共同性外斜视眼外肌病理学和MyoD、myogenin表达的研究
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摘要
目的：本课题旨在研究共同性外斜视眼外肌中MyoD1+和myogenin+活性肌卫星细胞数量、成肌调节因子MyoD和myogenin的mRNA表达与眼外肌病理学变化的关系，探讨MyoD和myogenin在共同性外斜视病因中的作用。
方法：1．收集我院收治病人24例，并分为先天性外斜视儿童组、间歇性外斜视儿童组、共同性外斜视成人组、成人对照组，各6例。斜视术中截除的内直肌；对照组为眼球摘除术中取得的水平直肌，固定切片后行HE染色和MyoD1、myogenin免疫组织化学染色。观察肌肉形态；测量肌细胞横截面积；计数100个肌纤维中MyoD1+和myogenin+阳性肌卫星细胞核数。2．收集我院收治病人27例，分为先天性外斜视儿童组(5例)、间歇性外斜视儿童组(9例)、共同性外斜视成人组(6例)、成人对照组(7例)，取材同方法1，荧光定量PCR检测各眼外肌中MyoD和myogenin的mRNA相对表达量，GAPDH为内参对照。各定量指标行组间比较。
结果：共同性外斜视患者弱侧眼外肌，即内直肌病理改变明显，平均肌纤维横截面积(288．5±65．3μm~2)、MyoD1+(2．2％±1．2％)和myogenin+(2．4±2．3％)肌卫星细胞数量均较正常对照组(分别为820．0±42．7μm~2；4．0％±0．5％；4．4±0．38％)明显减小(P＜0．05)。先天性外斜视儿童组平均肌纤维横截面积、MyoD1+活性卫星细胞数(分别为241．0±28．9μm~2；2．0％±0．1％)较间歇性外斜视儿童组(分别为310．0±95．3μm~2；3．0％±1．5％)减小，但差异无明显统计学意义；而先天性外斜视儿童组myogenin+卫星细胞数(0．6±0．1％)较间歇性外斜视儿童组(4．0±2．3％)明显减少(P＜0．05)。共同性外斜视成人组平均肌纤维横截面积(303．5±35．6μm~2)、MyoD1+(1．6±1．1％)和myogenin+(2．5±1．6％)卫星细胞数，较间歇性外斜视儿童减小，但显著性差异；而较成人对照组明显减小(P＜0．05)。MyoD基因mRNA表达在先天性外斜视儿童组(0．52±0．19)与间歇性外斜视儿童组(0．96±0．55)间，共同性外斜视成人组(0．20±0．19)与间歇性外斜视儿童组间，共同性外斜视成人组和正常成人对照组间(0．42±0．10)有显著差别(P＜0．05)，且均为前者表达低于后者。MyoD的mRNA表达与共同性外斜视患者斜视度、年龄呈负性相关性(分别为r=-0．528，P=0．014；r=-0．467，P=0．033)。各组间myogenin的mRNA表达无明显差异，且与斜视患者斜视度、年龄无相关性。
结论：共同性外斜视患者弱侧眼外肌病理性改变为：肌小节破坏，胶原组织增生，并且可见肌纤维数量减少、横截面积减小的萎缩变性。MyoD在成肌特异性基因调控中具有总开关的作用。眼外肌MyoD的mRNA表达与共同性外斜视患者斜视度和年龄呈负性相关，其表达的降低使Myogenin+和MyoD+的肌卫星细胞进行性减少，眼外肌自我更新和修复机制受到抑制，可能是肌纤维出现进行性病理改变，患者眼位控制程度渐差，斜视角度加大的重要的肌源性因素。MyoD的mRNA表达和MyoD+的肌卫星细胞减少是否为先天性外斜视发病的始动因素尚不明确，但其使眼外肌生后肌细胞增殖发育受到影响，可能是其眼外肌病理改变、临床上发病早且斜视度大的肌源性因素之一。Myogenin的mRNA表达在各斜视组间无明显差异并且与斜视度无相关性，其原因可能与维持保护共同性斜视眼外肌中Ⅰ型肌纤维类型有关，或是与肌肉内环境变化使基因转录后mRNA未能翻译成功能性的蛋白质有关。
Objective:To probe the number of the MyoD-and myogenin-positiveactivated satellite cells,mRNA expression of MyoD and myogenin gene,which are the members of MRFs,and pathological changes.in patients withconcomitant extropia.To investigate the relationship of these factors and therole of MyoD and myogenin in the etiology of concomitant extropia.
