线粒体ND基因单核苷酸多态性与肝细胞肝癌发病风险的相关性研究
发布时间：2018-08-04 12:29
【摘要】：目的:肝细胞肝癌(HCC)是最常见的恶性肿瘤之一,其死亡率居全球恶性肿瘤的第三位。在中国,肝细胞肝癌的发病率正在上升,目前占世界肝细胞肝癌总数的55%,这给人民身体素质的提升及国民经济的发展造成了严重的威胁。近年来很多研究发现线粒体DNA(mitochondrial DNA)突变和单核苷酸多态性(single nucleotide polymorphis ms)与肿瘤的发生相关。不同于核DNA(nuclear DNA),线粒体DNA(mitochondrial DNA,mtDNA)由于缺乏组蛋白保护、它的修复能力差以及处于富含活性氧(ROS)环境中,所以容易产生氧化应激损伤,因而容易发生基因改变。在我们实验团队前期的研究中,我们已经发现线粒体D-loop区基因的多态性和肝细胞肝癌的发病风险存在相关性。线粒体基因的D-loop区通过影响线粒体基因转录和复制从而改变线粒体基因,然而线粒体编码区的基因几乎不含内含子,所以线粒体编码区基因单核苷酸多态性可以通过改变编码的氨基酸类型改变氧化磷酸化的功能从而影响线粒体功能。线粒体编码区基因与肝细胞肝癌的相关性很少有人研究,所以开展线粒体编码区基因与肝细胞肝癌的相关性研究具有重要的意义。本研究通过对ND基因进行扩增测序,并与标准序列进行比对从而找出多态性基因位点,利用病例-对照统计学方法分析多态性基因位点与HCC发病风险的相关性,这为HCC易患人群早期筛查及防治工作提供新思路。方法:1研究对象及标本采集:病例组的80例肝细胞肝癌患者为2007年9月至2008年3月期间在河北医科大学第四医院肝胆外科住院病人,这些病人术前经免疫学检查和影像学方法临床诊断为肝细胞肝癌,并且这些病人术后病理证实为肝细胞肝癌。对照组是2008年10月至2009年4月期间河北医科大学第四医院采集的正常人抗凝静脉血标本。2基因组DNA提取:分离提取肝细胞肝癌组病人血样及对照组血样基因组DNA,提取基因组DNA后将其置于4℃的恒温冰箱中保存备用。3 PCR扩增ND基因:从NCBI数据库中查找ND基因,ND基因包含七个基因片段,即ND1、ND2、ND3、ND4L、ND4、ND5和ND6,将ND基因按大约600bps的长度划分为13段小的基因片段,根据引物设计原则为每段小基因片段设计PCR上游及下游引物,然后用PCR预混试剂盒进行基因扩增,扩增的产物经过琼脂糖凝胶电泳后,再放入凝胶成像系统中(美国Image公司)证实扩增出DNA条带,最后把扩增的产物送至生工生物工程技术有限公司(Shanghai)进行测序。3统计学分析:采用t检验比较计量资料,采用χ2检验或Fisher确切概率法比较计数资料,并计算P值。所有实验数据均使用SPSS21.0统计软件进行统计分析,P0.05时被认为有统计学意义。结果:1肝细胞肝癌组和对照组临床资料对比:两组间性别、年龄无明显差异(P0.05)。2从肝细胞肝癌组中随机的选出30例标本DNA进行扩增测序,分析后发现4820G/A(P=0.237),4824A/G(P=0.112),5301A/G(P=0.237)的SNP分布频率与对照组相比在肝细胞肝癌发病风险中具有潜在意义。3对4820G/A,4824A/G,5301A/G所属基因片段的病例数量进行扩大(80例),发现5301A/G位点的SNP在病例组与对照组间的分布频率存在统计学差异(χ2=4.783,P=0.029)。ND2基因的5301A/G位点SNP可导致ND2编码蛋白的第278位氨基酸ILE(异亮氨酸)转换为LEU(亮氨酸),这可能影响ND2编码蛋白的功能进而影响整个呼吸链的功能,最终增加肝细胞肝癌的发病风险。4将5301A/G位点SNP分布与肝细胞肝癌患者的临床特征进行比较分析,结果显示,5301A/G位点SNP和肝细胞肝癌患者的临床特征,包括年龄、性别、肿瘤数目、肿瘤长径、Child分级、临床分期及门脉瘤栓等都无相关性(P0.05)。结论:1 mtDNA编码区ND基因上的5301A/G位点SNP与肝细胞肝癌的发病风险相关,该位点SNP可导致ND2编码蛋白的第278位氨基酸ILE(异亮氨酸)转换为LEU(亮氨酸),这可能影响ND2编码蛋白的功能进而影响整个呼吸链的功能,最终提高了肝细胞肝癌的发病风险。2 ND基因5301A/G位点的多态性改变与肝细胞肝癌患者的临床特征没有相关性,提示ND区5301A/G位点的多态性改变与肝细胞肝癌的发生相关。
[Abstract]:Objective: hepatocellular carcinoma (HCC) is one of the most common malignant tumors, and its mortality rate is the third most malignant tumor in the world. In China, the incidence of hepatocellular carcinoma is rising and now accounts for 55% of the total number of hepatocellular carcinoma in the world, which poses a serious threat to the improvement of the people's physical quality and the development of the people's economy. The study found that mitochondrial DNA (mitochondrial DNA) mutation and single nucleotide polymorphisms (single nucleotide polymorphis MS) are related to the occurrence of tumors. Unlike nuclear DNA (nuclear DNA), mitochondrial DNA (mitochondrial), due to the lack of histone protection, is poor in repair ability and in the environment rich in reactive oxygen species. In our previous research team, we have found that there is a correlation between the polymorphism of the mitochondrial D-loop gene and the risk of hepatocellular carcinoma. The D-loop region of the mitochondrial gene changes the mitochondrial gene by affecting the mitochondrial gene transcription and replication. However, there are almost no introns in the mitochondrial encoded region, so the single nucleotide polymorphisms of the mitochondrial coding region gene can alter the function of the mitochondria by changing the type of encoded amino acids to change the function of oxidative phosphorylation. The correlation between the mitochondrial coding region gene and liver cell liver cancer is rarely studied. The study of the correlation between body coding region gene and hepatocellular carcinoma is of great significance. In this study, the ND gene was