of molecular probes and tumor tissues in the two groups was compared.Results The expression of NRP-1 in U87-MG and CHG-5 cell lines was significantly higher than that in HA.In addition,the expression of NRP-1 in U87-MG was higher than that in CHG-5 cell(P＜0.01).MRI results showed that R2 values of tumor tissues in the two groups were compared,and the difference was not statistically significant before the injection of molecular probe(U87-MG group(10.35±0.52)vs CHG-5 group(9.86±0.43),t=1.779,P=0.106).The R2 value of tumor tissue in the U87-MG group was higher than that in the CHG-5 group after the injection of molecular probe (6 h:U87-MG group (11.63±0.85)vs CHG-5 group (10.51 ±0.49),t=2.796,P=0.019;12h:U87-MG group(14.23±0.68)vs CHG-5 group(12.29±0.28),t=6.462,P=0.000;24 h:U87-MG group (13.36±0.92) vs CHG-5 group(11.32±0.64),t=4.459,P=0.001).The results of Prussian blue staining showed that there were significantly more blue staining particles in tumor tissues of the U87-MG group than that of the CHG-5 group,and the difference was statistically significant(P＜0.01).Conclusion The NRP-1 targeted molecular probe can be used for grading diagnosis of high and low grade heterotopic brain glioma in nude mice.