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Objective To observe the effects of the intercellular gap junction (GJIC) composed of connexin 43(Cx43) in mesenchymal stem cells (MSCs) from different sources and their signals on the biological behavior of multiple myeloma (MM) lateral population cells (SP cells), and to explore its possible mechanism. Methods Mesenchymal stem cells (MSCs) from different sources were isolated and cultured. SP cells of MM cell line RPMI 8266 were sorted by flow cytometry. RT-PCR and Western blot were used to detect the expression of Cx43 gene and protein in MSCs, RPMI 8266 and SP cells from different sources. The effects of MSCs from different sources on SP cell cycle, Cx43 protein expression, colony formation ability in vitro, stem cell related gene expression, cytokine secretion and drug resistance were observed. Results There was no significant difference in morphology and phenotype between MM-MSCs and ND-MSCs. Both MM-MSCs and RPMI 8266 cells expressed a higher level of Cx43. Co-culture with MM-MSCs induced more SP cells to enter G0 phase (P<0.001). The expressions of c-myc, Kif4 and Sox2 genes in SP cells were significantly up-regulated, while the expression of Oct-4 gene was down-regulated. After adding α-GA, c-myc, Kif4 and Sox2 were down-regulated in varying degrees, but there was no significant difference. The expression of Cx43 was up-regulated by (31.00±2)% and (39.00±2)%, respectively. The colony formation ability in vitro was up-regulated, and the addition of α-GA could partially inhibit this effect. A small amount of c-myc, Kif4, Sox2 and Oct-4 genes were expressed in RPMI 8266. These genes were significantly up-regulated in SP cell subpopulation. MM-MSCs secreted high levels of interleukin (IL)-6. After co-culture with SP cells, the expressions of IL-6, IL-10 and TGF-β in the supernatant of MM-MSCs were up-regulated (P=0.0072, P=0.037). bFGF and IL-17 had no significant change. After adding α-GA, the levels of IL-6, IL-10 and TGF-β in the supernatant decreased. MM cells were sensitive to bortezomib (BTZ) induced apoptosis, but SP cells were less sensitive. Co-culture with MM-MSCs significantly reduced BTZ-mediated apoptosis. The addition of α-GA partially restored the sensitivity of MM cells to bortezomib. Conclusion MM-MSCs and multiple myeloma SP cells up-regulate the expression of Cx43 protein, form more GJIC, and promote the proliferation and drug resistance of SP cells by changing the cytokine secretion profile of MSCs, which may be one of the reasons for the recurrence of MM.
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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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<p><b>OBJECTIVE</b>To observe the effect of rosiglitazone (RGZ) on the mRNA expression of HIF1α and IGF1 genes in the myeloma cells and explore possible mechanism of angiogenesis inhibition.</p><p><b>METHODS</b>Human myeloma cell line RPMI8226 and primary myeloma cells from five patients enriched by using CD138 immunomagnetic beads were treated with different concentrations (10, 20, 40 μmol/L) of RGZ. The mRNA expression of HIF1α and IGF1 was analyzed in cells treated with RGZ after 48h by RT-PCR, The levels of phosphorylated AKT and ERK proteins were detected by Western blotting. Bone marrow mononuclear cells from five patients with iron deficiency anemia were regarded as control.</p><p><b>RESULTS</b>Higher mRNA expression of HIF1α and IGF1 genes in RPMI8226 and in primary myeloma cells was showed as compared to those in control. Treated with RGZ of 20 μmol/L after 48 h, the mRNA expression of HIF1α (1.21 ± 0.08 vs 0.75 ± 0.06) and IGF1 (0.62 ± 0.06 vs 0.32 ± 0.04) in RPMI8226 cells was declined as compared to those without RGC treatment. The same declination was also seen in primary myeloma cells (HIF1α: 2.02 ± 0.16 vs 0.53 ± 0.04; IGF1: 1.92 ± 0.13 vs 0.58±0.03). RGZ could inhibit the expression of pAKT and pERK, nor the total AKT and ERK proteins, in RPMI8226 cells in a dose-dependent manner at the concentration of 10 μmol/L, 20 μmol/L, and 40 μmol/L.</p><p><b>CONCLUSION</b>RGZ could inhibit the mRNA expression of HIF1α and IGF1. Inhibition of angiogenesis by RGZ may be associated with down-regulation of pAKT and pERK expression.</p>