amplified and sequenced and compared with the standard sequence to find the polymorphic loci. The correlation of polymorphic loci with the risk of HCC was analyzed by case control statistics, which was the susceptible population of HCC. Early screening and prevention and control work provide new ideas. Methods: 1 subjects and specimen collection: 80 cases of hepatocellular carcinoma patients in the case group were hospitalized in the Department of hepatobiliary surgery, Fourth Hospital of Hebei Medical University from September 2007 to March 2008. These patients were diagnosed as hepatocellular carcinoma by immunological examination and imaging methods before operation, and the patients were diagnosed as hepatocellular carcinoma by immunological examination and imaging methods. And these patients were confirmed to be liver cell liver cancer after operation. The control group was extracted from the normal people of the fourth hospital of Hebei Medical University from October 2008 to April 2009. The.2 genome DNA was extracted from the normal human anticoagulant blood samples: the blood sample and the control group DNA were isolated and extracted from the liver cell liver cancer group, and the genomic DNA was extracted after the extraction of genomic DNA. In the constant temperature refrigerator, the ND gene is amplified by.3 PCR: the ND gene is found from the NCBI database. The ND gene contains seven gene fragments, namely, ND1, ND2, ND3, ND4L, ND4, ND5, and the fragments. The upstream and downstream primers are designed for each fragment according to the primer design principle. Then the PCR premixed kit was used to amplify the gene. After the agarose gel electrophoresis, the amplified product was then put into the gel imaging system (Image company, USA) to amplify the DNA band. Finally, the amplified products were sent to the Bioengineering Technology Co., Ltd. (Shanghai) for sequencing.3 statistics analysis: t test was used to compare the measurement capital. Material, using the chi 2 test or the Fisher exact probability method to compare the count data and calculate the P value. All the experimental data were statistically analyzed using SPSS21.0 statistical software, and P0.05 was considered to have statistical significance. Results: the clinical data of 1 liver cell hepatocellular carcinoma group and control group were compared: there was no significant difference between the two groups of sex and age (P0.05).2 from liver cell liver cancer 30 specimens of DNA were randomly selected to be amplified and sequenced. After analysis, the frequency of SNP distribution in 4820G/A (P=0.237), 4824A/G (P=0.112) and 5301A/G (P=0.237) was compared with the control group in the risk of hepatocellular carcinoma, and the number of cases of 4820G/A, 4824A/G, 5301A/ dependent gene fragments were expanded (80 cases). The frequency of SNP in the G site was statistically different between the case group and the control group (x 2=4.783, P=0.029) the 5301A/G site SNP of the.ND2 gene could lead to the conversion of the 278th bit amino acid ILE (isoleucine) to LEU (leucine) of the ND2 encoded protein, which may affect the function of the ND2 encoded egg white and then affect the function of the whole respiratory chain and eventually increase. The risk of hepatocellular carcinoma (HCC).4 compares the SNP distribution of 5301A/G site with the clinical features of HCC patients. The results show that the clinical features of 5301A/G site SNP and HCC patients, including age, sex, number of tumors, tumor length, Child classification, clinical staging and portal vein tumor suppository, are not related (P0.05). 