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Objective To observe the impact of mild hypothermia (MH)on the reactive oxygen species (ROS)and expression of cacpase-3mRNA and light chain 3 (LC3,a subunit of immunoglobulin)in hippocampus nerve cells of rats after cardiopulmonary resuscitation (CPR).Methods A total of 65 healthy male Sprague Dawley (SD)adult rats were randomly (random number)divided into 2 groups:blank control group (n =5)and CPR group (n =60).Cardiac arrest (CA)was induced in rats of CPR group by asphyxia.The survival rats after CPR were randomly (random number)divided into 2 groups:normothermia CPR group (NT)and hypothermia CPR group (HT).Homeothermia of 37 ℃ was maintained in NT group after restoration of spontaneous circulation (ROSC),and hypothermal intervention to 32 ℃ was carried out in HT group for 4 hours immediately after ROSC.Both NT group and HT group were then randomly divided to 2 subgroups 12 hours and 24 hours after ROSC (NT-12,NT-24,HT-12,HT-24 subgroups).During observation,the neurological deficit (NDS)of rats was scored,then the bilateral hippocampi were obtained from rats'head,and monoplast suspension of fresh hippocampus tissue was made immediately to determine the level of intracellular ROS by flow cytometry.Transmission electron microscope was used to observe the ultrastructure changes of cellular nucleus and mitochondria.Reverse transcription-polymerase chain reaction (RT-PCR)was used to determine the expression of caspase-3mRNA and Western-blotting (WB)was used to determine the level of LC3 in frozen hippocampus tissue.Measured data were analyzed with paired sample T test and One-Way ANOVA.Results Of 60 rats with CA,44 were successfully resuscitated (73%)and 33 survived until the end of the experiment (55%).The NDSs of rats in NT and HT groups were significantly reduced in comparison with BC group (F=8.107,P<0.05),while the NDSs of rats in HT-12 subgroup and HT-24 subgroup were significantly increased in comparison with NDSs of rats in NT-12 subgroup and NT-24 subgroup,respectively (t=9.692,P<0.01;t=14.374,P<0.01 ).The ROS in hippocampus nerve cells of rats in NT group and HT group were significantly increased compared to BC group (F=16.824,P<0.05 ),whereas the ROS in HT-12 and HT-24 subgroups were significantly reduced compared to ROS in NT-12 and NT-24 subgroups,respectively (t =9.836,P<0.01;t =7.499,P<0.01).The expressions of caspase-3 mRNA in hippocampus nerve cells of rats in NT and HT groups were significantly increased compared to BC group (F=24.527,P<0.05),while the expressions of caspase-3 mRNA in rats of HT-12 and HT-24 subgroups were significantly reduced compared to NT-12 and NT-24 subgroups,respectively (t =6.935,P <0.01;t =4.317,P <0.01 ).The level of LC3B-II/I in hippocampus nerve cells of rats in NT and HT groups were significantly increased compared to BC group (F=6.584,P<0.05),while the levels of LC3B-II/I in rats of HT-12 and HT-24 subgroups were significantly reduced compared to NT-12 and NT-24 subgroups,respectively (t=10.836,P<0.001;t=2.653,P=0.02).Ultrastructure damage of nucleus and mitochondria in NT group was more evident compared to BC group,and eumorphism of nucleus and mitochondria were maintained in rats of HT group compared to NT group.Conclusions The mild hypothermia reduced the injury of nerve cells and improved the neurological function of rats survived from cardiac arrest likely by reducing ROS production of nerve cells and inhibition the expression of caspase-3mRNA and lowering the level of LC3 leading to reducing cellular apoptosis and massive autophagy in rats survived from cardiac arrest after CPR.
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AIM: To investigate the CdTe quantum dots coated with MPA and explore its biocompatibility with living cells. METHODS: CdTe quantum dots coated with MPA were prepared in aqueous phase and MPA CdTe QDs were Characterized with TEM, fluorospectrophotometer and ultraviolet spectrophotometer. QDs were Modified with with avidin, purified and prepared as flurescent probe. LSCM was used to observe the expression of MHC Ⅱ antigen on PMφ cells, which was labeled by QDs. Cell culture and MTT assays were used to determine the biocompatibility of MPA coated CdTe quantum dots with the B-16 cells as target cells. RESULTS: The particle diameter of CdTe quantum dots prepared in aqueous phase was well distributed. They had good photological performance and greater stability after coated with MPA. MHC Ⅱ antigen on PMφ was labeled with the QDs-Avidin fluorescent probe showed great fluorescence intensity, which was easy to be detected by fluorescence microscope and LSCM. MPA CdTe QDs showed cytotoxicity when its density was very high, but they showed little cytotoxicity during the normal use of influence label density limit. CONCLUSION: MPA CdTe QDs can be used as new fluorescent lable as they are of even size, not easy to bleach or quench, have good photological performance and stability and good biocompatibility.