Methods:In part 1:to collect 24 cases with concomitant extropia and thendivide them into 4 groups which are the group of children with congenitalextropia,the group of children with intermittent extropia,the group of adultswith concomitant extropia and adults control group.Medial rectus muscles wereobtained with consent from patients undergoing surgical resection and were preparedfor histologic examination by fixation and paraffin embedding.Control muscles wereobtained from patients undergoing ophthalmectomy who had no history of strabismusor neuromuscular disease.Cross sections were obtained and stained by HE methodfor observing the pathological changes and measuring the cross-sectional area ofmyofibers.Cross sections were stained simmunohistochemically for the presence ofactivated satellite cells,as identified by MyoD and myogenin immunoreactivity.Thepercentages of MyoD-and myogenin-positive satellite cells per 100 myofibers incross section were calculated.In part 2:to collect 27 cases with concomitantextropia and then divide them into the same groups as that in part 1 and obtainthe muscles Using semiquantitative real-time PCR with SYBR Green,mRNA expression of MyoD and myogenin gene were calculated.To compareall the quantified data in different groups.
Results:The medial rectus muscles from the patients with extropia showedthe remarkable pathological changes,the decreased cross-sectional area ofmyofibers (288.5±65.3μm~2) and the decreased numbers of MyoD-(2.2%±1.2%) and myogenin-positive (2.4±2.3%) satellite cells comparingwith control group (820.0±42.7μm~2,4.0%±0.5%,4.4±0.38%,respectively)(P＜0.05).The cross-sectional area of myofibers and the number of MyoD-satellite cells were decreased in the group of children with congenital extropia (241.0±28.9μm~2,2.0%±0.1%,respectively),comparing with the group ofchildren with intermittent extropia (310.0±95.3μm~2,3.0%±1.5%,respectively).However,there was no significant differences between twogroups.The number of myogenin-positive satellite cells (0.6±0.1%) weredecreased in the former comparing with that (4.0±2.3%) in the latter(P＜0.05) The group of adults with extropia had decreased thecross-sectional area of myofibers (303.5±35.6μm~2) and the numbers ofMyoD-(1.6±1.1%) and myogenin-positive (2.5±1.6%) satellitecells,comparing with the group of children with intermittent extropia,however,there is no significant difference.The group of adults with extropiahad significantly decreased the cross-sectional area of myofibers and thenumbers of MyoD-and myogenin-positive satellite cells,comparing withadults control group(P＜0.05).There were significant differences (P＜0.05)in mRNA expression of MyoD gene between the group of children withcongenital extropia (0.52±0.19) and the group of children with intermittentextropia (0.96±0.55),the group of adults with extropia (0.20±0.19) and thegroup of children with intermittent extropia,and between the group of adultswith extropia and control group (0.42±0.10).The formers had lower levelthan the latters.There were negative correlation between mRNA expressionof MyoD gene and prism diopter of deviation and between mRNA expressionofMyoD gene and patients' age (r=-0.528,P=0.014;r=-0.467,P=0.033,respectively).There was no difference in mRNA expression of myogenin geneamong the groups.
Conclusions:There were muscular artrophy with decreased the numberof myofibers and cross-sectional area of that,accompanied with sarcomeredamaging and hyperplastic collagen tissue.MyoD has the role of masterswitch in the processing of myoblastic specific gene regulation.Thedecreased level of mRNA expression of MyoD gene may cause the decreasedthe numbers of MyoD-and myogenin-positive satellite cells,whichsuppressed the self-renewal and repairing process in extraocular muscle.Weconsidered it may be the muscular eitiology of concomitant exotropia with deteriorating process.Although it was not defined as the first cause forcongenital exotropia,the decreased level of mRNA expression of MyoD genemay affect the muscular development after birth.So we considered it shouldbe one of the factors which cause early onset and large deviation in congenitalexotropia.Unchanged mRNA expression level of myogenin gene mayberelate with sustaining existing MHC-I in the media rectus,or may becaused by mRNA not being translated to functional protein.
