肌肉特异性microRNAs在失神经性萎缩骨骼肌中表达变化的研究
摘要
目的:探讨miR-1、miR-133和miR-206等3种肌肉特异性microRNAs在失神经支配所致骨骼肌萎缩过程中的表达变化情况,为进一步研究它们在失神经支配肌肉萎缩中的作用机制及其对法医身源学的意义等奠定基础。
方法:
实验动物健康清洁级12周龄C57小鼠,体重20±1g,雄性,由中国人民解放军第三军医大学动物中心提供。
分组及模型建立C57小鼠共30只,随机分为2组,其中一组为正常对照组不做任何处理(n=6),另外一组为实验组(n=24),该组又分为4个时相点,每个时相点为6只小鼠,采用1%戊巴比妥钠40mg/Kg腹腔注射麻醉后在无菌条件下手术:右后肢背侧部纵向切口,于股二头肌与股外侧肌之间钝性分离,游离坐骨神经,切除距梨状肌下缘2mm以下长约5mm的坐骨神经,近端游离,远侧断端返转180°后缝合于腓肠肌及股二头肌肌膜上,然后逐层缝合,建立小鼠失神经腓肠肌萎缩模型;左后肢游离坐骨神经后不做手术处理。
腓肠肌相对湿重百分比检测分别于手术后0d(正常对照组)、3d、7d、14d、28d断颈处死小鼠,用眼科剪逐层剪开小鼠双后肢皮肤及皮下组织,小心剥离腓肠肌肌膜,然后完整取下双侧腓肠肌,立即用电子分析天平称量腓肠肌湿重,计算腓肠肌相对湿重及其百分比,绘制湿重百分比变化曲线。
镜下观察切出用于Northern blot检测的肌肉后,将剩余肌肉经4%的多聚甲醛溶液固定,常规石蜡包埋,选择腓肠肌肌腹部连续切片,制成厚5μm的组织切片,HE染色后,于光镜高倍视野下观察。
肌纤维横截面积百分比的检测用HPIAS-1000图像分析系统进行测量。每张切片中测量50根肌纤维的横截面积,自动计算均数。用电子计算机计算腓肠肌肌纤维横截面积百分比,绘制横截面积百分比变化曲线。
Northern blots分析采用RNAiso reagent提取组织总RNA(Takara),具体方法参照说明书进行。以mir-1、133、206和U6等分子的反向互补的寡核苷酸DNA序列为探针(miR-1:5′-TACATACTTCTTTACATTCCA-3′;miR-133:5′-CAGCTGGTTGAAGGGGACCAAA-3′;miR-206 : 5′-CCACACACTTCCTTACATTCCA -3′;U6 : 5′-ATATGGAACGCTTCAATT-3′),采用γ-32P-ATP末端转移法标记探针。取20μg总RNA上样,行15%尿素变性聚丙烯酰胺凝胶电泳进行分离,半干转印恒流400mA转尼龙膜1h。紫外交联1200μJ.1 min,而后80℃烘烤30 min。加入放射性标记探针后于42℃杂交24h,洗膜后,-70℃放射自显影48h。成像结果以Quantity One软件进行灰度扫描,3分子不同时相点的灰度与内参U6进行比对,以失神经支配第0天结果为1,各分子各时相点的量化值以其为失神经支配第0天的倍数表示。
结果:
正常小鼠双侧腓肠肌存在差异
正常C57小鼠双侧腓肠肌湿重及肌纤维横截面积存在明显差异,根据统计学分析,P<0.05,具有统计学意义。
失神经支配对腓肠肌相对湿重及肌纤维横截面积百分比的影响
神经离断三天后腓肠肌相对湿重百分比明显下降,其比值随着失神经支配时间的延长,呈逐渐下降的趋势,表明小鼠坐骨神经离断后,随着失神经支配时间的延长,腓肠肌萎缩程度逐渐加重(P<0.001)。同理,腓肠肌肌纤维的CSA百分比也随着失神经支配时间的延长逐渐下降,其下降程度比肌肉相对湿重百分比的下降程度更为显著(P<0.001)。
失神经支配对腓肠肌形态学影响
病理切片结果显示:失神经早期肌细胞胞浆开始丧失,直径减小;细胞核表现出代谢活跃的征象,如核变大、核仁清晰、核居中等,此现象可能为失神经支配后肌细胞的代偿性反应。随着失神经支配时间的延长,肌纤维直径及横截面积明显减小,胞浆明显固缩,核内移现象愈来愈普遍,胞核增多,胶原纤维增生明显,炎性细胞大量浸润等。
失神经支配对肌肉特异性microRNAs表达的影响
肌肉特异性microRNAs随着失神经支配时间的延长发生一系列改变,与正常对照组相比,miR-206随着失神经支配时间的延长其表达明显上调,第28天其丰度达到正常的5倍以上;miR-1、miR-133随着失神经支配时间的延长其表达先迅速下调,而后逐渐有所回升,但至失神经支配第28天其表达仍明显低于正常水平。
结论
1.随着失神经支配时间的延长,肌肉特异性microRNAs的表达发生明显变化,推测肌肉特异性microRNAs可能在失神经介导的骨骼肌萎缩过程中发挥了一定作用,为从microRNAs角度去研究失神经支配所致骨骼肌萎缩的机制提供了新的思路。
2.随着失神经支配时间的延长,腓肠肌相对湿重及肌纤维横截面积百分比明显下降,表明我们建立的失神经支配腓肠肌萎缩模型是成功的。
3.正常小鼠双侧腓肠肌湿重及肌纤维横截面积存在明显差异,根据统计学分析,具有统计学意义。
Objective:to explore the expression changes of muscle special microRNAs(miR-1、miR-133、miR-206 et al )in the atrophied skeletal muscle resulted in by the denervation,and to establish the foundation to futher to study their molecular mechanisms on the denervated skeletal muscle and the significance on forensic identity source science
Method:
