川芎嗪治疗大鼠急性脊髓损伤差异表达蛋白质的蛋白质组学分析
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摘要
目的利用蛋白质组学技术和大鼠急性脊髓损伤(acute spinal cord injury, ASCI)模型,比较川芎嗪治疗与生理盐水治疗后脊髓蛋白质组表达谱的差异,鉴定川芎嗪作用相关的差异蛋白质。并对差异蛋白质进行初步功能分析,从蛋白质分子水平揭示川芎嗪治疗大鼠急性脊髓损伤的机制。
方法正常成年SD大鼠12只,采用改良ALLEN氏重物打击法制作急性脊髓中度损伤模型12个。随机分成两组：(1)生理盐水治疗组(NS组,N组)(n=6)；(2)川芎嗪治疗组(TMP组,T组)(n=6)。两组大鼠按术后动物存活的时间再各分为3天组和7天组两个亚组,每亚组3只大鼠。TMP组大鼠伤后30分钟开始予以川芎秦注射液经腹腔内注射200mg/kg,每天给药一次,3天组大鼠用药3天,7天组大鼠共用药5天。NS组大鼠给予等量的生理盐水经腹腔内注射,给药次数和实验组相同。造模后在相应的时间点处死大鼠并切取脊髓标本,每只大鼠取以损伤段为中心的长约6mm的脊髓组织。加入裂解液提取脊髓蛋白质后进行蛋白质样品浓度测定。以1000ug的上样量进行双向凝胶电泳,蛋白质凝胶采用考马斯亮蓝染色后用PDQuest-8.0.1图象分析软件进行凝胶图像分析,寻找并切取差异表达的蛋白质斑点。用胰酶进行胶内原位酶解,获得的小肽片段采用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)进行质谱分析。获取肽质量指纹图谱后采用MascotWizard软件查询SWISS-PROT和NCBInr数据库进行检索,鉴定差异蛋白质。基于文献复习对检索鉴定的差异蛋白质进行初步功能分析。
结果通过固相PH梯度双向凝胶电泳获得了分辨率较高的双向凝胶电泳图谱,图像分析后发现两组图谱间存在较明显差异,应用基质辅助激光解吸电离飞行时间质谱成功鉴定出了差异蛋白质。3天组共鉴定出了15个差异蛋白质,表达上调的有血清白蛋白前体、低分子量神经丝蛋白、波形蛋白、神经纤维酸性蛋白、α-1A微管蛋白、β-2A微管蛋白、β-2C微管蛋白、亮氨酸氨基肽酶、二氢嘧啶酶相关蛋白2,表达下调的有α-互联蛋白、60kDa热休克蛋白、71kDa热休克蛋白同源蛋白、70kDa热休克蛋白相关蛋白2、β-1热休克蛋白、磷脂结合蛋白1；7天组共鉴定出了19个差异蛋白质,表达上调的有血清白蛋白前体、α-1A微管蛋白、β-2A微管蛋白、低分子量神经丝蛋白、中分子量神经丝蛋白、乙醛脱氢酶、鸟嘌呤核苷酸结合蛋白β1亚基、β肌动蛋白、波形蛋白、神经纤维酸性蛋白,表达下调的有H2异性核蛋白、帕金森病蛋白7同系物、囊泡相关膜蛋白B、泛素结合酶E2N、丙酮酸脱氢酶E1β亚基、异柠檬酸脱氢酶α亚基、脑型肌酸激酶、α-互联蛋白、60kDa热休克蛋白。结合文献复习,差异蛋白质主要涉及到神经细胞的增殖、凋亡、应激反应等过程。
结论急性脊髓损伤川芎嗪治疗组与生理盐水治疗组蛋白质组表达谱存在差异,差异蛋白质主要涉及到神经细胞的增殖、凋亡、应激反应等过程。为进一步阐明脊髓损伤修复机制及川芎嗪治疗大鼠急性脊髓损伤的机制提供了理论依据。
OBJECTIVE Compare the differential expression of proteins in spinal tissue between rats treated with tetramethylpyrazine(TMP) and rats treated with normal sodium(NS) after acute spinal cord injury(ASCI) by using proteomic technology and ASCI model.Identify the differential proteins related to the effect of TMP. Analyze the primary function of the differential proteins.Reveal protein molecular mechanism of the curative effect of TMP in rats after ASCI.
