活性氧、线粒体通透性转化孔介导肌细胞凋亡在失神经骨骼肌萎缩中的相关性

目的 观察活性氧(ROS)、线粒体通透性转化孔(MPTP)在失神经骨骼肌萎缩后的表达变化且与肌细胞凋亡的相关性,探讨ROS、MPTP参与失神经骨骼肌萎缩的具体分子机制.方法 将30只Vistar大鼠随机分为对照组、失神经2d组、失神经7d组、失神经14d组、失神经28d组,每组6只.制作坐骨神经切断后失神经支配的腓肠肌Vistar大鼠模型.应用流式细胞术(FCM)检测失神经支配后腓肠肌细胞ROS的含量,激光共聚焦显微镜检测MPTP的开放,脱氧核糖核苷酸转移酶介导的缺口末端标记(TUNEL)法检测肌细胞凋亡.结果 大鼠失神经支配后,肌细胞中的ROS、MPTP及凋亡率与正常对照组比较,表达随失神经支配时间的延长(<28d)而持续增加,且各组的表达均显著高于对照组(P<0.05),ROS的表达与MPTP的开放呈正相关(r=0.884,P<0.01),与肌细胞的凋亡率呈正相关(r=0.893,P<0.01),MPTP的开放与肌细胞凋亡率呈正相关(r=0.927,P<0.01)与肌细胞萎缩指标肌湿重比呈负相关(r=-0.907,P<0.01).结论 ROS、MPTP为调控失神经支配后骨骼肌萎缩的重要分子,其具体机制是通过线粒体介导的凋亡通路促进骨骼肌萎缩.

Abstract：

Objective To study the expression of reactive oxygen species (ROS) and mitochondrial permeability transition pore (MPTP) in denervated skeletal muscle atrophy and its correlation with cell apoptosis, and explore specific molecular mechanism in denervated skeletal muscle atrophy. MethodsThirty Vista rats were randomly divided into five group: control group, 2-days group, 7-days group, 14-days group, 28-days group. Standard model of denervated gastrocnemius muscle was established. The content of ROS and the opening of MPTP in the gastrocnemius were detected by flow cytometry (FCM) and fluorescence microscope respectively. The apoptotic cells in atrophic muscle were examined by TdT-mediated dUTP nick end labeling (TUNEL). Results As compared with the control group, the content of ROS, the opening of MPTP and the apoptosis of gastrocnemius were increased continuously (<28 days) in 2-days group, 7-days group, 14-days group, 28-days group (P<0.05). The content of ROS had a positive correlation with the opening of MPTP (r=0.884,P<0.01) and the apoptosis rate (r=0.893,P<0.01), and the opening of MPTP had a positive correlation with the apoptosis rate (r=0.927,P<0.01), but a negtive correlation with the ratio of muscle wet weight (r=-0.907,P<0.01). Conclusion ROS and MPTP are important elements in regulating skeletal muscle atrophy after denervation by the mitochondrial apoptosis pathway.     

经皮电刺激防治失神经骨骼肌萎缩机制研究

Objective To investigate the effect of electric stimulation on changes of nuclear factor-κB (NF-κB)and muscle Ring Finger-1(MuRF1)d denervated muscles,and explore the mechanism of muscle atrophy prevention bv electric stimulation. Methods Gastroonemius muscle denervation was created by transecting the right sciatic nerve in 54 Wister rats.The animals were then randomized to two groups,one with electric stimulation one without.Levels of NF-κB and MuRF1 mRNA and protein in the gastrocnemius muscle were detcetcd by RT-PCR and western Blot at different intervals after denervation.The ratio of muscle wet weight was analyzed for comparison. Results The level of mRNA and protein of increased correspondingly at different intervals after denervation(P<0.05).The group with electric stimulus had lower levels of NF-κB and MuRF1 of mRNA and protein,and higher muscle wet weight comparing to the denervation group.Conclusion Electric stimulation delays denervation muscle atrophy by inhibiting the NF-κB/MuRF1 pathways.     