Experimental animal:male healthy C57 mice weighing 20±1g of clean degree and of 12 weeks old were selected in this study. These mice were provided by the experimental animal center at Third Military Medical University.
Grouping and model building:30 C57 mice were used and divided randomly into 2 groups:one of which was the normal control group without mice be disposed(n=6);the other group was the control group(n=24)whichwas divided averagely into 4 time points(n=6),mice of the group were operated on sterile condition after they were anesthetized by injecting 1% pentobarbital sodium at the standard of 40 mg/kg drug dose: cutting longitudinally the pars dorsalis of the right hindlimb of mice, Seperating bluntly musles between the biceps femoris and vastus lateralis,exposing the sciatic nerve, cutting approximate 5mm sciatic nerve at the position of the piriformis, lower edge 2mm, exposing freely the proximal end,suturing the distal end after be returned 180°in the sarolemma of both the biceps femoris and the gastrocnemius muscle ,and suturing in turn each layer tissue, which built the model of the atrophying gastrocnemius muscle of mice from the denervation. The sciatic nerve of the left hindlimb of mice wes not cut after being exposed.
Detecting the percentage Of the relative wet weight of gastrocnemius muscle: Killing separately mice on 0d(the normal control group).3d 7d 14d and 28d after being operated,cuting and seperating in turn the skin and subcutaneous tissue of bilateral hindlimbs by ophthalmmic scissors, peeling carefully the sarolemma of gastrocnemius muscle,cutting and obtaining integral bilateral gastrocnemius muscle,balancing their wet weight immediately by the electronic analytical balance,computing the relative wet weight of muscle and its percentage,and finally drawing the changing curve of percentage of the relative wet weight of muscle.
Observed by microscope:After the muscle which was used in Northern blot analysis was cut-out,the rest muscle was disposed by the following processes: fixed with 4% formaldehydum polymerisatum, paraffin-embedded, and making sections of 5μm thickness by selecting the serial Section of muscle belly of gastrocnemius muscle. The sections were dyed with HE and observed by microscope.
Detecting the percentage of CSA of muscle fibers: We measured CSA of 50 muscle fibers in each section and automatically computered their average with HPIAS-1000 image analysis system.After the percentage of CSA was computered with the computer, the changing curve of percentage of .it was drawn.
