大鼠股薄肌原位游离移植模型的建立及相关肌萎缩指标的检测

目的建立大鼠股薄肌原位游离移植的模型,评价术后肌萎缩与肌肉收缩功能的相关性。方法显露、游离大鼠股薄肌,在肌肉起止点处切断肌肉附着部,支配神经入肌点前0.5cm处切断该神经,并夹闭该肌的主要供血动静脉约50min,在此期间将肌肉原位重新缝合固定,并行神经端端吻合,复通肌肉血循环。分别在术后不同时段观测肌肉萎缩和肌肉收缩功能的相关指标,并比较其相关性。结果成功构建了吻合血管神经的大鼠股薄肌游离移植动物模型,各分组大鼠成活良好。肌肉移植后,肌湿重维持率先降低后增加,术后第4周时最低,为59.3±5.2%,术后晚期时仍低于对照组的水平(p<0.05);肌纤维横截面积呈现相同的变化趋势。计算术后移植肌肉的最大单或强直收缩力的维持率,术后早期(术后1～3周,失神经期),肌肉功能维持率下降较快,3周时最大单收缩力仅存对照侧的39.53±3.63%,随后逐渐上升,至术后30周时恢复为正常对照的83.16±8.19%;而最大强直收缩力则分别降至39.09±5.07%,然后恢复至86.74±2.88%。结论所构建的吻合血管神经的大鼠股薄肌游离移植动物模型符合研究需要;肌肉移植后肌湿重维持率和肌细胞横截面积先呈现典型肌萎缩后表现,后随神经的再支配,萎缩逐渐好转,但至术后30周时仍未恢复到术前对照组水平;肌湿重维持率和肌细胞横截面积的变化具有较好的一致性,与肌肉功能的变化具有良好的相关性。肌肉萎缩是游离肌肉移植后功能降低的主要原因之一,从蛋白质代谢角度出发,移植后肌肉内蛋白含量尤其是收缩蛋白含量的变化,是肌萎缩与肌肉功能的降低密切相关的重要联系点。     

肌肉游离移植后烟碱型乙酰胆碱受体ε和γ亚单位表达的变化及意义

目的在基因水平上定量的观察肌肉游离移植后冲经肌肉接头烟碱型乙酰胆碱受体(nAChR)ε和γ亚单位基因表达的动态变化情况,探讨移植后肌肉功能减退与nAChRγ和ε亚单位基因变化之间的可能关系。方法以正常Wistar大鼠股薄肌为对照,利用荧光定量RT-PCR的方法分别检测肌肉移植术后不同时间肌肉样本内ε和γ亚单位mRNA的表达。结果肌肉移植后ε和γ亚单位mRNA表达的拷贝数均是先增高后减少。ε亚单位mRNA表达的拷贝数存术后第3周降到最低后又逐渐增高,但到术后晚期仍低于对照组的水平,差异有统计学意义(P<0.05),而γ亚单位mRNA的拷贝数自术后第2周后开始减少,到术后30周时未完全消失,仍保持低水平的表达。结论终板区亚单位基因的表达变化可能与肌肉游离移植后nAChR的数量减少及功能降低有关系。     

聚集蛋白基因转染促进移植肌肉功能恢复的实验研究

目的 研究转染神经源性聚集蛋白基因对游离移植肌肉功能恢复的影响。方法 选大鼠股薄肌原位移植模型，按移植术后即刻肌肉内注射溶液的不同分为pCS2＋．-agrin基因转染实验组。pCS2＋空质粒组以及生理盐水注射组，后两组作为手术对照组，基因电转染术后不同时间检测各组肌肉收缩功能、神经源性agrin蛋白表达，以及终板区烟碱型乙酰胆碱受体（nAChR）数量的变化。结果 术后早期agrin基因转染组肌肉功能恢复明显优于空质粒转染组以及生理盐水注射组（4、5、10周，P〈0．05），但术后晚期15及20周时各组肌肉功能间差异无统计学意义。免疫组化显示agrin基因转染后，术后1周到5周，有大量棕黄色抗原抗体复合物沉积在终板及其邻近区域，但到第10周时表达明显减少，接近对照组水平。agrin基因转染组终板区的nAChR数量在各个时间点均高于实验对照组。结论 移植肌肉终板区转染神经源性agrin基因，促进了移植肌肉早期功能的恢复。     