经皮电刺激防治失神经骨骼肌萎缩机制研究

Objective To investigate the effect of electric stimulation on changes of nuclear factor-κB (NF-κB)and muscle Ring Finger-1(MuRF1)d denervated muscles,and explore the mechanism of muscle atrophy prevention bv electric stimulation. Methods Gastroonemius muscle denervation was created by transecting the right sciatic nerve in 54 Wister rats.The animals were then randomized to two groups,one with electric stimulation one without.Levels of NF-κB and MuRF1 mRNA and protein in the gastrocnemius muscle were detcetcd by RT-PCR and western Blot at different intervals after denervation.The ratio of muscle wet weight was analyzed for comparison. Results The level of mRNA and protein of increased correspondingly at different intervals after denervation(P<0.05).The group with electric stimulus had lower levels of NF-κB and MuRF1 of mRNA and protein,and higher muscle wet weight comparing to the denervation group.Conclusion Electric stimulation delays denervation muscle atrophy by inhibiting the NF-κB/MuRF1 pathways.     

周围神经损伤修复后早期骨骼肌细胞凋亡的初步研究

Objective To explore skeletal muscle apoptosis at the early stage of peripheral never regeneration in rats.Methods A total of 54 male SD rats were randomly assigned to 3 groups ( n = 18/group): denervation group (A), neurorrhaphy group (B), normal control group (C).In group A, a 1 cm segment of the left sciatic nerve was removed.In group B, the left sciatic nerve was transected above the bifurcation and immediately repaired with 10-0 sutures.No surgery was done in group C.The gastrocnemius muscle wet weight served as an indicator of the degree of muscle atrophy.Marker of apoptosis, nuclear DNA fragmentation, was detected using terminal deoxyribnudeotidyl transferase mediated dUTP nick end labeling (the TUNEL method) and observed under confocal microscopy at 2, 14 and 28 days postoperatively.Another portion of the gastrocnemius muscle was homogenized to analyze the activity of caspase-3 and -8 by spectrophotometry.Results TUNEL labeling of fragmented DNA on histological sections in the neuorrhaphy group revealed levels of apoptotic nuclei higher than the control group and lower than the denervation group at the early stage ( ＜ 28days ).The activity of caspase-3 and -8 in the neuorrhaphy group was also higher than the control group but lower than the denervation group.Conclusion At the initial stage of peripheral never regeneration, apoptosis may contribute to muscle atrophy and extrinsic apoptotic pathways may take part in it.     

周围神经损伤修复后早期骨骼肌细胞凋亡的初步研究

目的 探讨神经修复后早期大鼠骨骼肌的细胞凋亡情况.方法 取SD大鼠54只,随机分为3组:失神经组(A组)18只,神经缝合组(B组)18只,健康对照组(C组)18只.A组大鼠切除左侧1 cm长坐骨神经,B组大鼠横断左侧坐骨神经后立即用10-0医用尼龙线行外膜缝合,C组大鼠不做任何处理.以腓肠肌肌湿重作为骨骼肌萎缩指标.分别应用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)和分光光度法检测术后2 d、14 d、28 d时骨骼肌凋亡细胞核和天冬氨酸特异性半胱氨酸蛋白酶(Caspase)-3与Caspase8活性.结果 神经缝合后早期骨骼肌与正常骨骼肌比较,细胞凋亡现象增加,凋亡相关蛋白Caspase-3和Caspase-8活性上升,但程度弱于失神经骨骼肌.结论 细胞凋亡可能是神经修复后早期骨骼肌萎缩原因之一,死亡受体信号通路参与神经修复后早期骨骼肌凋亡过程中.