Northern blot analysis:Total RNA samples were extracted using RNAiso reagent(Takara). (concrete methods refering to the specification).The used probes were oligonucleotide DNA sequences of reverse complementary of mir-1、133、206 and U6( ( miR-1:5′-TACATACTTCTTTACATTCCA -3′;miR-133: 5′-CAGCTGGTTGAAGGGGACCAAA-3′;miR-206 : 5′-CCACACACTTCCTTACATTCCA -3′;U6 : 5′-ATATGGAACGCTTCAATT-3′)and were labeled by usingγ-32P-ATP endlabeled method. 20μg of total RNA samples were electrophoresed on denaturing 15% polyacrylamide gels and electroblotted for 1h onto Hybond-N+ membranes(Amersham) on the condition of constant-current(400mA). The membranes were UV-crosslinked on the condition of 1200μJ for 1min,baked on the condition of 80℃for 1h, hybridized withγ-32P-ATP endlabeled oligonucleotide DNA probes on the condition of 42℃for 24 h,and autoradiographied on the condition of -70℃for 48 h after being washed.Images were captured on film.and executed gray scale scanning by Quantity One. Grayscales of 3 moleculars in different time points were compareed with that of internal reference(U6) ( the result was expressed using 1 on 0 day of denervation, quantized value of each molecular in each time points was expressed using multiple of the former).
Result:
Differences of bilateral gastrocnemius muscle in normal control group:
There were significant differences in wet weight and CSA of muscle fibers of bilateral gastrocnemius muscle in normal C57 mice.According to statistic analysis, the differences had statistical significance(P﹤0.05).
Effects of denervation on the percentage of relative wet weight and CSA of muscle fibers of gastrocnemius muscle:
After the sciatic nerve had been cut for 3 days,the percentage of relative wet weight of gastrocnemius muscle decreased significantly compared with the control group, and the ratio decreased gradually with the time of denervation prolonging,which the atrlphic degree of gastrocnemius muscle of mice aggravated gradually with time of denervation prolonging(P﹤0.001).The same holds ture with effects of CSA of muscle fibers. The percentage of CSA of muscle fibers decreased also gradually with the time of denervation prolonging,and its decreased degree was more significant than that of relative wet weight of gastrocnemius muscle(P﹤0.001).
Effects of denervation on morphology of gastrocnemius muscle:
High power microscopic(200) ,pathological sections showed that cytoplasm of myocyte started to lose in the early stage of the denervation,the diameter decreased,and Nucleus showed signs of active metabolism(forexample, volume of Nucleus changed big, clear nucleolus, Nucleus lied in the center of cells,and so on),which could be the compensatory reaction of myocyte of denervation. With the time of denervation prolonging,the diameter and CSA of muscle fibers decreaded significantly, cytoplasm pyknosis obviously, the phenomenon of nuclear ingression was more and more widespread,nucleus increased, collagen fibers hyperplasiaed obviously, inflammatory cells invaded generally,and so on.
Effects of denervation on expressions of muscle specific microRNAs:
Compared with the normal control group,expressions of muscle specific microRNAs appeared a series of changes with the time of denervation prolonging. The expression of miR-206 was obviously up-regulated with the time denervation prolonging, and on the twenty-eighth days, its abundance was over 5 times higher than that of normal muscle; however, those of miR-1 and miR-133 decreased firstly and then gradually recovered with the time elapsing,and on the twenty-eighth days,their abundance are still lower than that of normal muscle.
Conclusion:
1.With the time denervation prolonging, the expressions of muscle specific microRNAs appeared significant changes,therefore,we speculate that they could play a role to a certain degree during the denervated atrophy of skeletal muscle,which offered new view to explore the mechanisms of skeletal muscle atrophy in the level of microRNAs.