生物电刺激对游离肌肉移植的影响

为了探讨电刺激对游离肌肉移植的影响，采用新西兰大白兔１５只，以兔股直肌为实验模型肌，实验侧埋入不锈钢环式电极，运用模拟正常运动终板的电刺激模式直接刺激肌肉，３个月后检查肌肉的湿重、最大切面积和肌肉的收缩特性。结果显示：实验侧肌肉的湿重、最大切面积和收缩特性均明显优于对照侧，经统计学处理有显著性差异（Ｐ＜０．０５）。说明电刺激使肌肉的形态和功能得到了良好的恢复，为临床肌肉移植后防止肌肉萎缩提供了依据。     

失神经萎缩肌组织中卫星细胞池的耗竭与Pax7的相关性研究

目的 探讨长期失神经骨骼肌在反复增殖分化过程中,肌卫星细胞池耗竭可能的信号通路.方法 选用2月龄SD雄性大鼠9只,体重180～200 g.以大鼠右侧为实验侧,制备腓肠肌失神经支配模型;左侧为对照侧,分离显示坐骨神经后缝合肌肉皮肤.观察大鼠一般情况,于术后3、7、21 d各取3只大鼠,切取两侧腓肠肌进行大体观察后,行Pax7和MyoD免疫荧光染色观察及mRNA表达检测.结果 9只大鼠术后均存活.实验侧后肢僵直,活动受限;对照侧肢体活动自如.术后各时间点实验侧腓肠肌均出现不同程度萎缩;对照侧腓肠肌较饱满有光泽.荧光染色观察显示,随失神经时间延长,实验侧MyoD阳性细胞数逐渐增多,21 d达高峰,与对照侧比较,差异均有统计学意义(P<0.05).实验侧Pax7阳性细胞逐渐降低,3、7 d与对照侧比较,差异均有统计学意义(P<0.05).实验侧MyoDmRNA表达逐渐升高,21 d达高峰,各时间点实验侧均显著高于对照侧(P<0.05);实验侧Pax7 mRNA表达逐渐降低,仅出现于3、7 d,与对照侧比较差异有统计学意义(P<0.05).结论 长期失神经肌萎缩引发的Pax7短暂上调提示卫星细胞池耗竭的原因之一在于细胞难以返回静止状态.     

不同时间神经修复后失神经肌肉及运动终板变化的实验研究

目的 研究失神经支配肌肉不同时间神经修复后,肌肉及运动终板变化.探讨神经修复的最佳时机.方法 建立失神经支配腓肠肌实验模型,以损伤后不同的神经修复时间(0、2、4、6、8、10周)随机分为6组,每组6只.右后肢为实验侧,左后肢不作任何处理为对照侧(对照组).神经修复手术后第6周取材,测定各项检测指标.结果 肌肉失神经支配0～4周组行神经修复后肌肉湿重呈下降趋势,但各组间比较差异无统计学意义(P>0.05);4周后神经修复组肌肉湿重下降尤为明显,维持于一定水平,肌细胞直径及截面积呈持续性下降.2周内神经修复组,运动终板降钙素基因相关肽(calcitonin gene-related peptide,CGRP)灰度值与对照组比较差异无统计学意义(P>0.05);4,6周组,CGRP灰度值明显低于2周内神经修复组(P<0.05);8周后进行神经修复其灰度值进一步下降,与6周内神经修复组比较差异有统计学意义(P<0.05).超微结构的变化趋势与透射电镜观察基本一致.结论 实验提示神经损伤后2～4周内修复效果较好,6周后修复萎缩肌肉逆转可能性降低,但其各项指标仍能维持在一定水平,有指征尝试手术修复.     

吻合血管的小肌肉游离移植动态修复晚期面瘫

目的探讨应用吻合血管的小肌肉游离移植动态修复晚期面瘫的疗效.方法自2003年3月至2005年3月,采用吻合血管去神经的拇、趾短伸肌游离移植修复晚期面瘫患者共26例,将肌肉的肌腱端悬吊于患侧的鼻翼、鼻唇沟中点、口角及颏窝上,肌腹放置于咬肌的支配神经周围,并进行跗外侧动、静脉与颞浅动、静脉的吻合,修复由面瘫引起的口角畸形及运动障碍.结果术后即刻可矫正面部静态外观;1个月后可活动患侧口角;3个月后经训练可恢复微笑;6个月后口部活动恢复正常者23例占88%,较正常者2例占8%,略有改善者1例占4%.结论吻合血管的去神经小肌肉游离移植动态修复晚期面瘫,其肌肉无液化、萎缩少、成活可靠、神经再生确实,是重建面部表情及恢复口角功能的较成熟的修复方法.     