Abstract：

Objective To explore skeletal muscle apoptosis at the early stage of peripheral never regeneration in rats.Methods A total of 54 male SD rats were randomly assigned to 3 groups ( n = 18/group): denervation group (A), neurorrhaphy group (B), normal control group (C).In group A, a 1 cm segment of the left sciatic nerve was removed.In group B, the left sciatic nerve was transected above the bifurcation and immediately repaired with 10-0 sutures.No surgery was done in group C.The gastrocnemius muscle wet weight served as an indicator of the degree of muscle atrophy.Marker of apoptosis, nuclear DNA fragmentation, was detected using terminal deoxyribnudeotidyl transferase mediated dUTP nick end labeling (the TUNEL method) and observed under confocal microscopy at 2, 14 and 28 days postoperatively.Another portion of the gastrocnemius muscle was homogenized to analyze the activity of caspase-3 and -8 by spectrophotometry.Results TUNEL labeling of fragmented DNA on histological sections in the neuorrhaphy group revealed levels of apoptotic nuclei higher than the control group and lower than the denervation group at the early stage ( ＜ 28days ).The activity of caspase-3 and -8 in the neuorrhaphy group was also higher than the control group but lower than the denervation group.Conclusion At the initial stage of peripheral never regeneration, apoptosis may contribute to muscle atrophy and extrinsic apoptotic pathways may take part in it.     

周围神经损伤修复后早期骨骼肌细胞凋亡的初步研究

Objective To explore skeletal muscle apoptosis at the early stage of peripheral never regeneration in rats.Methods A total of 54 male SD rats were randomly assigned to 3 groups ( n = 18/group): denervation group (A), neurorrhaphy group (B), normal control group (C).In group A, a 1 cm segment of the left sciatic nerve was removed.In group B, the left sciatic nerve was transected above the bifurcation and immediately repaired with 10-0 sutures.No surgery was done in group C.The gastrocnemius muscle wet weight served as an indicator of the degree of muscle atrophy.Marker of apoptosis, nuclear DNA fragmentation, was detected using terminal deoxyribnudeotidyl transferase mediated dUTP nick end labeling (the TUNEL method) and observed under confocal microscopy at 2, 14 and 28 days postoperatively.Another portion of the gastrocnemius muscle was homogenized to analyze the activity of caspase-3 and -8 by spectrophotometry.Results TUNEL labeling of fragmented DNA on histological sections in the neuorrhaphy group revealed levels of apoptotic nuclei higher than the control group and lower than the denervation group at the early stage ( ＜ 28days ).The activity of caspase-3 and -8 in the neuorrhaphy group was also higher than the control group but lower than the denervation group.Conclusion At the initial stage of peripheral never regeneration, apoptosis may contribute to muscle atrophy and extrinsic apoptotic pathways may take part in it.     

脑死亡时猪肝脏功能及组织学改变的研究

Objective To observe how brain death affects the hepatic morphology and function of pigs and explore the roles of NF-κB. Methods Under general anaesthesia twelve healthy pigs were allocated randomly to two groups:control group(6 pigs),with non-inflacted Foley balloon catheter placed in the cerebral ventricle for 24 h,and brain death group,6 pigs,with estabhshment of brain death for 24 h.The serum and hepatic tissues in the same locus were taken at 6 h,12 h,and 24 h after the initial conformation of brain death.AST and ALT were determined by automatic biochemistry analyzer.IL-1βwas determined by ELISA.The NF-κB mRNA was determined by Real-time PCR and the NF-κB p65 by immunohistochemistry. Results The AST,ALT,IL-1β in serum,the NF-κB mRNA and the NF-κB p65 in hepatic tissues in brain death group were higher than those in control group and they all increased with the time(P<0.05).In brain death group,hepatocytes were edematous lightly after 12 hours,and the swelling progressively deteriorated after 24 hours,but there were no necrosis. Conclusion The activated NF-κB by brain death promoted the synthesis and release of inflammatory mediators,resulting in the hepatic dysfunction.     

