RESUMO
Objective:To inhibit the Itch gene expression of T-lymphocytes and investigate the immune activity of T lymphocytes using ultrasound-targeted microbubble destruction (UTMD).Methods:T lymphocytes were separated by magnetic bead, and to establish an Itch gene targeted short hairpin RNA (shRNA) plasmid. There were three groups in this study: ①experimental group, Itch-shRNA plasmid-SonoVue microbubbles combined with ultrasound irradiation; ②control group, negative control shRNA plasmid-SonoVue microbubbles combined with ultrasound irradiation; ③blank group, untreated. Forty-eight hours after UTMD transfection, transfection efficiency was detected and analyzed by fluorescence microscopy and flow cytometry, the expression of Itch protein was measured with Western blotting.Seventy-two hours after UTMD transfection, the secretion of interleukin 2 (IL-2) and interferon γ (IFN-γ) in the cell supernatant were measured by enzyme-linked immunosorbent assay (ELISA). One-way ANOVA was used to compare the differences between groups, and LSD- t test was used to compare the differences between groups. Results:The UTMD mediated shRNA transfection rate was 52.3%, and the relative expression levels of Itch protein in the experimental group, control group and blank group were 0.301±0.080, 0.773±0.101 and 0.719±0.090, respectively, and the difference was statistically significant( F=24.441, P<0.01). The Itch gene expression can be effectively suppressed in the experimental group. Seventy-two hours after transfection, the concentrations of IL-2 in the experimental group, control group and blank group were (417.3±37.1)ng/L, (158.7±17.3)ng/L and (147.0±10.2)ng/L, respectively, and the difference was statistically significant( F=118.701, P<0.001) and the concentrations of IFN-γ were (168.3±12.1)ng/L, (74.3±3.7)ng/L and (74.6±7.1)ng/L, respectively, and the difference was statistically significant ( F=126.833, P<0.001). The expression levels of IL-2 and IFN-γ in the experimental group were significantly higher than those in the control group and the blank group. Conclusions:UTMD mediated shRNA transfection can significantly decrease the expression of Itch and promote immune activity of T lymphocyte.
RESUMO
Objective To evaluate the effect of dexmedetomidine on retinal ischemia-reperfusion (I/R) injury in mice.Metbods Forty-eight C57BL/6 male mice,aged 8 weeks,weighing 20-24 g,were divided into 3 groups (n =16 each) using a random number table:control group (C group),I/R group and dexmedetomidine group (D group).The model of retinal I/R injury was established by elevating intraocular pressure for 60 min using anterior chamber cannulation followed by 24 h of reperfusion.At 15 min before ischemia and 5 min before reperfusion,dexmedetomidine 25 μg/kg was intraperitoneally injected in group D,and the equal volume of normal saline was intraperitoneally injected in C and I/R groups.Eight mice were sacrificed at 24 h of reperfusion,and the retina was removed for microscopic examination of pathologic changes (with light microscope) after haematoxylin and eosin staining and for determination of cell apoptosis in retinal tissues (by TUNEL).Apoptosis index (AI) was calculated.Eight mice were sacrificed at 24 h of reperfusion,and the retina was removed for determination of the expression of Bcl-2,Bax,caspase-3 and CCAAT/enhancer-binding protein homologous protein (CHOP) in retinal tissues (by Western blot).Bcl-2/Bax ratio was calculated.Results Compared with group C,AI was significantly increased,the expression of Bax,caspase-3 and CHOP was up-regulated,the expression of Bcl-2 was down-regulated,Bcl-2/Bax ratio was decreased (P＜0.05),and pathologic changes were found in retinal tissues in group I/R.Compared with group I/R,AI was significantly decreased,the expression of Bax,caspase-3 and CHOP was down-regulated,the expression of Bcl-2 was up-regulated,Bcl-2/Bax ratio was increased (P＜0.05),and pathologic changes of retinal tissues were significantly attenuated in group D (P＜0.05).Conclusion Dexmedetomidine can reduce retinal I/R injury,and the mechanism mav be related to inhibiting cell apoptosis in mice.