自体移植静脉中sonic hedgehog表达水平的动物实验研究

目的 研究细胞周期相关因子sonic hedgehog(SHH)在大鼠自体移植静脉中的表达变化及其与内膜增生的关系.方法 雄性Wistar大鼠24只,8周龄,体质量140 g.建立颈静脉-腹主动脉移植模型,分别于术后14 d、28 d各处死12只大鼠,取材移植血管,免疫组化检测SHH蛋白在移植血管新生内膜中的表达,Western blot法观察SHH及细胞增殖核抗原(PCNA)的表达,Real-time PCR定量检测SHH mRNA的表达;以对侧颈静脉作为对照.结果 免疫组化结果显示,正常静脉、术后14 d和术后28 d移植静脉SHH+细胞数比例分别为(2.0±0.5)%、(39.4±0.4)%和(63.0±0.3)%,正常静脉与移植静脉差异有统计学意义(P<0.01).Western blot法显示术后SHH表达水平增加并与PCNA表达呈正相关(r=0.808,P<0.01).Real-time PCR结果显示术后14 d组和术后28 d组的SHH mRNA表达增加,分别为对照组含量的9.5和23.8倍.结论 自体静脉移植术后,SHH在新生内膜中高表达并可能与细胞增殖相关.     

肌肉游离移植后运动终板乙酰胆碱受体分布数量及肌肉功能的观察

目的观察肌肉游离移植后运动终板乙酰胆碱受体（AChR）分布数量的变化以及肌肉功能的动态恢复情况，并进一步探讨两者之间的可能关系。方法以正常SD大鼠股薄肌为对照，动态观察肌肉移植后收缩功能的恢复以及运动终板区AChR分布和数量的变化。结果肌肉游离移植后4周，个别肌肉样本检测到单收缩，大部分肌肉到5周时恢复不完全的强直收缩，术后10周到20周期间肌肉收缩功能进一步的恢复，但最终到30周时仍不能恢复到术前的水平。终板区AChR的数量从最初的失神经支配期急剧下降，到神经再支配初期短时期内骤然上升，随后平缓上升形成平台，但术后30周时AChR的数量仍低于对照组的水平。正常运动终板区AChR二维和三维图像为连续光滑、精细分支的网络状结构。术后早期终板呈弥散模糊的片状荧光图像；随神经再支配的完善，新生的运动终板图像为不规则的斑点。之后斑点面积逐渐增大、数量逐渐增多，相互衔接形成分支样立体结构，到术后30周时，新生运动终板的图像仍达不到移植前的精细程度。结论运动终板区AChR的分布和数量是影响肌肉功能的一个因素，并且只有在影响神经肌肉兴奋传递后才可能影响到肌肉的收缩功能。     

黄芪通过核转录因子/肌肉环状指蛋白1通路延缓失神经骨骼肌萎缩

目的 研究黄芪对核转录因子kappaB(nuclear factor of kappaB,NF-kappaB)p65和肌肉环状指蛋白1(muscle RING finger protein 1,MuRF1)活性的影响,探讨黄芪防治肌萎缩的作用及其机制.方法 54只Wistar大鼠,建立右下肢腓肠肌失神经支配模型,随机分为黄芪治疗组和对照组.采用逆转录聚合酶链反应(RT-PCR)和Western bloting检测术后2 d、7 d、14 d和28 d时大鼠腓肠肌NF-kappB p65和MuRF1的mRNA和蛋白表达水平,并结合肌肉湿重比分析其相关性.结果 大鼠腓肠肌失神经支配后NF-lcappaB p65和MuRF1的mRNA和蛋白表达在各时间点持续增加(P<0.05).术后7 d、14 d和28 d黄芪治疗组腓肠肌湿重比高于同期失神经对照组(P<0.05),而其NF-kappaB p65和MuRF1的表达则低于相应失神经对照组(P<0.05).结论 黄芪可以有效延缓去神经骨骼肌的萎缩,其作用机制与拮抗NF-kappaB p65、下调MuRF1的表达有关.     

