失血性休克大鼠肠上皮细胞线粒体DNA COX基因、表达和功能改变及Rg1干预的研究
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摘要
失血性休克可导致机体各组织器官损伤，其中肠道是最容易受损伤的主要靶器官。失血性休克后肠黏膜上皮细胞缺血缺氧，能量代谢障碍是不可逆性休克和多器官功能衰竭(MSOF)的重要原因。线粒体是能量转换的细胞器，在维持细胞正常的能量代谢、结构和功能方面具有重要作用。同时，线粒体又是真核细胞内含有核外遗传物质——线粒体DNA(mitochondrial DNA,mtDNA)的特殊细胞器，有一套不同于核基因表达的复制、转录和翻译体系。mtDNA为一16596bp的闭环双链DNA分子，编码参与氧化磷酸化(Oxidative phosphorylation，OXPHOS)过程的13种多肽，细胞通过OXPHOS产生的ATP占细胞生命活动所需能量的90％以上，因此mtDNA对维持细胞的正常功能起重要作用。线粒体编码基因中最大的3个亚基(COX Ⅰ、COX Ⅱ、COX Ⅲ)由mtDNA编码。细胞色素氧化酶(Cytochrome oxidase，COX)是位于线粒体内膜上呼吸链末端的限速酶，是唯一能将电子传给氧分子的细胞色素复合物，是线粒体呼吸链OXPHOS过程中的关键酶，其损伤可直接影响线粒体功能。然而这些损伤是发生在何环节?是基因水平、表达水平、还是蛋白质水平?是否是新的药物治疗靶点?至今尚不清楚。损伤后对其蛋白质结构、功能有无影响和三七皂甙单体Rg1的调节保护作用如何也未见报道。
目的：
一、研究失血性休克大鼠缺血缺氧后肠上皮细胞线粒体DNA COX Ⅰ、COX Ⅱ、COX Ⅲ基因损伤(突变)及其表达改变和突变后对其编码的COX酶蛋白质空间结构的改变及其对酶功能的影响，从基因、转录、翻译水平探讨缺血缺氧后COX在线粒体有氧呼吸功能改变中的作用，阐明其结构和功能的关系，揭示细胞缺血缺氧性损害中能量代谢障碍的分子机制。
二、研究三七皂甙单体Rg1对失血性休克大鼠缺血缺氧后肠上皮细胞线粒体损伤的保护作用及其对COX活性和相关基因表达的影响，从线粒体的形态、功能、基因表达及失血性休克大鼠存活率等多方面探讨Rg1对缺血缺氧性线粒体损伤的保护作用
第三军医大学博士研究生论文
及其机理。
方法:
Wistar大鼠120只，按改良Wigger’s法制作失血性休克模型，动物随机分为失
血性休克组、治疗组、对照组，于失血性休克后不同时相提取肠上皮细胞线粒体、
mtDNA、线粒体蛋白、总RNA及制备电镜标本，采用PCR产物直招狈峙法街贝明易上皮细
胞mONA COXI、COXn、COXlll基因损伤(突勿，并不佣生物信息学技术、计算机同源
模建的方法进行肠上皮细胞mtDNA COX基因突变位点与其编码蛋白质结构的改变及
其对功能的影响;采用RFPCR场劫卸明元勺彭田胞川ONA COXI、COXn、COXllllllRNA白年友
达变化;采用Westem一lot法检测肠上皮细胞线粒体COXI蛋白的表达变化;采用电镜体
视学图象定量法分析检测线粒体形态特征变化;采用Clark氧电极法检测线粒体呼吸
功能;采用紫外分光光度法检测COX活性;从线粒体的形态、功能、基因表达及失
血性休克大鼠存活率等多方面探讨三七皂贰单体Rgl对缺血缺氧性线粒体损伤的保护
作用。
结果:
一、mtDNA基因测序结果显示，失血性休克缺血缺氧5h，大鼠肠上皮细胞
mtDNA COxl序列，从5545到6245出现了13个散在性点突变，5692与5693之间出
现一插入突变(5 692 t 5693)，从6260到6838出现了较多的散在性点突变，且突变类型
多为G、A转换;eoX 11亚基基因在7 191(T~C)、7212(T一e)、7235(o一A)、 7356(A
~G)、7483(A一G))、7542(C一G)出现点突变;COXm亚基基因未出现突变。
二、计算机模拟构建COX基因突变后的空间结构显示，mtDNA COXI亚基基因
的突变，使突变区的30一32肤段发生了明显改变;COXn亚基基因的突变，使其二级
结构片段之间的连接区域(肤段52一59)发生了较大的改变，从而导致整个cox的空间
结构改变。
三、Rl，一PCR结果显示，失血性休克缺血缺氧lh肠上皮细胞COX 1 mRNA表达
量开始逐渐增加，3h达高峰，之后逐渐降低，到休克晚期sh时，非常显著低于正常
对照组(P<0.01)。cOX n mRNA的表达量Zh达高峰，维持到3h，4h时其表达逐渐减
少，sh时非常显著低于正常对照组(P<0.01)。COX nl mRNA表达变化不大，在休克
晚期表达呈减少趋势。Rgl能非常显著提高CoXI、CoX 11 mRNA的表达量(p<0.01)。
四、大鼠失血性休克th时肠上皮细胞线粒体COXI蛋白表达量已增高，休克2h
时表达开始下降，休克5h时，其蛋白表达量非常显著低于正常对照组(P<0.01)。Rgl
能非常显著提高其表达(P<0.01)。
五、COX活性测定结果显示，失血性休克后1h大鼠肠上皮细胞线粒体COX活
性有所增加，休克2h后COX活性开始下降，之后逐渐降低，到休克晚期sh时，非
第三军医大学博士研究生论文
常显著低于正常对照组(P<0.01)，Rgl治疗能非常显著提高其活性(P<0.01)。
六、失血性休克2h大鼠肠上皮细胞线粒体三态呼吸(S T3)降低，四态呼吸(S T4)升