2.With the time denervation prolonging,the percentage of relative wet weight of gastrocnemius muscle and CSA of muscle fibers degreased significantly,which proved that the mouse model of sciatic nerve resection was successfully established;
3.There were significant differences in wet weight and CSA of muscle fibers of bilateral gastrocnemius muscle in normal C57 mice.According to statistic analysis, the differences had statistical significance..
方法:
实验动物健康清洁级12周龄C57小鼠,体重20±1g,雄性,由中国人民解放军第三军医大学动物中心提供。
分组及模型建立C57小鼠共30只,随机分为2组,其中一组为正常对照组不做任何处理(n=6),另外一组为实验组(n=24),该组又分为4个时相点,每个时相点为6只小鼠,采用1%戊巴比妥钠40mg/Kg腹腔注射麻醉后在无菌条件下手术:右后肢背侧部纵向切口,于股二头肌与股外侧肌之间钝性分离,游离坐骨神经,切除距梨状肌下缘2mm以下长约5mm的坐骨神经,近端游离,远侧断端返转180°后缝合于腓肠肌及股二头肌肌膜上,然后逐层缝合,建立小鼠失神经腓肠肌萎缩模型;左后肢游离坐骨神经后不做手术处理。
腓肠肌相对湿重百分比检测分别于手术后0d(正常对照组)、3d、7d、14d、28d断颈处死小鼠,用眼科剪逐层剪开小鼠双后肢皮肤及皮下组织,小心剥离腓肠肌肌膜,然后完整取下双侧腓肠肌,立即用电子分析天平称量腓肠肌湿重,计算腓肠肌相对湿重及其百分比,绘制湿重百分比变化曲线。
镜下观察切出用于Northern blot检测的肌肉后,将剩余肌肉经4%的多聚甲醛溶液固定,常规石蜡包埋,选择腓肠肌肌腹部连续切片,制成厚5μm的组织切片,HE染色后,于光镜高倍视野下观察。
肌纤维横截面积百分比的检测用HPIAS-1000图像分析系统进行测量。每张切片中测量50根肌纤维的横截面积,自动计算均数。用电子计算机计算腓肠肌肌纤维横截面积百分比,绘制横截面积百分比变化曲线。
Northern blots分析采用RNAiso reagent提取组织总RNA(Takara),具体方法参照说明书进行。以mir-1、133、206和U6等分子的反向互补的寡核苷酸DNA序列为探针(miR-1:5′-TACATACTTCTTTACATTCCA-3′;miR-133:5′-CAGCTGGTTGAAGGGGACCAAA-3′;miR-206 : 5′-CCACACACTTCCTTACATTCCA -3′;U6 : 5′-ATATGGAACGCTTCAATT-3′),采用γ-32P-ATP末端转移法标记探针。取20μg总RNA上样,行15%尿素变性聚丙烯酰胺凝胶电泳进行分离,半干转印恒流400mA转尼龙膜1h。紫外交联1200μJ.1 min,而后80℃烘烤30 min。加入放射性标记探针后于42℃杂交24h,洗膜后,-70℃放射自显影48h。成像结果以Quantity One软件进行灰度扫描,3分子不同时相点的灰度与内参U6进行比对,以失神经支配第0天结果为1,各分子各时相点的量化值以其为失神经支配第0天的倍数表示。
结果:
正常小鼠双侧腓肠肌存在差异
正常C57小鼠双侧腓肠肌湿重及肌纤维横截面积存在明显差异,根据统计学分析,P<0.05,具有统计学意义。
失神经支配对腓肠肌相对湿重及肌纤维横截面积百分比的影响
神经离断三天后腓肠肌相对湿重百分比明显下降,其比值随着失神经支配时间的延长,呈逐渐下降的趋势,表明小鼠坐骨神经离断后,随着失神经支配时间的延长,腓肠肌萎缩程度逐渐加重(P<0.001)。同理,腓肠肌肌纤维的CSA百分比也随着失神经支配时间的延长逐渐下降,其下降程度比肌肉相对湿重百分比的下降程度更为显著(P<0.001)。
失神经支配对腓肠肌形态学影响
病理切片结果显示:失神经早期肌细胞胞浆开始丧失,直径减小;细胞核表现出代谢活跃的征象,如核变大、核仁清晰、核居中等,此现象可能为失神经支配后肌细胞的代偿性反应。随着失神经支配时间的延长,肌纤维直径及横截面积明显减小,胞浆明显固缩,核内移现象愈来愈普遍,胞核增多,胶原纤维增生明显,炎性细胞大量浸润等。
失神经支配对肌肉特异性microRNAs表达的影响
肌肉特异性microRNAs随着失神经支配时间的延长发生一系列改变,与正常对照组相比,miR-206随着失神经支配时间的延长其表达明显上调,第28天其丰度达到正常的5倍以上;miR-1、miR-133随着失神经支配时间的延长其表达先迅速下调,而后逐渐有所回升,但至失神经支配第28天其表达仍明显低于正常水平。
结论
1.随着失神经支配时间的延长,肌肉特异性microRNAs的表达发生明显变化,推测肌肉特异性microRNAs可能在失神经介导的骨骼肌萎缩过程中发挥了一定作用,为从microRNAs角度去研究失神经支配所致骨骼肌萎缩的机制提供了新的思路。
2.随着失神经支配时间的延长,腓肠肌相对湿重及肌纤维横截面积百分比明显下降,表明我们建立的失神经支配腓肠肌萎缩模型是成功的。
3.正常小鼠双侧腓肠肌湿重及肌纤维横截面积存在明显差异,根据统计学分析,具有统计学意义。
Objective:to explore the expression changes of muscle special microRNAs(miR-1、miR-133、miR-206 et al )in the atrophied skeletal muscle resulted in by the denervation,and to establish the foundation to futher to study their molecular mechanisms on the denervated skeletal muscle and the significance on forensic identity source science
