失神经骨骼肌NF-kappa B的表达变化与线粒体通透性转换孔开放及细胞凋亡的关系

目的 通过研究去神经骨骼肌细胞核转录因子kappa B(nuclear factor of kappa B,NF-kappa B)p65与线粒体通透性转换孔(mitochondrion permeability transition pore,MPTP)及细胞凋亡的关系,探讨失神经肌萎缩的相关机制.方法 Wistar大鼠30只,随机分为健康对照组、去神经2 d组、去神经7 d组、去神经14 d组、去神经28 d组.建立右下肢腓肠肌失神经支配模型,对照组仅做假手术.采用Western Bloting检测大鼠腓肠肌NF-kappa B p65蛋白表达水平,共聚焦荧光显微镜观察MPTP的开放情况,脱氧核糖核苷酸转移酶介导的缺口末端标记(Tunnel)技术检测细胞凋亡情况,并结合肌湿重进行相关性分析.结果 大鼠失神经支配后随着肌湿重比的下降,NF-kappa B p65蛋白、MPTP的开放以及骨骼肌细胞的凋亡率在各个时间点持续增加,且明显高于对照组,差异有统计学意义(P＜0.05),NF-kappa B与MPTP的开放(r=-0.896,P＜0.01)以及凋亡率(r=0.890,P＜0.01)密切相关,并且NF-kappa B和凋亡率的升高与肌湿重比呈负相关关系(r分别为-0.919、-0.924,P均＜0.01).结论 NF-kappa B在失神经肌萎缩中起重要作用,其作用机制与MPTP的开放及细胞凋亡有关.

Abstract：

Objective To analyze the relationship of nuclear factor of kappaB (NF-κB) p65 to mitochondrion permeability transition pore (MPTP) and apoptosis during denervation atrophy of the gastrocnemius muscle, and delineate their potential roles in skeletal muscle atrophy. Methods Thirty Wistar rats were randomly divided into 5 groups: control group, 2 day denervation group, 7 day denervation group, 14 day denervation group, and 28 day denervation group. The gastrocnemius muscle was denervated by transection of the sciatic nerve. Sham operation was done in the control group. Levels of NF-κB p65 protein and the opening of the mitochondrial permeability transition pore in the gastrocnemius muscle were detected respectively by Westem Bloting and confocal fluorescence microscopy. The apoptotic cells in atrophic muscles were quantitated with Tunel. Muscle wet weight was also measure for comparison. Results At different time points after denervation, the levels of NF-κB p65, the opening of MPTP and apoptosis of gastrocnemius were significantly higher than those in the normal group ( P ＜ 0.05). Furthermore NF-κB p65 had a positive correlation with the opening of MPTP (r= - 0.896, P ＜ 0.01) and with the ratio of apoptosis (r = 0.890,P ＜0.01 ), while the increase of NF-κB p65 and apoptosis was negatively correlated with the ratio of muscle wet weight ( r = - 0. 919,-0.924, P ＜ 0.01 ). Conclusion NF-κ3 plays an important role in denervation skeletal muscle atrophy.This effect is related to the opening of MPTP and apoptosis.     

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.     

骨骼肌线粒体通透转换孔在骨质疏松症中的变化

目的 观察骨骼肌线粒体通透转换孔(MPTP)的变化,探讨骨骼肌与骨质疏松症的关系.方法 选择因腰椎间盘突出或者腰椎管狭窄症行手术治疗的女性骨质疏松患者13例,另选同时期需上述手术治疗的女性非骨质疏松患者13例.于术中收集肌肉组织,提取骨骼肌线粒体,进行细胞色素C氧化酶(COX)和MPTP的活性检测,并进行相关分析.结果 在MPTP活性变化的比较方面,两组差异有统计学意义(P<0.05).在线粒体COX的活性比较方面,两组差异有统计学意义(P<0.05).在MPTP与COX的相关分析中,MPTP的A比值与COX的活性呈正相关 (r=0.405,P<0.05).结论 骨质疏松骨骼肌的MPTP的通透性高于非骨质疏松,而COX的活性对MPTP的通透性有重要影响,揭示只要维持COX的活性,就能有效控制MPTP的变化,从而有效防治细胞的过早衰老.

Abstract：

Objective To investigate the relationship between osteoporosis and skeletal muscle by studying the change mechanism of the mitochondrial permeability transition pore (MPTP) of skeletal muscle. Methods Twenty-six female patients were selected between May 2008 and December 2008, who were diagnosed as having lumbar intervertebral disc herniation or lumbar spinal stenosis receiving surgical operation. They were divided into osteoporosis group (n=13) and non-osteoporosis group (n=13). Mitochondrions were extracted from the muscle tissue that were collected during the operation.Cytochrome Coxidase (COX) and MPTP were detected. Results The absorbance (A) ratio of MPTP in non-osteoporosis and osteoporosis groups was 0.994±0.007 and 0.985±0.006 respectively (P<0.05). The COX activity in non-osteoporosis and osteoporosis groups was 0.338±0.142 and 0.124±0.093 respectively (P<0.05). The correlation analysis revealed that there was a positive correlation between the A ratio of MPTP and COX activity (r=0.405,P<0.05). Conclusion The permeability of MMPT in osteoporotic muscle is greater than non-osteoporosis group, and COX is important for the permeability of MPTP, indicating that as long as maintaining the activity of COX, the change of MPTP could be effectively controlled, and cell aging would be prevented.     

