ABSTRACT
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.
ABSTRACT
Objective To investigate the effect of nerve cells apoptosis induced by endoplasmic reticulum stress (ERS) on brain injury after asphyxiating cardiac arrest and cardiopulmonary resuscitation (CA/CPR) in rats.Methods A total of 40 male Sprague-Dawley rats were randomly divided into control group and CA/CPR group (n=20).The CA/CPR models were established by asphyxia method/CPR.The levels of neuron specific enolization enzyme (NSE) and S100 beta protein (S100β protein) in serum at baseline and 0,3 and 6 h after CPR were detected by double antibody sandwich enzyme-linked immunosorbent assay (ELISA).The mRNA expressions of CCAAT-enhancer binding protein homologous protein (CHOP),activate transcription factor 4 (A TF4) and X-4 box binding protein 1 (XBP1) in the hippocampus were detected by reverse transcription-polymerase chain reaction (RT-PCR).The protein expressions of CHOP,B-cell lymphoma-2 (Bcl-2),Bcl-2 associated X protein (Bax) and cysteinyl aspartate specific proteinase 3 (caspase-3) in the hippocampus were measured by Western boltting.Neuronal apoptosis were detected by terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL).Morphological and ultrastructural changes of the hippocampi were observed by light microscopy and electron microscopy.Results As compared to that in the control group,S100β protein in the CA/CPR group at 0,3 and 6 h after resuscitation was statistically different (P＜0.05);NSE protein level in the CA/CPR group at 6 h after resuscitation was significantly higher than that in the control group (P＜0.05).As compared with those in the control group,the mRNA expressions of CHOP,A TF4 and XBP-1 in the hippocampus of the CA/CPR group were obviously increased (P＜0.05).Significantly increased protein expressions of CHOP,Bax,and caspase-3,and statistically decreased Bcl-2 expression in the CA/CPR group were noted as compared with those in the control group (P＜0.05).The neuronal apoptosis rate in the CA/CPR group (29.74％±6.26％) was significantly higher than that in the control group (7.48％±4.34％,P＜0.05).The morphology changes and ultramicrostructure injuries of the hippocampus in the CA/CPR group were more obvious as compared with those in the control group.Conclusion CA/CPR in rats causes significant damage to brain tissues,and brain injury is aggravated gradually along with the prolongation of time,and the mechanism of brain injury may be connected with ERS-induced apoptosis of nerve cells.