脑死亡时猪肝脏功能及组织学改变的研究

Objective To observe how brain death affects the hepatic morphology and function of pigs and explore the roles of NF-κB. Methods Under general anaesthesia twelve healthy pigs were allocated randomly to two groups:control group(6 pigs),with non-inflacted Foley balloon catheter placed in the cerebral ventricle for 24 h,and brain death group,6 pigs,with estabhshment of brain death for 24 h.The serum and hepatic tissues in the same locus were taken at 6 h,12 h,and 24 h after the initial conformation of brain death.AST and ALT were determined by automatic biochemistry analyzer.IL-1βwas determined by ELISA.The NF-κB mRNA was determined by Real-time PCR and the NF-κB p65 by immunohistochemistry. Results The AST,ALT,IL-1β in serum,the NF-κB mRNA and the NF-κB p65 in hepatic tissues in brain death group were higher than those in control group and they all increased with the time(P<0.05).In brain death group,hepatocytes were edematous lightly after 12 hours,and the swelling progressively deteriorated after 24 hours,but there were no necrosis. Conclusion The activated NF-κB by brain death promoted the synthesis and release of inflammatory mediators,resulting in the hepatic dysfunction.     

硼替佐米对小鼠急性移植物抗宿主病的抑制作用

目的 探讨硼替佐米对小鼠急性移植物抗宿主病(aGVHD)的抑制作用及其可能机制.方法 以C57BL/6小鼠为供鼠,获取骨髓细胞及脾细胞.以Balb/c小鼠为受鼠,共分为5组:空白对照组(n=17)小鼠不予任何处理;单纯照射组(n=17)小鼠仅接受7.0 Gy X射线全身照射(TBI);药物对照组(n=17)小鼠也接受TBI,并且由尾静脉注射硼替佐米;aGVHD组(n=8)小鼠TBI后注射供鼠骨髓细胞及脾细胞;实验组(n=8)小鼠TBI后输注供鼠骨髓细胞及脾细胞,并予以硼替佐米.观察各组小鼠aGVHD的发生情况、存活时间及嵌合状态,蛋白印迹法测定空白对照组、单纯照射组和药物对照组小鼠肝脏及小肠组织细胞核中核因子κB(NF-κB)p65的表达.结果 aGVHD组和实验组的临床aGVHD评分分别为7.37±0.32和5.85±0.40,实验组明显低于aGVHD组(P＜0.05).aGVHD组受鼠肝脏、小肠及皮肤组织为Thomas GVHD病理分级Ⅲ～Ⅳ级改变.实验组受鼠肝脏、小肠及皮肤组织为Ⅰ～Ⅲ级GVHD改变,较aGVHD组有所减轻.实验组存活时间长于aGVHD组(P＜0.05).aGVHD组和实验组小鼠移植后12 d时外周血细胞中H-2Kb分子阳性细胞的百分率均＞90%.药物对照组肝脏及小肠组织细胞核内NF-κB p65表达均高于单纯照射组(P＜0.05).结论 硼替佐米可能通过在一定程度上抑制照射预处理损伤所致肝脏及小肠组织中NF-κB的激活起到减轻aGVHD的作用.

Abstract：

Objective To observe the effect of bortezomib on acute graft-versus-host disease (aGVHD) in an aGVHD model of mice and investigate the related mechanism. Methods Male C57BL/6( H-2Kb)mice were used as donors and female Balb/c (H-2Kd) mice used as recipients. Balb/c mice received total body irradiation (TBI) by 7.0 Gy X-radiation, and randomly divided into five groups. normal (group A), TBI (group B), TBI + bortezomib (group C), TBI + bone marrow cells (BMC) + spleen cells (SC) (group D) and TBI + bortezomib + BMC + SC (group E). The physical signs and the pathological damage of aGVHD, mean survival time, and chimerism were observed in recipients. The NF-κB p65 levels in nuclei of the liver and small intestine tissues of groups A,B and C were analyzed by Western blot. Results ( 1 ) The clinical aGVHD score in group D was (7.37±0. 32), significantly higher than in group E (5.85 ± 0.40) (P＜0. 05). Histopathology of the gut, liver and skin illuminated that the Ⅲ-Ⅳ degree GVHD occurred in group D. The occurrence of aGVHD in group E was later than in group D. The symptoms and the pathological damage of aGVHD in group E were milder than in group D. The average survival time in group E was significantly longer than that in group D (P＜0.05). The percentage of donor-derived cells in recipient mice was above 90% at day 12 after transplantation; (2) NF-κB p65 levels in nuclei of the liver and small intestine tissues in group B was significantly higher than in group C on the day 1,3 and 5 (P＜0. 05). Conclusion Bortezomib can inhibit the activation and expression of NF-κB,which may be the underlying mechanism for it to relieve aGVHD.     