1 mtDNA coding region ND gene 5301A/G locus SNP is associated with the risk of hepatocellular carcinoma, the site SNP can lead to the conversion of the 278th bit amino acid ILE (isoleucine) to LEU (leucine) of the ND2 encoded protein, which may affect the function of the ND2 encoded protein and then affect the function of the whole respiratory chain and ultimately improve the liver cell liver cancer. There is no correlation between the polymorphism of the.2 ND gene 5301A/G locus and the clinical characteristics of HCC patients, suggesting that the polymorphism of the 5301A/G site in the ND region is associated with the occurrence of hepatocellular carcinoma.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7
本文编号：2163883
[Abstract]:Objective: hepatocellular carcinoma (HCC) is one of the most common malignant tumors, and its mortality rate is the third most malignant tumor in the world. In China, the incidence of hepatocellular carcinoma is rising and now accounts for 55% of the total number of hepatocellular carcinoma in the world, which poses a serious threat to the improvement of the people's physical quality and the development of the people's economy. The study found that mitochondrial DNA (mitochondrial DNA) mutation and single nucleotide polymorphisms (single nucleotide polymorphis MS) are related to the occurrence of tumors. Unlike nuclear DNA (nuclear DNA), mitochondrial DNA (mitochondrial), due to the lack of histone protection, is poor in repair ability and in the environment rich in reactive oxygen species. In our previous research team, we have found that there is a correlation between the polymorphism of the mitochondrial D-loop gene and the risk of hepatocellular carcinoma. The D-loop region of the mitochondrial gene changes the mitochondrial gene by affecting the mitochondrial gene transcription and replication. However, there are almost no introns in the mitochondrial encoded region, so the single nucleotide polymorphisms of the mitochondrial coding region gene can alter the function of the mitochondria by changing the type of encoded amino acids to change the function of oxidative phosphorylation. The correlation between the mitochondrial coding region gene and liver cell liver cancer is rarely studied. The study of the correlation between body coding region gene and hepatocellular carcinoma is of great significance. In this study, the ND gene was amplified and sequenced and compared with the standard sequence to find the polymorphic loci. The correlation of polymorphic loci with the risk of HCC was analyzed by case control statistics, which was the susceptible population of HCC. Early screening and prevention and control work provide new ideas. Methods: 1 subjects and specimen collection: 80 cases of hepatocellular carcinoma patients in the case group were hospitalized in the Department of hepatobiliary surgery, Fourth Hospital of Hebei Medical University from September 2007 to March 2008. These patients were diagnosed as hepatocellular carcinoma by immunological examination and imaging methods before operation, and the patients were diagnosed as hepatocellular carcinoma by immunological examination and imaging methods. And these patients were confirmed to be liver cell liver cancer after operation. The control group was extracted from the normal people of the fourth hospital of Hebei Medical University from October 2008 to April 2009. The.2 genome DNA was extracted from the normal human anticoagulant blood samples: the blood sample and the