摘要
Objective To investigate the effect and mechanism of transplantation of neuregulin1(NRG1)gene-modified bone marrow mesenchymal stem cells(BMSCs)on the repair of hemi-transected spinal cord injury(SCI)in rats.Methods Isolated and cultured rat BMSCs,followed by transfection with the NRG1 gene.The levels of NRG1 in BMSCs lysate and culture supernatant was deected by ELISA method,and the proliferation activity of the BMSCs was detected by cell counting method.Forty-three healthy 8-week-old SD rats were randomly divided into control group(n=10),SCI model group(n=10),BMSCs group(n=10),and NRG1-BMSCs group(n=13).After establishing the spinal cord hemisection model,animals received in-situ transplantation of BMSCs or NRG1-BMSCs.On the 1,7,14,21,and 28 days after transplantation,the hind limb motor function was evaluated using BBB score and inclined plate test;on the 7th day after transplantation,the migration and distribution of transplanted cells was monitored using a fluorescence microscope;on the 28th day after transplantation,the pathological changes of rat spinal cord tissues was examined using HE staining and Nissl staining;cell apoptosis using TUNEL staining,and levels of endoplasmic reticulum stress-related proteins[X-box binding protein 1(XBP1),C/EBP homologous protein(CHOP),activating transcription factor 4(ATF4),ATF6,glucose-regulated protein 78(GRP78)]and apoptosis-related proteins[B-cell lymphoma-2(Bcl-2)and Bcl-2-associated protein X(Bax)]in rat spinal cord tissues using Western blotting.Results BMSCs were successfully isolated,cultured,and transfected with the NRG1 gene.ELISA method results showed that the NRG1 contents in the NRG1-BMSCs lysate and culture supernatant were significantly higher than that of BMSCs in a time-dependent manner(P<0.05).The proliferation activity of NRG1-BMSCs was significantly higher than that of BMSCs(P<0.05).On the 21 and 28 days after transplantation,the BBB score and the slope angle of the inclined plate in NRG1-BMSCs group were higher than those in SCI model group or BMSCs group(P<0.05).However,it did not reverse to the level in control group(P<0.05).On the 28th day after transplantation,compared with the SCI model group and BMSCs group,neuronal pyknosis reduced,the Nissl body density increased,the expression levels of XBP1,CHOP,ATF4,ATF6,GRP78,and Bax,and the rate of TUNEL-positive cells significantly reduced in NRG1-BMSCs group(P<0.05),and the expression level of Bcl-2 significantly increased(P<0.05).Conclusion Transplantation of NRG1 gene-modified BMSCs can alleviate SCI and improve the recovery of motor function in rats.The mechanism may be related to promoting the proliferation activity of BMSCs,inhibiting cell apoptosis,and mitigating endoplasmic reticulum stress.