Involvement of nuclear factor-kappa B on corticosterone- induced rat Leydig cell apoptosis

Aim:To investigate the activation of nuclear factor-kappa B(NF-kappa B)and its function in glucocorticoid-inducedLeydig cell apoptosis.Methods:The Leydig cells were isolated from male Sprague-Dawley rats(90 days of age)andwere incubated with corticosterone(CORT,glucocorticoid in rat)for 6 h,12 h and 24 h,respectively.The P65subunit of NF-kappa B(NF-kappa B/P65)in nuclei and the inhibitor of NF-kappa B(Ikappa B)in cytoplasm wereanalyzed by Western-blotting.The Leydig cells were treated with anti-Fas antibody for 3 h followed by Westernblotting to assay the changes of NF-kappa B/P65 in nuclei and in cytoplasm.The role of NF-kappa B in CORT-induced Leydig cell apoptosis was evaluated by observing the effects of NF-kappa B/P65 overexpression and inhibit-ing activation of NF-kappa B by 100μmol/L Pyrrolidine dithiocarbamate(PDTC)on this apoptosis.Results:Thetreatment of Leydig cells with CORT increased the levels of NF-kappa B/P65 in nuclei and decreased the levels ofIkappa B in cytoplasm.Following the Leydig cells were treated with anti-Fas antibody,the levels of NF-kappaB/P65was increased in nuclei and decreased in cytoplasm.The CORT-induced Leydig cell apoptosis was inhibited byoverexpressed NF-kappaB/P65 and was enhanced by incubation with PDTC.Conclusion:NF-kappa B is activatedby increased FasL/Fas in CORT-induced Leydig cell apoptosis.NF-kappa B may play an anti-apoptotic role in thisapoptosis.(Asian J Androl 2006 Nov;8:693-702)     

胰岛素对烧伤血清诱导血管内皮细胞核因子κB核移位的抑制作用

Objective To study the inhibitory effects of insulin on nuclear factor-kappa B (NF-κB) nuclear translocation of vascular endothelial cells induced by burn serum and its correlative mechanism. Methods Human umbilical vein endothelial cells (HUVECs) were cultured in vitro and divided into 5 groups: blank control group (BC, ordinary culture without any stimulation) , normal serum control group (NS, cultured with nutrient solution containing 20% healthy human serum) , burn serum stimulation group (BS, cultured with nutrient solution containing 20% burn human serum) , burn serum + insulin treatment group (BI, cultured with nutrient solution containing 20% burn human serum and 1 × 10-7 mol/L insulin) ,inhibitor pretreatment group [IP, pretreated with 50 μmol/L protein kinase B (Akt) specific inhibitor LY-294002, then cultured with the same medium as used in BI group 30 minutes later] according to the random number table. Six hours later, the injury and apoptosis of HUVECs was respectively observed by the scanning electron microscope and determined by the flow cytometry. Meanwhile, the phosphorylation of inhibitor kappa B-α (p-IκB-α) and Akt (p-Akt) in cytoplasm, and the content of NF-κB-p65 in nucleus were determined with Western blot. Results (1) Compared with those in BC group, HUVECs in BS group shrank obviously with irregular nuclear structure, and intercellular links jagged or vanished. Slight change was observed in HUVECs structure in NS and BI groups, with the cell ductility and nuclear structure much better than those in BS group. (2) The apoptosis rates of HUVECs in BS group [(28. 5 ± 2. 3) %] , BI group [(22. 3 ±1.8)%], and IP group [(29. 7 ± 2. 4) %] were all obviously higher than that in BC group [(15.7 ±2.2)% , F =14.288, P <0.05otP <0.01]. There was no significant statistical difference between NS group [(17. 0 ± 2. 5) %] and BC group in apoptosis rate (F = 14. 288 , P > 0. 05). The apoptosis rate of HUVECs in BI group was obviously lower than that in BS group (F = 14. 288 , P <0.05). (3)Compared with those in BC group, the protein expressions of p-IκB-α in cytoplasm and NF-κB-p65 in nucleus were up-regulated, and the protein expression of p-Akt in cytoplasm was down-regulated in BS and IP groups. The expression levels of the three proteins in NS and BI groups were close to those in BC group. Conclusions Insulin could inhibit the IκB phosphorylation, and then restrict NF-κB nuclear translocation and improve the vascular endothelial cells function accordingly through regulating phosphatidylinositol 3 ki-nase/Akt pathway.     