control group DNA were isolated and extracted from the liver cell liver cancer group, and the genomic DNA was extracted after the extraction of genomic DNA. In the constant temperature refrigerator, the ND gene is amplified by.3 PCR: the ND gene is found from the NCBI database. The ND gene contains seven gene fragments, namely, ND1, ND2, ND3, ND4L, ND4, ND5, and the fragments. The upstream and downstream primers are designed for each fragment according to the primer design principle. Then the PCR premixed kit was used to amplify the gene. After the agarose gel electrophoresis, the amplified product was then put into the gel imaging system (Image company, USA) to amplify the DNA band. Finally, the amplified products were sent to the Bioengineering Technology Co., Ltd. (Shanghai) for sequencing.3 statistics analysis: t test was used to compare the measurement capital. Material, using the chi 2 test or the Fisher exact probability method to compare the count data and calculate the P value. All the experimental data were statistically analyzed using SPSS21.0 statistical software, and P0.05 was considered to have statistical significance. Results: the clinical data of 1 liver cell hepatocellular carcinoma group and control group were compared: there was no significant difference between the two groups of sex and age (P0.05).2 from liver cell liver cancer 30 specimens of DNA were randomly selected to be amplified and sequenced. After analysis, the frequency of SNP distribution in 4820G/A (P=0.237), 4824A/G (P=0.112) and 5301A/G (P=0.237) was compared with the control group in the risk of hepatocellular carcinoma, and the number of cases of 4820G/A, 4824A/G, 5301A/ dependent gene fragments were expanded (80 cases). The frequency of SNP in the G site was statistically different between the case group and the control group (x 2=4.783, P=0.029) the 5301A/G site SNP of the.ND2 gene could lead to the conversion of the 278th bit amino acid ILE (isoleucine) to LEU (leucine) of the ND2 encoded protein, which may affect the function of the ND2 encoded egg white and then affect the function of the whole respiratory chain and eventually increase. The risk of hepatocellular carcinoma (HCC).4 compares the SNP distribution of 5301A/G site with the clinical features of HCC patients. The results show that the clinical features of 5301A/G site SNP and HCC patients, including age, sex, number of tumors, tumor length, Child classification, clinical staging and portal vein tumor suppository, are not related (P0.05). 1 mtDNA coding region ND gene 5301A/G locus SNP is associated with the risk of hepatocellular carcinoma, the site SNP can lead to the conversion of the 278th bit amino acid ILE (isoleucine) to LEU (leucine) of the ND2 encoded protein, which may affect the function of the ND2 encoded protein and then affect the function of the whole respiratory chain and ultimately improve the liver cell liver cancer. There is no correlation between the polymorphism of the.2 ND gene 5301A/G locus and the clinical characteristics of HCC patients, suggesting that the polymorphism of the 5301A/G site in the ND region is associated with the occurrence of hepatocellular carcinoma.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7
【参考文献】
相关期刊论文 前2条
1 Asmaa Ibrahim Gomaa;Shahid A Khan;Mireille B Toledano;Imam Waked;Simon D Taylor- Robinson;;Hepatocellular carcinoma: Epidemiology, risk factors and pathogenesis[J];World Journal of Gastroenterology;2008年27期
2 孙玉兰;胡义德;;线粒体DNA与肿瘤关系的研究进展[J];中华肿瘤防治杂志;2006年12期
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