摘要
Background The hyperplasia of human Tenon capsule fibroblasts (HTFs) is a common cause of filtering surgery failure in glaucomous eye.Researches demonstrated that hydroxycamptothecin is a cell cycle arresting drug and induce apoptosis of cancer and fibroblasts.However,its mechanism is currently less understood.Objective This study was to investigate whether hydroxycamptothecin induce the apoptosis of HTFs and explore the possible mechanism.Methods Human Tenon capsule tissue was obtained from EyeBank of Jiangsu Province Hospital.HTFs were cultured using explant method in vivo and passaged in DMEM containing 10％ FBS.The cells were identified using vimentin and keratin by immunochemistry,and the cells of generation 3-6 in the logarithmic growth phase were used in the experiment.The cells were incubated with 0.01,0.05 or 0.10 g/L hydroxycamptothecin for 5 minutes respectively,and the cells without any hydroxycamptothecin were served as the control group.Cell viability then was assessed by cell counting kit-8 (CCK8) for the optimal inhibition concentration.The cells were treated by 0.10 g/L hydroxycamptothecin for 24 hours,and the apoptotic rate of the cells were assayed with annexin V/PI double-staining.Mitochondrial membrane potential of HTFs was assessed using JC-1 staining.The expressions of caspase-3,caspase-9 and cytochrome C (cyt C) in mitochondria and cytoplasm of HTFs were detected by Western blot.Results The proliferative value (A450) of the HTFs 0,0.01,0.05,0.10 g/L was 0.9716±0.0608,0.8035 ± 0.0346,0.7048 ±0.0446,0.6265 ±0.0286,with a significant difference (F =26.372,P =0.002).A450 of HTFs in the 0.01,0.05,0.10 g/L groups was significantly lower than the control group (P＜0.05),with the lowest A450 value in the 0.10 g/L group.The apoptotic percentage of HTFs was (18.72±1.41)％,in the 0.10 g/L hydroxycamptothecin group and that of the control group was (3.67 ±0.36)％,showing a significant difference between them (t =-10.374,P=0.001).The expression intensity of caspase-3 and caspase-9 protein in HTFs was higher in the 0.10 g/L hydroxycamptothecin group than that in the control group.JC-1 staining showed that the green fluorescence of the monomer JC-1 in cytoplasm was stronger in the 0.10 g/L hydroxycamptothecin group than that in the control group,but the red fluorescence of the polymer JC-1 in the 0.10 g/L hydroxycamptothecin group was weaker than that in the control group.The grey scale of cyt C protein in HTFs in mitochondrion was 0.0605±0.0022 in the 0.10 g/L hydroxycamptothecin group,showing a significant increase in comparison with 0.0301 ±0.0016 of the control group (t=4.865,P=0.014).However,the grey scale of cyt C protein in cytoplasm was declined in the 0.10 g/L hydroxycamptothecin group than that in the control group (0.0605 ±0.0022 vs.0.0301 ±0.0016) (t =-11.177,P =0.001).Conclusions Hydroxycamptothecin can induce the apoptosis of HTFs through activating the mitochondrial apoptosis pathway.
摘要
Objective To investigate the influence of coxsackie B virus (CVB) and mycoplasma pneumoniae (MP) superinfection on immunological manifestation of recurrent respiratory tract inflammations(RRI) in children.Methods One hundred and thirty-two hospitalized children with RRI between Jan.to Dec.2005 were divided into negative control group,MP infection group,CVB infection group and superintection group by determining anti-MP IgM and anti-CVB IgM.Blood sedimentation,C-reactive protein,IgG,IgA,IgM and T lymphocyte subpopulation etc.were determined in four groups.The anti-MP IgM was determined by specificity immune agglutination test.Enzyme linked immunosorbent assay was used to detect anti-CVB IgM.The IgG,IgA,IgM were determined by simple agar diffusion method.T lymphocyte subpopulation was tested by flow cytometry.Results The percentage of CVB infection was 32.1%,mainly expressed in the 1-3 years old children;the percentage of MP infection was 22.7%,mainly expressed in children over 3 years old.In both CVB and MP infection group,the ten-dency of IgG increased and that of IgA decreased.IgM in the CVB and MP superinfection group was obviously higher than that in negative control group and MP infection group (Pa
摘要
Objective To study the different effects between intermittent eliminating white blood cells and immunosuppressive agents pulse therapy in children′s anaphylatoid purpura nephritis on the base of hormone′s treatment.Methods Twelve cases of anaphylatoid purpura nephritis were enrolled in the study and divided into 2 groups.After failure in the hormone′s treatment,the observation group was applied with intermittent eliminating white blood cells(5 mL/kg,1-2 times/week),and the control group was treated with immunosuppressive agents[cyclophosphamide(CTX) 0.8-1.0 g/m~2,or methotrexate(MTX) 8-10 g/m~2].Results There were no significant differences between the 2 groups.The observation group had no obvious side effects,but the control group all had different side effects.Conclusion The treatment of intermittent eliminating white blood cells can have better effects without the side effect resulting from immunosuppressive agents.