单肺通气吸入不同氧分量对兔肺组织NF-κB的影响

Objective To study the effects of fraction of inspired oxygen on nuclear factor-κB (NF-κB) of lung tissue in rabbit one-lung ventilation(OLV) models.Methods 12 white Japanese rabbits were randomly divided into 2 groups(n=6).Bronchial intubation was performed with artificial double-lumen tube,and right OLV was conducted for 2 h and then tollowed tow-lung ventilation(TLV) for 1 h.Fraction of inspired oxygen was set as 1.0(group A) or 0.6(group B).NF-κB in lung tissue was detected,and arterial blood gases were analyzed before OLV,at 30 min of OLV and 30 min after TLV reversion.Pathology of lung tissue was examined and wet/dry(W/D) of lung weight was measured.Results After TLV restore,the oxygenation index was higher and lower than 300 in group B and group A,respectively.The W/D,the activation and the level of NF-κB in left lung tissue was less in group B than in group A (P＜0.01),and pathological change in left lung tissue was lighter in group B than in group A.Conclusion OLV with 60% oxygen may attenuate lung injury by decreasing the activation and the level of NF-κB in lung tissue.     

神经干细胞移植延缓失神经肌萎的作用机制

Objective To study the effect and mechanism of transplanting spinal fetal neural stem cells (NSCs) into the peripheral nerve for delaying muscle denervation atrophy. Methods Spinal. fetal NSCs were separated from spinal cord of enceinte 10 to 12 days SD rats, cultured and purified. After three passages, the formed NSC spheres were blew into single cell suspension ( 106/μl×5 μl) and transplanted into the distal part of the transected tibial nerve. 5 μl of cell culture medium was injected into the distal tibial nerve in the control group. Three and 5 months after the transplantation, the distal part of the tibial nerve and the triceps suraes were harvested and identified with specific markers, by means of indirect immunofluorescent staining to evaluate survival and differentiation of transplanted NSCs in the nerve, and to observe the neuromuscular junctions.Results Compare to the control group, atrophy of the triceps suraes muscle was less severe 3 and 5 months after NSCs transplantation. Postsynaptic membrane was also better preserved in NSCs transplanted group. Five months after NSCs transplantation, new synapses (neuromuscular junction) formed in the denervated muscle.Conclusion NSCs transplantation can delay atrophy denervated muscles. NSCs transplantation can not only maintain the structure of postsynaptic membrane, but also form new synapse with the denervated muscle.     

神经干细胞移植延缓失神经肌萎的作用机制

Objective To study the effect and mechanism of transplanting spinal fetal neural stem cells (NSCs) into the peripheral nerve for delaying muscle denervation atrophy. Methods Spinal. fetal NSCs were separated from spinal cord of enceinte 10 to 12 days SD rats, cultured and purified. After three passages, the formed NSC spheres were blew into single cell suspension ( 106/μl×5 μl) and transplanted into the distal part of the transected tibial nerve. 5 μl of cell culture medium was injected into the distal tibial nerve in the control group. Three and 5 months after the transplantation, the distal part of the tibial nerve and the triceps suraes were harvested and identified with specific markers, by means of indirect immunofluorescent staining to evaluate survival and differentiation of transplanted NSCs in the nerve, and to observe the neuromuscular junctions.Results Compare to the control group, atrophy of the triceps suraes muscle was less severe 3 and 5 months after NSCs transplantation. Postsynaptic membrane was also better preserved in NSCs transplanted group. Five months after NSCs transplantation, new synapses (neuromuscular junction) formed in the denervated muscle.Conclusion NSCs transplantation can delay atrophy denervated muscles. NSCs transplantation can not only maintain the structure of postsynaptic membrane, but also form new synapse with the denervated muscle.     