高，呼吸控制率(RCR)，氧化磷酸化效率(OPR)均下降，到休克晚期sh时，呼吸控制
率非常显著降低(P<住01)。Rgl治疗组ST:升高，ST4降低，RCR和OPR非常显著提
高(p<0 .01)。
七、失血性休克缺血缺氧sh，大鼠肠上皮细胞线粒体
Hemorragic shock can introduce the damages of tissue and Organs , especially,the small intestine. The small intestine served as an important target organ in hemorragic shock. Ischemia- hypoxia and the energy metabolism disorders played an important role in the pathogenesis of irreversible shock and multipe system organ failure. Mitochondria was organelle for energy transform. It can maintain the energy metabolism of cell and the structure and the function. Mitochondria contain their own DNA(mtDNA) and replication , transcription and translation system differented from nuclear DNA. Mitochondrial DNA(mtDNA) consist of 16596bp closed-loop doublestranded DNA which encodes 13 polypeptides of oxidative phosphorylation.The major function of mitochondria in human cells is to provide more than 90% ATP. The three largest subunits of cytochrome c oxidases (COX) are encoded by mitochondria DNA. COX is the terminal complex of the mitochondria respiratory chain. It is located in the mitochondrial inner membrane where it transfers electrons from ferrocytochrome c to molecular oxygen. It is the most important enzyme and the functions of mitochondria were impaired when COX was injuried. But what link cause the damages ? Is there the changes of gene expression or protein ? Is there any drug to treat ? The mechanism of the damages is unknow. It remains a matter for further investigation.
Objective: (1) To study the damage and the change of expressions of mtDNA encoding COX genes(COX I , COX II and COX III subunits) of intestinal epithelial cells in hemorrhagic shock rats and to explore the relationship between the damages of structure and function induced by mutation of COX genes and to explain the mechanism of the energy metabolism disorder of mitochondria and the damages of cells induced by ischemia- hypoxia of intestinal epithelial cells.(2) To observe the protective effects of Panax notoginseng saponins Rg 1 on mitochondria and to study the effects of Panax notoginseng saponins Rgl on COX activity, and COX genes expressions, and mitochondrial morphology,
and function, and survival rate of hemorrhagic shock rats.To explain the mechanism of effects of Panax notoginseng saponins Rg1.