METHODS A total of 12 healthy adult Sprague Dawley rats were used to establish the ASCI models according to Allen's weight drop methed. These models were divided randomly into two groups:(1) Normal sodium treatment group(group NS,n=6);(2)Tetramethylpyrazine treatment group (group TMP, n=6).Two groups animals were further divided into two sub-groups(n=3 per sub-group), each at 3 and 7 days of survival before euthanasia.200mg/kg of TMP was administered in the TMP group intraperitoneally and the NS group were treated with the same volume of normal solution 30 minutes after injury. Each rat was given the same dosage once a day until euthanasia or survived for 5 days following the spinal cord injury if the rats survived for more than 5 days. At the corresponding time points following the injury, the rats of two groups were killed and the spinal cord tissue samples were collected from the injured segment with the length of 0.6cm. The spinal cord protein's lysate was extracted and the concentrations of proteins were detected by Bradford method. Then the proteins were separated by two-dimensional gel electrophoresis(2-DE).The differential expression proteins were detected by PDQuest-8.0.1 software after the gels were stained with colloidal coomassie blue.These proteins were digested with trypsin into peptides.And the resulting tryptic peptides were analyzed by matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-Q-TOF).The mass spectrometric data were used to identify the proteins by searching SWISS-PROT and NCBInr protein sequence database through MASCOT software.The primary function of the differential proteins was analyzed by literature reviewing.
RESULTS High resolution gel maps were produced by 2-DE.The difference of gel maps between two groups were significant. The differential expression proteins were successfully identified by MALDI-Q-TOF.3 days after ASCI, a total of 15 differential expression proteins were identified. Up-regulated proteins were serum albumin、neurofilament light polypeptide、vimentin、glial fibrillary acidic protein、tubulin alpha-1A chain、tubulin beta-2A chain、tubulin beta-2C chain、leucyl aminopeptidase、dihydropyrimidinase-related protein 2; down-regulated proteins were alpha-internexin、60 kDa heat shock protein、heat shock cognate 71 kDa protein、heat shock related 70 kDa protein 2、heat shock protein beta-1、phosphatidylethanolamine-binding protein 1.7 days after ASCI, a total of 19 differential expression proteins were identified. Up-regulated proteins were serum albumin、tubulin alpha-1A chain、tubulin beta-2A chain、neurofilament light polypeptide、neurofilament medium polypeptide、aldehyde dehydrogenase、guanine nucleotide binding protein subunit beta-1、beta-actin、vimentin、glial fibrillary acidic protein;down-regulated proteins were heterogeneous nuclear ribonucleoprotein H2、parkinson disease protein 7 homolog、vesicle-associated membrane protein-associated protein B、ubiquitin-conjugating enzyme E2N、pyruvate dehydrogenase E1 component subunit beta、isocitrate dehydrogen ase [NAD] subunit alpha、creatine kinase B-type、alpha-internexin、60 kDa heat shock protein. By literature reviewing, the majority of differential expression proteins have been reported to participate in neuron growth, apoptosis and stress-reaction.
CONCULUSION There are proteomic difference between the expression proteins of TMP group and NS group.The majority of differential expression proteins have been reported to participate in neuron growth, apoptosis and stress-reaction. The data will be helpful to illuminate the mechanism involved in the recovery of ASCI and the curative effect of TMP in rats after ASCI.
方法正常成年SD大鼠12只,采用改良ALLEN氏重物打击法制作急性脊髓中度损伤模型12个。随机分成两组：(1)生理盐水治疗组(NS组,N组)(n=6)；(2)川芎嗪治疗组(TMP组,T组)(n=6)。两组大鼠按术后动物存活的时间再各分为3天组和7天组两个亚组,每亚组3只大鼠。TMP组大鼠伤后30分钟开始予以川芎秦注射液经腹腔内注射200mg/kg,每天给药一次,3天组大鼠用药3天,7天组大鼠共用药5天。NS组大鼠给予等量的生理盐水经腹腔内注射,给药次数和实验组相同。造模后在相应的时间点处死大鼠并切取脊髓标本,每只大鼠取以损伤段为中心的长约6mm的脊髓组织。加入裂解液提取脊髓蛋白质后进行蛋白质样品浓度测定。以1000ug的上样量进行双向凝胶电泳,蛋白质凝胶采用考马斯亮蓝染色后用PDQuest-8.0.1图象分析软件进行凝胶图像分析,寻找并切取差异表达的蛋白质斑点。用胰酶进行胶内原位酶解,获得的小肽片段采用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)进行质谱分析。获取肽质量指纹图谱后采用MascotWizard软件查询SWISS-PROT和NCBInr数据库进行检索,鉴定差异蛋白质。基于文献复习对检索鉴定的差异蛋白质进行初步功能分析。
结果通过固相PH梯度双向凝胶电泳获得了分辨率较高的双向凝胶电泳图谱,图像分析后发现两组图谱间存在较明显差异,应用基质辅助激光解吸电离飞行时间质谱成功鉴定出了差异蛋白质。3天组共鉴定出了15个差异蛋白质,表达上调的有血清白蛋白前体、低分子量神经丝蛋白、波形蛋白、神经纤维酸性蛋白、α-1A微管蛋白、β-2A微管蛋白、β-2C微管蛋白、亮氨酸氨基肽酶、二氢嘧啶酶相关蛋白2,表达下调的有α-互联蛋白、60kDa热休克蛋白、71kDa热休克蛋白同源蛋白、70kDa热休克蛋白相关蛋白2、β-1热休克蛋白、磷脂结合蛋白1；7天组共鉴定出了19个差异蛋白质,表达上调的有血清白蛋白前体、α-1A微管蛋白、β-2A微管蛋白、低分子量神经丝蛋白、中分子量神经丝蛋白、乙醛脱氢酶、鸟嘌呤核苷酸结合蛋白β1亚基、β肌动蛋白、波形蛋白、神经纤维酸性蛋白,表达下调的有H2异性核蛋白、帕金森病蛋白7同系物、囊泡相关膜蛋白B、泛素结合酶E2N、丙酮酸脱氢酶E1β亚基、异柠檬酸脱氢酶α亚基、脑型肌酸激酶、α-互联蛋白、60kDa热休克蛋白。结合文献复习,差异蛋白质主要涉及到神经细胞的增殖、凋亡、应激反应等过程。