神经干细胞移植延缓失神经肌萎的作用机制

Objective To study the effect and mechanism of transplanting spinal fetal neural stem cells (NSCs) into the peripheral nerve for delaying muscle denervation atrophy. Methods Spinal. fetal NSCs were separated from spinal cord of enceinte 10 to 12 days SD rats, cultured and purified. After three passages, the formed NSC spheres were blew into single cell suspension ( 106/μl×5 μl) and transplanted into the distal part of the transected tibial nerve. 5 μl of cell culture medium was injected into the distal tibial nerve in the control group. Three and 5 months after the transplantation, the distal part of the tibial nerve and the triceps suraes were harvested and identified with specific markers, by means of indirect immunofluorescent staining to evaluate survival and differentiation of transplanted NSCs in the nerve, and to observe the neuromuscular junctions.Results Compare to the control group, atrophy of the triceps suraes muscle was less severe 3 and 5 months after NSCs transplantation. Postsynaptic membrane was also better preserved in NSCs transplanted group. Five months after NSCs transplantation, new synapses (neuromuscular junction) formed in the denervated muscle.Conclusion NSCs transplantation can delay atrophy denervated muscles. NSCs transplantation can not only maintain the structure of postsynaptic membrane, but also form new synapse with the denervated muscle.     

吡咯烷二硫代氨基甲酸酯延缓失神经肌萎缩的实验研究

目的研究NF-κB抑制剂吡咯烷二硫代氨基甲酸酯(pyrrolidine dithiocarbamate,PDTC)延缓失神经肌萎缩的效果及其机制。方法健康成年Wistar大鼠30只,雌雄不限,体重(200±10)g。实验动物随机分为3组,即正常对照组(A组,n=6)、失神经对照组(B组,n=12)和PDTC治疗组(C组,n=12)。A组仅暴露右侧坐骨神经并不切断;B、C组切断右侧坐骨神经0.7～1.0 cm,建立右下肢腓肠肌失神经支配模型后,C组采用PDTC 100 mg/(kg?d)腹腔注射,B组以等量生理盐水腹腔注射。A组术后当天和B、C组术后14、28 d,完整分离双侧腓肠肌检测肌肉湿重维持率,采用Western blot检测腓肠肌NF-κB p65表达水平,激光共聚焦扫描显微镜观察线粒体通透性转换孔(mitochondrial permeability transition pore,MPTP)开放情况,TUNEL法检测细胞凋亡情况。结果 A组腓肠肌湿重维持率为1.039±0.115;与A组比较,B、C组各时间点腓肠肌湿重维持率均明显下降,且C组腓肠肌湿重维持率明显高于同期B组,差异均有统计学意义(P<0.05)。A组NF-κB p65表达为0.224±0.041;与A组比较,B、C组各时间点NF-κB p65表达明显升高,且C组NF-κB p65表达明显低于同期B组,差异均有统计学意义(P<0.05)。A组MPTP荧光强度为31.582±1.754;与A组比较,B、C组各时间点MPTP荧光强度均降低,且C组MPTP荧光强度明显高于同期B组,差异均有统计学意义(P<0.05)。A组有少量细胞凋亡,细胞凋亡率为4.542%±0.722%;与A组比较,B、C组各时间点细胞凋亡率均明显增加,且C组细胞凋亡率明显低于同期B组,差异均有统计学意义(P<0.05)。结论 PDTC可有效延缓失神经肌萎缩,其作用机制与抑制NF-κB表达、降低骨骼肌细胞MPTP开放及抑制骨骼肌细胞凋亡有关。     