Methods: One hundred twenty Wistar rats were used and divided into three groups randomizely: hemorragic shock group, resuscitation group, resuscitation and Rgl treatment group. The present study has been carried using Wigger' s hemorrhagic shock model. Intestinal epithelial cells,mtDNA, mitochondria protein and RNA were isolated. The mutation of COX I, COX n and COX III genes were determined by polymerase chain reaction(PCR) with different primers of COX I, COX II and COX III and directly sequenced the products of PCR. Bioinformatics and computer homology modeling method were used to predict the trimetric space structure of the proteins coded by the normal genes and the mutant genes(COX I, COX II and COX III). Using experiment data and modeling resusts, the relationship between the structure and the function were analyzed.The changes of expressions of mtDNA encoding COX genes(COX I, COX II and COX III subunits) mRNA were determined by Reverse-transcriptional polymerase chain reaction(RT-PCR). The changes of expressions of COX I protein was examined by Western-blot. The mitochondrial morphologic changes were observed by an electron microscope and analysed quantitatively by an electron microscope image analysis system. Mitochondrial respiratory function was measured with Clark oxygen electrode. Cytochrome c oxidase activity was determined by ultraviolet spectrophotometer.
Results: (l)The sequenced results of mtDNA COX I gene showed that there were thirteen diffuse point mutation from 5545 to 6245 and a insert mutation between 5692 and 5693 (5692 t 5693)and a lot of diffuse point mutation from 6260-6838 in hemorragic shock rats 5h group, and most of the mutation was G→ A. The sequenced results of mtDNA COX II gene showed that there were the point mutations at 7191(T→C), 7212(T→C), 7235(G →A), 7386(A→G), 7483(A→G)), 7542(C→G)in hemor
目的：
一、研究失血性休克大鼠缺血缺氧后肠上皮细胞线粒体DNA COX Ⅰ、COX Ⅱ、COX Ⅲ基因损伤(突变)及其表达改变和突变后对其编码的COX酶蛋白质空间结构的改变及其对酶功能的影响，从基因、转录、翻译水平探讨缺血缺氧后COX在线粒体有氧呼吸功能改变中的作用，阐明其结构和功能的关系，揭示细胞缺血缺氧性损害中能量代谢障碍的分子机制。
二、研究三七皂甙单体Rg1对失血性休克大鼠缺血缺氧后肠上皮细胞线粒体损伤的保护作用及其对COX活性和相关基因表达的影响，从线粒体的形态、功能、基因表达及失血性休克大鼠存活率等多方面探讨Rg1对缺血缺氧性线粒体损伤的保护作用
第三军医大学博士研究生论文
及其机理。
方法:
Wistar大鼠120只，按改良Wigger’s法制作失血性休克模型，动物随机分为失
血性休克组、治疗组、对照组，于失血性休克后不同时相提取肠上皮细胞线粒体、
mtDNA、线粒体蛋白、总RNA及制备电镜标本，采用PCR产物直招狈峙法街贝明易上皮细
胞mONA COXI、COXn、COXlll基因损伤(突勿，并不佣生物信息学技术、计算机同源
模建的方法进行肠上皮细胞mtDNA COX基因突变位点与其编码蛋白质结构的改变及
其对功能的影响;采用RFPCR场劫卸明元勺彭田胞川ONA COXI、COXn、COXllllllRNA白年友
达变化;采用Westem一lot法检测肠上皮细胞线粒体COXI蛋白的表达变化;采用电镜体