方法：1．收集我院收治病人24例，并分为先天性外斜视儿童组、间歇性外斜视儿童组、共同性外斜视成人组、成人对照组，各6例。斜视术中截除的内直肌；对照组为眼球摘除术中取得的水平直肌，固定切片后行HE染色和MyoD1、myogenin免疫组织化学染色。观察肌肉形态；测量肌细胞横截面积；计数100个肌纤维中MyoD1+和myogenin+阳性肌卫星细胞核数。2．收集我院收治病人27例，分为先天性外斜视儿童组(5例)、间歇性外斜视儿童组(9例)、共同性外斜视成人组(6例)、成人对照组(7例)，取材同方法1，荧光定量PCR检测各眼外肌中MyoD和myogenin的mRNA相对表达量，GAPDH为内参对照。各定量指标行组间比较。
结果：共同性外斜视患者弱侧眼外肌，即内直肌病理改变明显，平均肌纤维横截面积(288．5±65．3μm~2)、MyoD1+(2．2％±1．2％)和myogenin+(2．4±2．3％)肌卫星细胞数量均较正常对照组(分别为820．0±42．7μm~2；4．0％±0．5％；4．4±0．38％)明显减小(P＜0．05)。先天性外斜视儿童组平均肌纤维横截面积、MyoD1+活性卫星细胞数(分别为241．0±28．9μm~2；2．0％±0．1％)较间歇性外斜视儿童组(分别为310．0±95．3μm~2；3．0％±1．5％)减小，但差异无明显统计学意义；而先天性外斜视儿童组myogenin+卫星细胞数(0．6±0．1％)较间歇性外斜视儿童组(4．0±2．3％)明显减少(P＜0．05)。共同性外斜视成人组平均肌纤维横截面积(303．5±35．6μm~2)、MyoD1+(1．6±1．1％)和myogenin+(2．5±1．6％)卫星细胞数，较间歇性外斜视儿童减小，但显著性差异；而较成人对照组明显减小(P＜0．05)。MyoD基因mRNA表达在先天性外斜视儿童组(0．52±0．19)与间歇性外斜视儿童组(0．96±0．55)间，共同性外斜视成人组(0．20±0．19)与间歇性外斜视儿童组间，共同性外斜视成人组和正常成人对照组间(0．42±0．10)有显著差别(P＜0．05)，且均为前者表达低于后者。MyoD的mRNA表达与共同性外斜视患者斜视度、年龄呈负性相关性(分别为r=-0．528，P=0．014；r=-0．467，P=0．033)。各组间myogenin的mRNA表达无明显差异，且与斜视患者斜视度、年龄无相关性。
结论：共同性外斜视患者弱侧眼外肌病理性改变为：肌小节破坏，胶原组织增生，并且可见肌纤维数量减少、横截面积减小的萎缩变性。MyoD在成肌特异性基因调控中具有总开关的作用。眼外肌MyoD的mRNA表达与共同性外斜视患者斜视度和年龄呈负性相关，其表达的降低使Myogenin+和MyoD+的肌卫星细胞进行性减少，眼外肌自我更新和修复机制受到抑制，可能是肌纤维出现进行性病理改变，患者眼位控制程度渐差，斜视角度加大的重要的肌源性因素。MyoD的mRNA表达和MyoD+的肌卫星细胞减少是否为先天性外斜视发病的始动因素尚不明确，但其使眼外肌生后肌细胞增殖发育受到影响，可能是其眼外肌病理改变、临床上发病早且斜视度大的肌源性因素之一。Myogenin的mRNA表达在各斜视组间无明显差异并且与斜视度无相关性，其原因可能与维持保护共同性斜视眼外肌中Ⅰ型肌纤维类型有关，或是与肌肉内环境变化使基因转录后mRNA未能翻译成功能性的蛋白质有关。
Objective:To probe the number of the MyoD-and myogenin-positiveactivated satellite cells,mRNA expression of MyoD and myogenin gene,which are the members of MRFs,and pathological changes.in patients withconcomitant extropia.To investigate the relationship of these factors and therole of MyoD and myogenin in the etiology of concomitant extropia.