结论急性脊髓损伤川芎嗪治疗组与生理盐水治疗组蛋白质组表达谱存在差异,差异蛋白质主要涉及到神经细胞的增殖、凋亡、应激反应等过程。为进一步阐明脊髓损伤修复机制及川芎嗪治疗大鼠急性脊髓损伤的机制提供了理论依据。
OBJECTIVE Compare the differential expression of proteins in spinal tissue between rats treated with tetramethylpyrazine(TMP) and rats treated with normal sodium(NS) after acute spinal cord injury(ASCI) by using proteomic technology and ASCI model.Identify the differential proteins related to the effect of TMP. Analyze the primary function of the differential proteins.Reveal protein molecular mechanism of the curative effect of TMP in rats after ASCI.
METHODS A total of 12 healthy adult Sprague Dawley rats were used to establish the ASCI models according to Allen's weight drop methed. These models were divided randomly into two groups:(1) Normal sodium treatment group(group NS,n=6);(2)Tetramethylpyrazine treatment group (group TMP, n=6).Two groups animals were further divided into two sub-groups(n=3 per sub-group), each at 3 and 7 days of survival before euthanasia.200mg/kg of TMP was administered in the TMP group intraperitoneally and the NS group were treated with the same volume of normal solution 30 minutes after injury. Each rat was given the same dosage once a day until euthanasia or survived for 5 days following the spinal cord injury if the rats survived for more than 5 days. At the corresponding time points following the injury, the rats of two groups were killed and the spinal cord tissue samples were collected from the injured segment with the length of 0.6cm. The spinal cord protein's lysate was extracted and the concentrations of proteins were detected by Bradford method. Then the proteins were separated by two-dimensional gel electrophoresis(2-DE).The differential expression proteins were detected by PDQuest-8.0.1 software after the gels were stained with colloidal coomassie blue.These proteins were digested with trypsin into peptides.And the resulting tryptic peptides were analyzed by matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-Q-TOF).The mass spectrometric data were used to identify the proteins by searching SWISS-PROT and NCBInr protein sequence database through MASCOT software.The primary function of the differential proteins was analyzed by literature reviewing.
RESULTS High resolution gel maps were produced by 2-DE.The difference of gel maps between two groups were significant. The differential expression proteins were successfully identified by MALDI-Q-TOF.3 days after ASCI, a total of 15 differential expression proteins were identified. Up-regulated proteins were serum albumin、neurofilament light polypeptide、vimentin、glial fibrillary acidic protein、tubulin alpha-1A chain、tubulin beta-2A chain、tubulin beta-2C chain、leucyl aminopeptidase、dihydropyrimidinase-related protein 2; down-regulated proteins were alpha-internexin、60 kDa heat shock protein、heat shock cognate 71 kDa protein、heat shock related 70 kDa protein 2、heat shock protein beta-1、phosphatidylethanolamine-binding protein 1.7 days after ASCI, a total of 19 differential expression proteins were identified. Up-regulated proteins were serum albumin、tubulin alpha-1A chain、tubulin beta-2A chain、neurofilament light polypeptide、neurofilament medium polypeptide、aldehyde dehydrogenase、guanine nucleotide binding protein subunit beta-1、beta-actin、vimentin、glial fibrillary acidic protein;down-regulated proteins were heterogeneous nuclear ribonucleoprotein H2、parkinson disease protein 7 homolog、vesicle-associated membrane protein-associated protein B、ubiquitin-conjugating enzyme E2N、pyruvate dehydrogenase E1 component subunit beta、isocitrate dehydrogen ase [NAD] subunit alpha、creatine kinase B-type、alpha-internexin、60 kDa heat shock protein. By literature reviewing, the majority of differential expression proteins have been reported to participate in neuron growth, apoptosis and stress-reaction.
CONCULUSION There are proteomic difference between the expression proteins of TMP group and NS group.The majority of differential expression proteins have been reported to participate in neuron growth, apoptosis and stress-reaction. The data will be helpful to illuminate the mechanism involved in the recovery of ASCI and the curative effect of TMP in rats after ASCI.
引文
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