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.     

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.     

核因子κB p65在肌肉萎缩中的表达与被动运动对其影响

目的 研究核因子κB(nuclearfactor kappa B,NF-κB)p65在失神经骨骼肌中的表达及被动运动对其影响作用,探讨失神经骨骼肌萎缩的分子机制及防治方法.方法 选Wistar大鼠42只,随机分成健康对照组(6只)、失神经对照组(18只)和被动运动组(18只).失神经对照组和被动运动组建立右下肢失神经腓肠肌动物模型.被动运动组大鼠采用右下肢被动运动300次/d.分别在术后2 d、14 d及28d取其双侧腓肠肌称重,计算肌湿重比,采用RT-PCR和Western Blot检测大鼠右侧失神经腓肠肌NF-κB p65的mRNA和蛋白表达水平,根据实验结果做统计处理数据得出结论.结果 大鼠腓肠肌失神经支配后肌肉湿重比持续下降,NF-κB p65的mRNA和蛋白表达在2 d、14 d、28d持续增加(P＜0.05),术后14 d、28d被动运动组腓肠肌湿重比高于同期失神经对照组(P＜0.05),被动运动组mRNA和蛋白的表达在各个时间点低于失神经对照组(P＜0.05),差异有统计学意义.结论 在失神经骨骼肌萎缩中,NF-κB p65的表达持续增高.被动运动可以通过干扰NF-κB p65的mRNA和蛋白质的表达来延缓失神经骨骼肌萎缩.     

川芎嗪对失神经骨骼肌萎缩大鼠FoXO3a、MAFbx以及MuRF1表达的影响

目的研究川芎嗪对失神经骨骼肌萎缩中FoXO3a、MAFbx及MuRF1表达的影响。方法 8周龄雌性SD大鼠54只,体重(200±10)g。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=24)、干预组(C组,n=24)。B、C组实验动物制备右下肢失神经腓肠肌动物模型,C组于模型制备后每日腹腔注射川芎嗪注射液80 mg/(kg.d),B组每日腹腔注射等剂量生理盐水;A组实验动物不作任何处理。取A组实验动物以及造模后2、7、14、28 d B、C组实验动物各6只,处死取其双侧腓肠肌称湿重,计算腓肠肌肌湿重比;另取右侧肌组织采用RT-PCR及Western blot检测各时间点FoXO3a、MAFbx、MuRF1 mRNA和蛋白表达水平。结果与A组比较,随着大鼠失神经时间延长,B、C组腓肠肌肌湿重比逐渐下降,差异均有统计学意义(P<0.05);造模后7、14、28 d,C组腓肠肌肌湿重比高于B组,且差异有统计学意义(P<0.05)。B、C组造模后7、14、28 d,FoXO3a、MAFbx、MuRF1 mRNA和蛋白表达水平均较A组高,C组各时间点均较B组降低,差异均有统计学意义(P<0.05)。结论川芎嗪可能通过降低FoXO3a、MAFbx以及MuRF1 mRNA和蛋白表达水平进而延缓失神经骨骼肌萎缩。     