Method:
Experimental animal:male healthy C57 mice weighing 20±1g of clean degree and of 12 weeks old were selected in this study. These mice were provided by the experimental animal center at Third Military Medical University.
Grouping and model building:30 C57 mice were used and divided randomly into 2 groups:one of which was the normal control group without mice be disposed(n=6);the other group was the control group(n=24)whichwas divided averagely into 4 time points(n=6),mice of the group were operated on sterile condition after they were anesthetized by injecting 1% pentobarbital sodium at the standard of 40 mg/kg drug dose: cutting longitudinally the pars dorsalis of the right hindlimb of mice, Seperating bluntly musles between the biceps femoris and vastus lateralis,exposing the sciatic nerve, cutting approximate 5mm sciatic nerve at the position of the piriformis, lower edge 2mm, exposing freely the proximal end,suturing the distal end after be returned 180°in the sarolemma of both the biceps femoris and the gastrocnemius muscle ,and suturing in turn each layer tissue, which built the model of the atrophying gastrocnemius muscle of mice from the denervation. The sciatic nerve of the left hindlimb of mice wes not cut after being exposed.
Detecting the percentage Of the relative wet weight of gastrocnemius muscle: Killing separately mice on 0d(the normal control group).3d 7d 14d and 28d after being operated,cuting and seperating in turn the skin and subcutaneous tissue of bilateral hindlimbs by ophthalmmic scissors, peeling carefully the sarolemma of gastrocnemius muscle,cutting and obtaining integral bilateral gastrocnemius muscle,balancing their wet weight immediately by the electronic analytical balance,computing the relative wet weight of muscle and its percentage,and finally drawing the changing curve of percentage of the relative wet weight of muscle.
Observed by microscope:After the muscle which was used in Northern blot analysis was cut-out,the rest muscle was disposed by the following processes: fixed with 4% formaldehydum polymerisatum, paraffin-embedded, and making sections of 5μm thickness by selecting the serial Section of muscle belly of gastrocnemius muscle. The sections were dyed with HE and observed by microscope.
Detecting the percentage of CSA of muscle fibers: We measured CSA of 50 muscle fibers in each section and automatically computered their average with HPIAS-1000 image analysis system.After the percentage of CSA was computered with the computer, the changing curve of percentage of .it was drawn.