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。     

Evaluation of Akt/mTOR activity in muscle atrophy after rotator cuff tears in a rat model

Atrophy of the rotator cuff muscles is a factor that complicates the treatment of a massive rotator cuff tear (RCT). However, the molecular mechanisms that govern the development of muscle atrophy after RCTs have not been well defined. The Akt/mammalian target of rapamycin (mTOR) signaling pathway plays a central role in maintaining muscle mass in response to mechanical loading. The role of this pathway in the development of muscle atrophy after a massive RCT remains unknown. The purpose of this study was to investigate the regulation of the Akt/mTOR pathway in the development of muscle atrophy after a RCT and suprascapular nerve (SSN) injury. We evaluated the activity of the Akt/mTOR signaling pathway and how this pathway interacts with two atrophy‐related genes, MuRF‐1 and MAFbx, in supraspinatus muscles of rats that underwent unilateral complete rotator cuff tendon transection or SSN transection. Akt/mTOR activity was significantly reduced after tendon rupture, but increased after nerve injury. MuRF‐1 and MAFbx were only up‐regulated following denervation. These results suggest that tendon transection leads to a decrease in protein synthesis with down‐regulation of the Akt/mTOR signaling pathway, whereas denervation leads to an increase in protein degradation via up‐regulation of expression of MuRF‐1 and MAFbx. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1440–1446, 2012     

Time-dependent expression of myostatin RNA transcript and protein in gastrocnemius muscle of mice after sciatic nerve resection

Myostatin, a member of the transforming growth factor-beta (TGF-beta) superfamily, has been identified as a negative regulator of skeletal muscle mass. To provide more data on the role of myostatin in denervation-induced muscle atrophy, we examined the time-dependent changes in myostatin mRNA and protein as well as Smad2 and phospho-Smad2 protein levels in the denervated gastrocnemius muscle of mice after sciatic neurectomy, using quantitative real-time RT-PCR and Western blotting, respectively. We conducted morphometric analyses to measure the wet weight ratio of the denervated muscle (the operated side/contralateral nonoperated side) and the cross-sectional area of muscle fibers, and observed the morphology of denervated muscle. The experimental results showed that in the early stage of denervation, the levels of myostatin mRNA and protein in the denervated gastrocnemius muscle increased instantly, reaching a peak at day 3 and day 7 after sciatic neurectomy, respectively, when compared with the normal values. In addition, the phospho-Smad2 protein was observed to have a similar expression profile to that of the myostatin mRNA. The present study perhaps opens a new window into myostatin modulation in muscle atrophy due to denervation.     

Correlation of contractile function recovery with acetylcholine receptor changes in a rat muscle flap model

The aim of this study was to investigate the correlation between contractile function recovery and changes of acetylcholine receptors (AChR) in a transferred muscle flap following reinnervation. Orthotopic transfer of the gracilis muscle flap with repair of its nerve was performed bilaterally in 48 rats. The rats were randomly divided into six experimental groups based on the time intervals for assessments (1, 4, 5, 10, 20, and 30 weeks). Sixteen gracilis muscle samples from eight rats without surgery were used as the controls. In each group, muscle contractile force and weight were measured (n = 16). The AChR numbers (n = 8) and subunits (? and γ) mRNA (n = 8) were examined using [¹²⁵I]‐α‐bungarotoxin and fluorescent quantitative‐PCR. The results showed the AChR numbers in the muscle flap increased from 4 to 20 weeks after reinnervation and correlated with recovery of the tetanic contraction force. However, correlation between the increase of AChR number with the specific tension (peak contractile force normalized to wet muscle weight) was only found from 4 to 10 weeks postoperatively. The expression of γ‐subunit mRNA increased at the early period after flap transfer and then decreased rapidly, whereas the ?‐subunit mRNA recovered gradually since fourth week postoperatively. A small amount of γ‐subunit mRNA could still be detected at 30 weeks after surgery. In conclusion, following reinnervation of the transferred muscle flap, the contractile functional recovery is partially correlated to increase of the AChR?. Our findings may provide evidence for further study of improving muscle function in functional reconstruction by targeting the AChR. © 2010 Wiley‐Liss, Inc. Microsurgery 2010.     

骨骼肌废用性萎缩信号通路及蛋白质代谢的研究进展

骨骼肌废用性萎缩是临床常见问题,其机制尚未完全明确。既往研究认为废用性萎缩的发生是通过一条或多条细胞信号通路的激活来实现,但也有研究指出废用性萎缩是泛素一蛋白酶体的活化而导致蛋白大量分解的结果。目前对于废用性萎缩的研究主要集中在NF—KB、IGF-1／P13K／Akt、TGF-β／Smad及MAPK信号通路对上游信号分子MuRF1和Atroginl／MAFbx的调控作用以及多条信号通路激活或抑制及其相互作用,进而通过泛素-蛋白酶体来影响蛋白质代谢。但对于MuRF1和Atroginl／MAFbx表达的调控机制还有待研究,参与废用性萎缩基因的确认和功能验证也需要深入研究。     

Early reattachment does not reverse atrophy and fat accumulation of the supraspinatus--an experimental study in rabbits.