视学图象定量法分析检测线粒体形态特征变化;采用Clark氧电极法检测线粒体呼吸
功能;采用紫外分光光度法检测COX活性;从线粒体的形态、功能、基因表达及失
血性休克大鼠存活率等多方面探讨三七皂贰单体Rgl对缺血缺氧性线粒体损伤的保护
作用。
结果:
一、mtDNA基因测序结果显示，失血性休克缺血缺氧5h，大鼠肠上皮细胞
mtDNA COxl序列，从5545到6245出现了13个散在性点突变，5692与5693之间出
现一插入突变(5 692 t 5693)，从6260到6838出现了较多的散在性点突变，且突变类型
多为G、A转换;eoX 11亚基基因在7 191(T~C)、7212(T一e)、7235(o一A)、 7356(A
~G)、7483(A一G))、7542(C一G)出现点突变;COXm亚基基因未出现突变。
二、计算机模拟构建COX基因突变后的空间结构显示，mtDNA COXI亚基基因
的突变，使突变区的30一32肤段发生了明显改变;COXn亚基基因的突变，使其二级
结构片段之间的连接区域(肤段52一59)发生了较大的改变，从而导致整个cox的空间
结构改变。
三、Rl，一PCR结果显示，失血性休克缺血缺氧lh肠上皮细胞COX 1 mRNA表达
量开始逐渐增加，3h达高峰，之后逐渐降低，到休克晚期sh时，非常显著低于正常
对照组(P<0.01)。cOX n mRNA的表达量Zh达高峰，维持到3h，4h时其表达逐渐减
少，sh时非常显著低于正常对照组(P<0.01)。COX nl mRNA表达变化不大，在休克
晚期表达呈减少趋势。Rgl能非常显著提高CoXI、CoX 11 mRNA的表达量(p<0.01)。
四、大鼠失血性休克th时肠上皮细胞线粒体COXI蛋白表达量已增高，休克2h
时表达开始下降，休克5h时，其蛋白表达量非常显著低于正常对照组(P<0.01)。Rgl
能非常显著提高其表达(P<0.01)。
五、COX活性测定结果显示，失血性休克后1h大鼠肠上皮细胞线粒体COX活
性有所增加，休克2h后COX活性开始下降，之后逐渐降低，到休克晚期sh时，非
第三军医大学博士研究生论文
常显著低于正常对照组(P<0.01)，Rgl治疗能非常显著提高其活性(P<0.01)。
六、失血性休克2h大鼠肠上皮细胞线粒体三态呼吸(S T3)降低，四态呼吸(S T4)升
高，呼吸控制率(RCR)，氧化磷酸化效率(OPR)均下降，到休克晚期sh时，呼吸控制
率非常显著降低(P<住01)。Rgl治疗组ST:升高，ST4降低，RCR和OPR非常显著提
高(p<0 .01)。
七、失血性休克缺血缺氧sh，大鼠肠上皮细胞线粒体
Hemorragic shock can introduce the damages of tissue and Organs , especially,the small intestine. The small intestine served as an important target organ in hemorragic shock. Ischemia- hypoxia and the energy metabolism disorders played an important role in the pathogenesis of irreversible shock and multipe system organ failure. Mitochondria was organelle for energy transform. It can maintain the energy metabolism of cell and the structure and the function. Mitochondria contain their own DNA(mtDNA) and replication , transcription and translation system differented from nuclear DNA. Mitochondrial DNA(mtDNA) consist of 16596bp closed-loop doublestranded DNA which encodes 13 polypeptides of oxidative phosphorylation.The major function of mitochondria in human cells is to provide more than 90% ATP. The three largest subunits of cytochrome c oxidases (COX) are encoded by mitochondria DNA. COX is the terminal complex of the mitochondria respiratory chain. It is located in the mitochondrial inner membrane where it transfers electrons from ferrocytochrome c to molecular oxygen. It is the most important enzyme and the functions of mitochondria were impaired when COX was injuried. But what link cause the damages ? Is there the changes of gene expression or protein ? Is there any drug to treat ? The mechanism of the damages is unknow. It remains a matter for further investigation.