神经干细胞移植延缓失神经肌萎的作用机制

Objective To study the effect and mechanism of transplanting spinal fetal neural stem cells (NSCs) into the peripheral nerve for delaying muscle denervation atrophy. Methods Spinal. fetal NSCs were separated from spinal cord of enceinte 10 to 12 days SD rats, cultured and purified. After three passages, the formed NSC spheres were blew into single cell suspension ( 106/μl×5 μl) and transplanted into the distal part of the transected tibial nerve. 5 μl of cell culture medium was injected into the distal tibial nerve in the control group. Three and 5 months after the transplantation, the distal part of the tibial nerve and the triceps suraes were harvested and identified with specific markers, by means of indirect immunofluorescent staining to evaluate survival and differentiation of transplanted NSCs in the nerve, and to observe the neuromuscular junctions.Results Compare to the control group, atrophy of the triceps suraes muscle was less severe 3 and 5 months after NSCs transplantation. Postsynaptic membrane was also better preserved in NSCs transplanted group. Five months after NSCs transplantation, new synapses (neuromuscular junction) formed in the denervated muscle.Conclusion NSCs transplantation can delay atrophy denervated muscles. NSCs transplantation can not only maintain the structure of postsynaptic membrane, but also form new synapse with the denervated muscle.     

胰岛素对烧伤血清诱导血管内皮细胞核因子κB核移位的抑制作用

Objective To study the inhibitory effects of insulin on nuclear factor-kappa B (NF-κB) nuclear translocation of vascular endothelial cells induced by burn serum and its correlative mechanism. Methods Human umbilical vein endothelial cells (HUVECs) were cultured in vitro and divided into 5 groups: blank control group (BC, ordinary culture without any stimulation) , normal serum control group (NS, cultured with nutrient solution containing 20% healthy human serum) , burn serum stimulation group (BS, cultured with nutrient solution containing 20% burn human serum) , burn serum + insulin treatment group (BI, cultured with nutrient solution containing 20% burn human serum and 1 × 10-7 mol/L insulin) ,inhibitor pretreatment group [IP, pretreated with 50 μmol/L protein kinase B (Akt) specific inhibitor LY-294002, then cultured with the same medium as used in BI group 30 minutes later] according to the random number table. Six hours later, the injury and apoptosis of HUVECs was respectively observed by the scanning electron microscope and determined by the flow cytometry. Meanwhile, the phosphorylation of inhibitor kappa B-α (p-IκB-α) and Akt (p-Akt) in cytoplasm, and the content of NF-κB-p65 in nucleus were determined with Western blot. Results (1) Compared with those in BC group, HUVECs in BS group shrank obviously with irregular nuclear structure, and intercellular links jagged or vanished. Slight change was observed in HUVECs structure in NS and BI groups, with the cell ductility and nuclear structure much better than those in BS group. (2) The apoptosis rates of HUVECs in BS group [(28. 5 ± 2. 3) %] , BI group [(22. 3 ±1.8)%], and IP group [(29. 7 ± 2. 4) %] were all obviously higher than that in BC group [(15.7 ±2.2)% , F =14.288, P <0.05otP <0.01]. There was no significant statistical difference between NS group [(17. 0 ± 2. 5) %] and BC group in apoptosis rate (F = 14. 288 , P > 0. 05). The apoptosis rate of HUVECs in BI group was obviously lower than that in BS group (F = 14. 288 , P <0.05). (3)Compared with those in BC group, the protein expressions of p-IκB-α in cytoplasm and NF-κB-p65 in nucleus were up-regulated, and the protein expression of p-Akt in cytoplasm was down-regulated in BS and IP groups. The expression levels of the three proteins in NS and BI groups were close to those in BC group. Conclusions Insulin could inhibit the IκB phosphorylation, and then restrict NF-κB nuclear translocation and improve the vascular endothelial cells function accordingly through regulating phosphatidylinositol 3 ki-nase/Akt pathway.