INTRODUCTION: Reattachment of the supraspinatus (SSP) tendon after spontaneous rupture leads to improved shoulder function. Whether this improvement of function is due to a reversal of muscle atrophy and fat accumulation known to occur after SSP rupture is still debated. Our previous study of late reattachment of SSP (12 weeks) failed to confirm a reversal of muscle atrophy and of fat accumulation. PURPOSE: To find out whether earlier reattachment (6 weeks) reverses atrophy and fat accumulation of the SSP. MATERIAL AND METHODS: Reattachment group: in seven rabbits unilateral supraspinatus detachment, reattachment after 6 weeks and killing 6 weeks later. Detachment group: in seven rabbits unilateral supraspinatus detachment and killing 12 weeks later. The contralateral shoulders served as controls (n=14). Determination of the supraspinatus constituents: muscle, extra- and intramuscular fat in volume and cross-sectional area. RESULTS: Muscle tissue in the reattachment group (8.6 ml+/-1s.d.=0.6) and in the detachment group (8.9 ml+/-0.9) were less than in control supraspinati (10.2 ml+/-0.9, both p<0.05). Extra- and intramuscular fat in the reattachment group (8.7%+/-3.2) was greater than in both, the detachment group (4.6%+/-3.5), and control supraspinati (2.8%+/-1.7, both p<0.05). CONCLUSION: In the rabbit, reattachment of the SSP at 6 weeks did neither reverse muscle atrophy nor fat accumulation during the ensuing 6 weeks. However, earlier reattachment (6 weeks) when compared with later reattachment (12 weeks) prevented an increase in fat accumulation. On the other hand, the delay before reattaching the tendon did not lead to an increase in muscle atrophy.     

Long-term morphometric and immunohistochemical findings in human free microvascular muscle flaps

Reinnervation, muscle regeneration, density of microvessels, and muscle-type specific atrophy were studied 3-4 years after surgery in surgically nonreinnervated free microvascular muscle flaps to 13 patients transplanted to the upper or lower extremities. Routine histology and immunohistochemistry for PGP 9.5 and S-100 (neuronal markers), Ki-67 (cell proliferation), myosin (muscle fiber types), and CD-31 (endothelium) were carried out, and results were analyzed morphometrically. Three to 4 years after surgery, severe atrophy of predominantly slow-type fibers was seen in 9 cases. In 4 cases, muscle-fiber diameter and fiber-type distribution were close to normal. Long intraoperative muscle ischemia and postoperative immobilization were associated with poor muscle bulk in flaps. The density of microvessels in flaps did not differ from control muscles. PGP 9.5 and S-100 immunopositive nerve fibers were detected in 7 patients. Reinnervation was associated with good muscle bulk. In 4 patients, activation of satellite cells was evident. The results suggest that in some cases, spontaneous reinnervation may occur in free muscle flaps, and that several years after microvascular free flap transfer, the muscle still attempts to regenerate.     

Fiber type morphometry and capillary geometry in free, vascularized muscle transfers.

In order to establish the relationship among intraoperative ischemia time, the recovery of contractile function, muscle fiber morphometry, and capillary geometry following free muscle transfer, 15 male dogs underwent unilateral, orthotopic free transfer of the left gracilis muscle with microneurovascular anastomosis. Intraoperative ischemia time varied from 0 to 4 hours. After a recovery period averaging 14 months, isometric contractile function was measured in the transfers and the contralateral, control muscles. Muscle fiber type composition, fiber size, and capillary geometry were quantified from histochemical sections. Transferred and control muscles had similar type I fiber percentages, type I fiber sizes, and capillary densities. Type II fiber sizes were reduced in transfers relative to controls. There was no significant effect of intraoperative ischemia on any morphometric variable. Although contractile function was generally reduced after free, vascularized transfer, there was only a weak correlation between the recovery of tetanic tension and type I fiber percentage (r = 0.53, P less than 0.05). It is concluded that free, vascularized muscle transfer results in minimal disturbance of muscle fiber type composition, fiber size, or capillary supply and that the diminished contractile function observed in this setting is still largely unexplained.