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Objective To prepare the chitosan-Mg membranes and to explore the biocompatibility of membranes with PC12 cells and its usage as biomaterials in tissue engineering.Methods The appearance of the chitosan-Mg membranes (CM) was observed under scanning electromicroscope (SEM) and the element of the membranes was analyzed by x-ray energy spectromete. PC12 cells were co-cultured with CM in vitro,the morphological changes of PC12 cells on membranes were observed under scanning electron microscope (SEM) and light microscope (LM),the cell vitality was detected by MTT assay. Results The surface of chitosan membranes (CS) was smoother than that of CM;but the CM groups were full of pore observed under SEM;The content of Mg element was related with the dosage of MgSO4 added into chitosan solution. Morphology observation showed that PC12 cells grew well on CM as compared with CS group. The cells were rich of microvilli and long processes on 7th day,and synapses-like structure was formed in many PC12 cells. In addition,the cell viability of experiment group was higher than that of control group(P
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AIM:To investigate the immunomodulatory effect of astragalus saponin(AS)on macrophages and explore the mechanism of its immunomodulation.METHODS:By adding different concentrations of AS into cultured mouse peritoneal macrophages in vitro,the influence of AS on the synthesis of nitro oxide(NO)was detected by NO kit(enzymatic method).MTT assay was used to determine the cytotoxicity of macrophages induced by AS.The morphological changes of macrophages were observed under transmission electron microscope.LSCM and specificity fluorescent probe Fluo-3/AM were applied to observe the change of Ca2+ in macrophages induced by AS.RESULTS:AS significantly increased NO synthesis,enhanced the effects of mouse peritoneal macrophages on killing carcinoma cells.Cell surface projection exhibited multiplication,becoming thickening and growth longer via transmission electron microscope.Increase in intracellular Ca2+ in macrophages was also observed.CONCLUSION:AS enhances the immune function of macrophages by increasing NO synthesis and enhancing cytotoxicity.The increased intracellular Ca2+ may be the mechanism of immunomodulatory effect of AS.
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AIM:To study the expressing variation of TNF-? and IFN-? mRNA in mouse splenocytes induced by H22 tumor cells derived heat shock protein gp96-peptide complexes in vitro,and to observe the morphologic change of H22 tumor cells which treated with the culture supernatant.METHODS:H22 tumor cells derived HSP gp96 was obtained by the techniques for protein extraction and purification and was identified by Western blotting method.The expression values of TNF-? and IFN-? mRNA in spleen lymphocytes were detected by semi-quantitative RT-PCR.The morphologic changes of H22 tumor cells induced by the culture supernatant were observed by laser scanning confocal microscopy(LSCM)and transmission electron microscope(TEM).RESULTS:Purified heat shock protein gp96 was identified by Western blotting.The expression value of TNF-? and IFN-? mRNA in activated spleen lymphocytes induced by gp96-peptide complexes was higher than that in control groups(P
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Objective: To evaluate the cytotoxic effect of PEM? induced by HSPgp96 on anti-tumor in vitro. Methods; PEM? separated from mice induced by thioglycolate were divided into three groups randomly: Culture medium in control; LPS-induced group; HSPgp96-induced group. The production of NO, the cytotoxic effect to H22 cells and the morphologic change of PEM? were investigated separately by enzyme method, MTT assay and scanning electron microscope. Results: In vitro, HSPgp96 can increased NO production from PEM? of mice and significantly enhance the cytotoxic effect of PEM? to H22 cells as well as LPS. Conclusion: HSPgp96 can effectively induce the cytotoxic effect of PEM? on anti-tumor in which NO is one of the capital effective molecules in vitro.
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Objective:To explore the availability for tumor-derived gp96 to induce specific CTL activity of splenocytes in vitro.Methods:gp96 purified by the techniques for protein extraction and identified by SDS-PAGE gel electrophoresis and Western blot method; CD8+T cell induced by gp96 and CTL activity detected by flow cytometry, immunofluorescence technic and CCK-8 assay.Results:gp96 was identified by SDS-PAGE and Western blot; Analysis with FCM showed that the number of CD8+T cells(nearly 70%) was obviously increased after pulsed with gp96-peptide complexes as compared with that of control groups(35%,26%, P