Objective: (1) To study the damage and the change of expressions of mtDNA encoding COX genes(COX I , COX II and COX III subunits) of intestinal epithelial cells in hemorrhagic shock rats and to explore the relationship between the damages of structure and function induced by mutation of COX genes and to explain the mechanism of the energy metabolism disorder of mitochondria and the damages of cells induced by ischemia- hypoxia of intestinal epithelial cells.(2) To observe the protective effects of Panax notoginseng saponins Rg 1 on mitochondria and to study the effects of Panax notoginseng saponins Rgl on COX activity, and COX genes expressions, and mitochondrial morphology,
and function, and survival rate of hemorrhagic shock rats.To explain the mechanism of effects of Panax notoginseng saponins Rg1.
Methods: One hundred twenty Wistar rats were used and divided into three groups randomizely: hemorragic shock group, resuscitation group, resuscitation and Rgl treatment group. The present study has been carried using Wigger' s hemorrhagic shock model. Intestinal epithelial cells,mtDNA, mitochondria protein and RNA were isolated. The mutation of COX I, COX n and COX III genes were determined by polymerase chain reaction(PCR) with different primers of COX I, COX II and COX III and directly sequenced the products of PCR. Bioinformatics and computer homology modeling method were used to predict the trimetric space structure of the proteins coded by the normal genes and the mutant genes(COX I, COX II and COX III). Using experiment data and modeling resusts, the relationship between the structure and the function were analyzed.The changes of expressions of mtDNA encoding COX genes(COX I, COX II and COX III subunits) mRNA were determined by Reverse-transcriptional polymerase chain reaction(RT-PCR). The changes of expressions of COX I protein was examined by Western-blot. The mitochondrial morphologic changes were observed by an electron microscope and analysed quantitatively by an electron microscope image analysis system. Mitochondrial respiratory function was measured with Clark oxygen electrode. Cytochrome c oxidase activity was determined by ultraviolet spectrophotometer.
Results: (l)The sequenced results of mtDNA COX I gene showed that there were thirteen diffuse point mutation from 5545 to 6245 and a insert mutation between 5692 and 5693 (5692 t 5693)and a lot of diffuse point mutation from 6260-6838 in hemorragic shock rats 5h group, and most of the mutation was G→ A. The sequenced results of mtDNA COX II gene showed that there were the point mutations at 7191(T→C), 7212(T→C), 7235(G →A), 7386(A→G), 7483(A→G)), 7542(C→G)in hemor
引文
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2． Wilmore DW.Smith RJ,Odwyer ST,et al. The gut: a central organ after surgical stress. Surgery, 1988, 104: 917-923
3． 刘志刚，肖南，刘韧等．失血性休克及复苏后不同组织代谢变化与组织损伤差异性的研究．中国病理生理杂志，2002，18(6) ：701-703
4． Koike K, Yamamoto Y. Splanchnic hypoperfusion and distant organ injury. Nippon Geka Gakkai Zasshi,1999,100:357-360
5． Moore FA. The role of the gastrointestinal tract in postinjury multiple organ failure. Am-J-Surg,1999,178(6) :449-53
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18． Bosetti F, Brizzi F, Barogi S, et al. Cytochrome c oxidase and mitochondrial FIFO-ATPase (ATP synthase) activities in platelets and brain from patients with Alzheimer's disease[J]. Neurobiol Aging, 2002,23(3) :371-376
19． Bruno C, Martinuzzi A, Tang Y, et al. A stop-codon mutation in the human mtDNA cytochrome c oxidase I gene disrupts the functional structure of complex IV[J]. Am J Hum Genet, 1999, 65(3) :611-620
20． Hirata Y, Taguchi T, Nakao M, et al. Therelati-onship between the adenine nucleotide metabolism and the conversion of the xanthine oxidase enzyme system in ischemia-reperfusion of the rat small intestine. J. Pediatr Surg, 1996, 31(9) : 1199-204
2． Wilmore DW.Smith RJ,Odwyer ST,et al. The gut: a central organ after surgical stress. Surgery, 1988, 104: 917-923
3． 刘志刚，肖南，刘韧等．失血性休克及复苏后不同组织代谢变化与组织损伤差异性的研究．中国病理生理杂志，2002，18(6) ：701-703
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