氯沙坦减少细胞凋亡延缓失神经骨骼肌萎缩的实验研究

目的 失神经骨骼肌萎缩是临床亟待解决的难题,研究旨在探讨氯沙坦能否减少细胞凋亡,以期寻找延缓失神经骨骼肌萎缩的新途径.方法 雄性SD大鼠42只,随机分成3组(n=14),分别为Ⅰ组(对照组)、Ⅱ组(失神经对照组)和Ⅲ组(氯沙坦治疗组).Ⅰ组不作处理,Ⅱ组和Ⅲ组制备失神经腓肠肌(gastrocnemius, GAS)动物模型.术后Ⅲ组大鼠采用氯沙坦以10mg/kg·d空腹灌胃4周;Ⅰ、Ⅱ组大鼠分别以等剂量生理盐水灌胃.术后4周处死大鼠,以GAS和体重(body mass, BM)之比(GAS/BM)作为骨骼肌萎缩指标;TUNEL法检测腓肠肌细胞凋亡;免疫组织化学和Western blot检测GAS Bcl-2及Bax的表达.结果 术后4周Ⅰ组腓肠肌正常粗细;Ⅱ、Ⅲ组腓肠肌明显萎缩,肌肉变细,与周围组织粘连明显.GAS/BM Ⅱ组为11.68 4±1.98,与Ⅰ组(12.86±0.74)比较,差异有统计学意义(P<0.05):Ⅲ组GAS/BM为12.11±0.65;与Ⅱ组比较,差异无统计学意义(P>0.05).TUNEL法检测:Ⅰ组罕见凋亡细胞核,凋亡率为0.56%±0.2l%;Ⅱ组凋亡细胞核较Ⅰ组明显增多,凋亡率为11.32%±4.51%,两组比较差异有统计学意义(P<0.05);Ⅲ组可见凋亡细胞核,凋亡率为7.21%±2.05%,与Ⅱ组比较差异有统计学意义(P<0.05).免疫组织化学染色观察:Bcl-2阳性率Ⅱ组为18.3%±4.9%,较Ⅰ组(27.5%±2.8%)和Ⅲ组(25.5%±3.5%)低,差异均有统计学意义(P<0.05);Ⅲ组与Ⅰ比较,差异无统计学意义(P>0.05);Bax阳性率:Ⅱ组为24.1%±3.1%,较Ⅰ组(22.1%±3.6%)和Ⅲ组(21.7%4±2.3%)高,差异均有统计学意义(P<0.05);Ⅲ组与Ⅰ组比较差异无统计学意义(P>0.05).Ⅰ、Ⅱ及Ⅲ组Bax/Bcl-2平均分别为0.8、1.3及0.8.Western blot检测:Bcl-2蛋白表达Ⅱ组为122.5±14.6,较Ⅰ组(135.3±6.2)下降(P<0.05),Ⅲ组为139.2±16.2,较Ⅱ组增加(P<0.05);Bax蛋白表达Ⅱ组为107.1±15.8,Ⅰ组为89.3±8.4,差异有统计学意义(P<0.05),Ⅲ组为94.2±9.5,较Ⅱ组下降(P<0.05);Bcl-2蛋白和Bax蛋白表达Ⅲ组与Ⅰ组比较,差异均无统计学意义(P>0.05).结论 细胞凋亡在失神经骨骼肌萎缩中发挥重要作用,可能是骨骼肌萎缩的原因之一;氯沙坦可通过抑制肌细胞凋亡延缓大鼠失神经骨骼肌的萎缩.     

重组慢病毒介导心脏营养素-1基因转染延缓小鼠失神经骨骼肌萎缩

目的 探讨重组慢病毒介导的心脏营养素-1(cardiotrophin-1,CT-1)基因转染延缓小鼠失神经骨骼肌萎缩的疗效.方法 将36只Swiss小鼠,随机分为实验组和对照组,每组18只,切断胫神经,建立右下失神经支配肌萎缩模型.于实验组和对照组失神经腓肠肌分别转染重组慢病毒载体Lenti-GFP-CT-1和Lenti-GFP溶液20μl(108TU/ml),转染后2、4周,每个时间点分别取3只小鼠,于荧光显微镜下观察绿色荧光蛋白(GFP)的表达、Western blot检测CT-1的表达;取6只小鼠行单次收缩力、强直收缩力的检测,测定肌湿重、肌纤维横截面积,并观察肌纤维超微结构的变化.结果 慢病毒转染2,4周,两组腓肠肌中均可见大量GFP表达.Western blot检测显示实验组失神经腓肠肌中有明显的CT-1表达(P均<0.01).转染2周,实验组肌肉单次收缩力恢复率、强直收缩力恢复率、肌湿重维持率和肌纤维横截面积分别为[(47.61±6.25)%,x-±s,下同]、(56.08±5.47)%、(63.02±5.23)%、(1372.42±149.73)μm2,均明显高于对照组,后者分别为(27.23±5.06)%、(30.78±4.67)%、(52.41±4.98)%、(1147.28±128.67)μm2(P均<0.01);4周时,实验组上述各项指标分别为(33.13±4.76)%、(36.59±5.67)%、(51.46±5.36)%、(1209.12±142.57)μm2,仍明显高于对照组,后者分别为(16.40±5.48)%、(15.35±4.08)%、(39.15±6.12)%、(989.45±136.12)μm2(P均<0.01).此外,实验组肌浆网的扩张程度明显减轻.结论 慢病毒介导的基因治疗有较高的转染效率,其介导的CT-1基因转染能有效延缓小鼠失神经骨骼肌的萎缩,其疗效至少可以维持4周.     