Methods:In part 1:to collect 24 cases with concomitant extropia and thendivide them into 4 groups which are the group of children with congenitalextropia,the group of children with intermittent extropia,the group of adultswith concomitant extropia and adults control group.Medial rectus muscles wereobtained with consent from patients undergoing surgical resection and were preparedfor histologic examination by fixation and paraffin embedding.Control muscles wereobtained from patients undergoing ophthalmectomy who had no history of strabismusor neuromuscular disease.Cross sections were obtained and stained by HE methodfor observing the pathological changes and measuring the cross-sectional area ofmyofibers.Cross sections were stained simmunohistochemically for the presence ofactivated satellite cells,as identified by MyoD and myogenin immunoreactivity.Thepercentages of MyoD-and myogenin-positive satellite cells per 100 myofibers incross section were calculated.In part 2:to collect 27 cases with concomitantextropia and then divide them into the same groups as that in part 1 and obtainthe muscles Using semiquantitative real-time PCR with SYBR Green,mRNA expression of MyoD and myogenin gene were calculated.To compareall the quantified data in different groups.
Results:The medial rectus muscles from the patients with extropia showedthe remarkable pathological changes,the decreased cross-sectional area ofmyofibers (288.5±65.3μm~2) and the decreased numbers of MyoD-(2.2%±1.2%) and myogenin-positive (2.4±2.3%) satellite cells comparingwith control group (820.0±42.7μm~2,4.0%±0.5%,4.4±0.38%,respectively)(P＜0.05).The cross-sectional area of myofibers and the number of MyoD-satellite cells were decreased in the group of children with congenital extropia (241.0±28.9μm~2,2.0%±0.1%,respectively),comparing with the group ofchildren with intermittent extropia (310.0±95.3μm~2,3.0%±1.5%,respectively).However,there was no significant differences between twogroups.The number of myogenin-positive satellite cells (0.6±0.1%) weredecreased in the former comparing with that (4.0±2.3%) in the latter(P＜0.05) The group of adults with extropia had decreased thecross-sectional area of myofibers (303.5±35.6μm~2) and the numbers ofMyoD-(1.6±1.1%) and myogenin-positive (2.5±1.6%) satellitecells,comparing with the group of children with intermittent extropia,however,there is no significant difference.The group of adults with extropiahad significantly decreased the cross-sectional area of myofibers and thenumbers of MyoD-and myogenin-positive satellite cells,comparing withadults control group(P＜0.05).There were significant differences (P＜0.05)in mRNA expression of MyoD gene between the group of children withcongenital extropia (0.52±0.19) and the group of children with intermittentextropia (0.96±0.55),the group of adults with extropia (0.20±0.19) and thegroup of children with intermittent extropia,and between the group of adultswith extropia and control group (0.42±0.10).The formers had lower levelthan the latters.There were negative correlation between mRNA expressionof MyoD gene and prism diopter of deviation and between mRNA expressionofMyoD gene and patients' age (r=-0.528,P=0.014;r=-0.467,P=0.033,respectively).There was no difference in mRNA expression of myogenin geneamong the groups.
Conclusions:There were muscular artrophy with decreased the numberof myofibers and cross-sectional area of that,accompanied with sarcomeredamaging and hyperplastic collagen tissue.MyoD has the role of masterswitch in the processing of myoblastic specific gene regulation.Thedecreased level of mRNA expression of MyoD gene may cause the decreasedthe numbers of MyoD-and myogenin-positive satellite cells,whichsuppressed the self-renewal and repairing process in extraocular muscle.Weconsidered it may be the muscular eitiology of concomitant exotropia with deteriorating process.Although it was not defined as the first cause forcongenital exotropia,the decreased level of mRNA expression of MyoD genemay affect the muscular development after birth.So we considered it shouldbe one of the factors which cause early onset and large deviation in congenitalexotropia.Unchanged mRNA expression level of myogenin gene mayberelate with sustaining existing MHC-I in the media rectus,or may becaused by mRNA not being translated to functional protein.
引文
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