Northern blot analysis:Total RNA samples were extracted using RNAiso reagent(Takara). (concrete methods refering to the specification).The used probes were oligonucleotide DNA sequences of reverse complementary of mir-1、133、206 and U6( ( miR-1:5′-TACATACTTCTTTACATTCCA -3′;miR-133: 5′-CAGCTGGTTGAAGGGGACCAAA-3′;miR-206 : 5′-CCACACACTTCCTTACATTCCA -3′;U6 : 5′-ATATGGAACGCTTCAATT-3′)and were labeled by usingγ-32P-ATP endlabeled method. 20μg of total RNA samples were electrophoresed on denaturing 15% polyacrylamide gels and electroblotted for 1h onto Hybond-N+ membranes(Amersham) on the condition of constant-current(400mA). The membranes were UV-crosslinked on the condition of 1200μJ for 1min,baked on the condition of 80℃for 1h, hybridized withγ-32P-ATP endlabeled oligonucleotide DNA probes on the condition of 42℃for 24 h,and autoradiographied on the condition of -70℃for 48 h after being washed.Images were captured on film.and executed gray scale scanning by Quantity One. Grayscales of 3 moleculars in different time points were compareed with that of internal reference(U6) ( the result was expressed using 1 on 0 day of denervation, quantized value of each molecular in each time points was expressed using multiple of the former).
Result:
Differences of bilateral gastrocnemius muscle in normal control group:
There were significant differences in wet weight and CSA of muscle fibers of bilateral gastrocnemius muscle in normal C57 mice.According to statistic analysis, the differences had statistical significance(P﹤0.05).
Effects of denervation on the percentage of relative wet weight and CSA of muscle fibers of gastrocnemius muscle:
After the sciatic nerve had been cut for 3 days,the percentage of relative wet weight of gastrocnemius muscle decreased significantly compared with the control group, and the ratio decreased gradually with the time of denervation prolonging,which the atrlphic degree of gastrocnemius muscle of mice aggravated gradually with time of denervation prolonging(P﹤0.001).The same holds ture with effects of CSA of muscle fibers. The percentage of CSA of muscle fibers decreased also gradually with the time of denervation prolonging,and its decreased degree was more significant than that of relative wet weight of gastrocnemius muscle(P﹤0.001).
Effects of denervation on morphology of gastrocnemius muscle:
High power microscopic(200) ,pathological sections showed that cytoplasm of myocyte started to lose in the early stage of the denervation,the diameter decreased,and Nucleus showed signs of active metabolism(forexample, volume of Nucleus changed big, clear nucleolus, Nucleus lied in the center of cells,and so on),which could be the compensatory reaction of myocyte of denervation. With the time of denervation prolonging,the diameter and CSA of muscle fibers decreaded significantly, cytoplasm pyknosis obviously, the phenomenon of nuclear ingression was more and more widespread,nucleus increased, collagen fibers hyperplasiaed obviously, inflammatory cells invaded generally,and so on.
Effects of denervation on expressions of muscle specific microRNAs:
Compared with the normal control group,expressions of muscle specific microRNAs appeared a series of changes with the time of denervation prolonging. The expression of miR-206 was obviously up-regulated with the time denervation prolonging, and on the twenty-eighth days, its abundance was over 5 times higher than that of normal muscle; however, those of miR-1 and miR-133 decreased firstly and then gradually recovered with the time elapsing,and on the twenty-eighth days,their abundance are still lower than that of normal muscle.
Conclusion:
1.With the time denervation prolonging, the expressions of muscle specific microRNAs appeared significant changes,therefore,we speculate that they could play a role to a certain degree during the denervated atrophy of skeletal muscle,which offered new view to explore the mechanisms of skeletal muscle atrophy in the level of microRNAs.
2.With the time denervation prolonging,the percentage of relative wet weight of gastrocnemius muscle and CSA of muscle fibers degreased significantly,which proved that the mouse model of sciatic nerve resection was successfully established;
3.There were significant differences in wet weight and CSA of muscle fibers of bilateral gastrocnemius muscle in normal C57 mice.According to statistic analysis, the differences had statistical significance..
引文
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