神经干细胞移植防治骨骼肌失神经肌萎缩的电生理研究

目的探讨采用神经干细胞移植的方法防治骨骼肌失神经萎缩的可行性。方法采用机械分离的方法从孕14～16 d的SD孕鼠中获取神经干细胞,并于神经元限定性培养基中进行传代培养,制备神经干细胞单细胞悬液。采用切断右侧胫神经的方法建立腓肠肌失神经支配的动物(SD大鼠)模型。将108只SD大鼠按注射药物的不同随机分为3组,每组36只大鼠。实验组:将神经干细胞悬液注射到切断的胫神经远端。损伤组:注射等量的生理盐水。对照组:注射等量的细胞培养液。术后8、12周采用HRP逆行示踪技术检测失神经骨骼肌重获神经再支配的情况,并应用肌肉电生理方法对重获神经再支配的骨骼肌进行功能评价。结果术后8、12周实验组用电刺激细胞移植部位的腓肠肌,均可引出肌肉收缩活动;且随着时间的延长,单次收缩的波幅、速度,和强直收缩的时间和强直收缩波幅的恢复率均进一步得到改善。对照组和损伤组均未能引出肌肉活动。结论神经干细胞移植能够实现失神经骨骼肌的神经再支配,并且能够与骨骼肌建立起功能性突触连接,有效预防骨骼肌的萎缩。     

大鼠失神经支配骨骼肌萎缩后肌细胞凋亡及Bcl-2、Bax表达的变化

目的 研究失神经支配骨骼肌萎缩后肌细胞凋亡及Bcl-2、Bax表达的变化规律.方法 实验以失神经支配腓肠肌为动物模型.选用体质量为200～250 g的雌性SD大鼠42只,随机分成2组,实验组36只,健康对照组6只.按术后取材时间的不同再将实验组分为6组,每组6只大鼠.分别运用Tunel法及免疫组织化学方法检测失神经后不同时间萎缩腓肠肌细胞凋亡及Bcl-2、Bax的表达变化,同时测量肌湿重比和肌纤维横截面积,透射电镜观察肌细胞的超微结构变化.结果 萎缩的腓肠肌湿重比在失神经4周内和第10周到第12周下降最快,而在第4周到10周和第12周到16周下降较慢,且变化差异无统计学意义(P＞0.05).Bcl-2仅在失神经早期表达增高,于第4周达高峰;而Bax分别于失神经第2周与第12周达高峰,增高幅度大于Bcl-2.Bcl-2/Bax的比值除对照组(1.522±0.215)和失神经第8周时(1.065±0.165)大于1外,其余各时间点均小于1.肌细胞凋亡率分别于失神经第4周和第12周达到高峰,其变化趋势与Bax相仿.结论 失神经腓肠肌萎缩过程呈现4个阶段;凋亡和凋亡相关基因Bax过量表达在失神经不可逆肌萎缩过程中发挥着重要的作用;靶向作用Bax基因,抑制Bax表达可能延缓失神经骨骼肌萎缩.     

班布特罗与当归补血汤对失神经肌肉萎缩防治作用的实验研究

目的：研究班布特罗及当归补血汤对防治失神经骨骼肌萎缩的作用效果及有无协同作用。方法 SD大鼠40只，随机分组，每组10只，建立失神经骨骼肌萎缩的实验模型。术后4周处死大鼠，双侧腓肠肌及心脏称重，测定肌肉SOD含量，测肌纤维直径，检测细胞凋亡情况。结果 各用药组与对照组肌总蛋白含量、SOD含量、肌纤维直径均无显著性差异（P〉0．05）。在排除个体体重差异后肌肉湿重亦无显著性差异。两种药物之间无协同作用。各组均可见凋亡细胞，荧光显微镜下肉眼观测凋亡细胞的数量无显著性差异。结论 班布特罗与当归补血汤在4周时无明显预防肌萎缩作用及协同作用；细胞凋亡在失神经肌萎缩中发挥一定的作用；本实验为探索药物长期应用防治失神经骨骼肌萎缩的研究提供借鉴。     

电穿孔介导睫状神经营养因子基因转染延缓失神经肌萎缩

目的　探索一次性电穿孔介导睫状神经营养因子 (ciliaryneurotrophicfactor ,CNTF)转染延缓失神经骨骼肌萎缩的疗效。方法　制备 3 6只SD大鼠右下肢腓肠肌失神经支配模型 ,按手术先后顺序随机分成失神经对照组 (A组 )和CNTF基因转染组 (B组 ) ,每组 18只大鼠。于术后 2、4、8周测定肌湿重维持率、肌细胞截面积、肌肉蛋白含量、胶原纤维与肌细胞面积比和细胞凋亡数。结果　术后 2、4周B组的肌湿重维持率、肌细胞截面积和肌肉蛋白含量均明显高于A组 (P <0 .0 5 ) ,但胶原纤维与肌细胞面积比和细胞凋亡数明显低于A组 (P <0 .0 5 )。术后 8周 ,两组间各参数无明显区别 (P >0 .0 5 )。结论　一次性电穿孔介导CNTF基因转染可延缓失神经骨骼肌萎缩四周。