線粒體ND基因單核苷酸多態(tài)性與肝細(xì)胞肝癌發(fā)病風(fēng)險(xiǎn)的相關(guān)性研究
發(fā)布時(shí)間：2018-08-04 12:29
【摘要】：目的:肝細(xì)胞肝癌(HCC)是最常見的惡性腫瘤之一,其死亡率居全球惡性腫瘤的第三位。在中國,肝細(xì)胞肝癌的發(fā)病率正在上升,目前占世界肝細(xì)胞肝癌總數(shù)的55%,這給人民身體素質(zhì)的提升及國民經(jīng)濟(jì)的發(fā)展造成了嚴(yán)重的威脅。近年來很多研究發(fā)現(xiàn)線粒體DNA(mitochondrial DNA)突變和單核苷酸多態(tài)性(single nucleotide polymorphis ms)與腫瘤的發(fā)生相關(guān)。不同于核DNA(nuclear DNA),線粒體DNA(mitochondrial DNA,mtDNA)由于缺乏組蛋白保護(hù)、它的修復(fù)能力差以及處于富含活性氧(ROS)環(huán)境中,所以容易產(chǎn)生氧化應(yīng)激損傷,因而容易發(fā)生基因改變。在我們實(shí)驗(yàn)團(tuán)隊(duì)前期的研究中,我們已經(jīng)發(fā)現(xiàn)線粒體D-loop區(qū)基因的多態(tài)性和肝細(xì)胞肝癌的發(fā)病風(fēng)險(xiǎn)存在相關(guān)性。線粒體基因的D-loop區(qū)通過影響線粒體基因轉(zhuǎn)錄和復(fù)制從而改變線粒體基因,然而線粒體編碼區(qū)的基因幾乎不含內(nèi)含子,所以線粒體編碼區(qū)基因單核苷酸多態(tài)性可以通過改變編碼的氨基酸類型改變氧化磷酸化的功能從而影響線粒體功能。線粒體編碼區(qū)基因與肝細(xì)胞肝癌的相關(guān)性很少有人研究,所以開展線粒體編碼區(qū)基因與肝細(xì)胞肝癌的相關(guān)性研究具有重要的意義。本研究通過對(duì)ND基因進(jìn)行擴(kuò)增測序,并與標(biāo)準(zhǔn)序列進(jìn)行比對(duì)從而找出多態(tài)性基因位點(diǎn),利用病例-對(duì)照統(tǒng)計(jì)學(xué)方法分析多態(tài)性基因位點(diǎn)與HCC發(fā)病風(fēng)險(xiǎn)的相關(guān)性,這為HCC易患人群早期篩查及防治工作提供新思路。方法:1研究對(duì)象及標(biāo)本采集:病例組的80例肝細(xì)胞肝癌患者為2007年9月至2008年3月期間在河北醫(yī)科大學(xué)第四醫(yī)院肝膽外科住院病人,這些病人術(shù)前經(jīng)免疫學(xué)檢查和影像學(xué)方法臨床診斷為肝細(xì)胞肝癌,并且這些病人術(shù)后病理證實(shí)為肝細(xì)胞肝癌。對(duì)照組是2008年10月至2009年4月期間河北醫(yī)科大學(xué)第四醫(yī)院采集的正常人抗凝靜脈血標(biāo)本。2基因組DNA提取:分離提取肝細(xì)胞肝癌組病人血樣及對(duì)照組血樣基因組DNA,提取基因組DNA后將其置于4℃的恒溫冰箱中保存?zhèn)溆谩? PCR擴(kuò)增ND基因:從NCBI數(shù)據(jù)庫中查找ND基因,ND基因包含七個(gè)基因片段,即ND1、ND2、ND3、ND4L、ND4、ND5和ND6,將ND基因按大約600bps的長度劃分為13段小的基因片段,根據(jù)引物設(shè)計(jì)原則為每段小基因片段設(shè)計(jì)PCR上游及下游引物,然后用PCR預(yù)混試劑盒進(jìn)行基因擴(kuò)增,擴(kuò)增的產(chǎn)物經(jīng)過瓊脂糖凝膠電泳后,再放入凝膠成像系統(tǒng)中(美國Image公司)證實(shí)擴(kuò)增出DNA條帶,最后把擴(kuò)增的產(chǎn)物送至生工生物工程技術(shù)有限公司(Shanghai)進(jìn)行測序。3統(tǒng)計(jì)學(xué)分析:采用t檢驗(yàn)比較計(jì)量資料,采用χ2檢驗(yàn)或Fisher確切概率法比較計(jì)數(shù)資料,并計(jì)算P值。所有實(shí)驗(yàn)數(shù)據(jù)均使用SPSS21.0統(tǒng)計(jì)軟件進(jìn)行統(tǒng)計(jì)分析,P0.05時(shí)被認(rèn)為有統(tǒng)計(jì)學(xué)意義。結(jié)果:1肝細(xì)胞肝癌組和對(duì)照組臨床資料對(duì)比:兩組間性別、年齡無明顯差異(P0.05)。2從肝細(xì)胞肝癌組中隨機(jī)的選出30例標(biāo)本DNA進(jìn)行擴(kuò)增測序,分析后發(fā)現(xiàn)4820G/A(P=0.237),4824A/G(P=0.112),5301A/G(P=0.237)的SNP分布頻率與對(duì)照組相比在肝細(xì)胞肝癌發(fā)病風(fēng)險(xiǎn)中具有潛在意義。3對(duì)4820G/A,4824A/G,5301A/G所屬基因片段的病例數(shù)量進(jìn)行擴(kuò)大(80例),發(fā)現(xiàn)5301A/G位點(diǎn)的SNP在病例組與對(duì)照組間的分布頻率存在統(tǒng)計(jì)學(xué)差異(χ2=4.783,P=0.029)。ND2基因的5301A/G位點(diǎn)SNP可導(dǎo)致ND2編碼蛋白的第278位氨基酸ILE(異亮氨酸)轉(zhuǎn)換為LEU(亮氨酸),這可能影響ND2編碼蛋白的功能進(jìn)而影響整個(gè)呼吸鏈的功能,最終增加肝細(xì)胞肝癌的發(fā)病風(fēng)險(xiǎn)。4將5301A/G位點(diǎn)SNP分布與肝細(xì)胞肝癌患者的臨床特征進(jìn)行比較分析,結(jié)果顯示,5301A/G位點(diǎn)SNP和肝細(xì)胞肝癌患者的臨床特征,包括年齡、性別、腫瘤數(shù)目、腫瘤長徑、Child分級(jí)、臨床分期及門脈瘤栓等都無相關(guān)性(P0.05)。結(jié)論:1 mtDNA編碼區(qū)ND基因上的5301A/G位點(diǎn)SNP與肝細(xì)胞肝癌的發(fā)病風(fēng)險(xiǎn)相關(guān),該位點(diǎn)SNP可導(dǎo)致ND2編碼蛋白的第278位氨基酸ILE(異亮氨酸)轉(zhuǎn)換為LEU(亮氨酸),這可能影響ND2編碼蛋白的功能進(jìn)而影響整個(gè)呼吸鏈的功能,最終提高了肝細(xì)胞肝癌的發(fā)病風(fēng)險(xiǎn)。2 ND基因5301A/G位點(diǎn)的多態(tài)性改變與肝細(xì)胞肝癌患者的臨床特征沒有相關(guān)性,提示ND區(qū)5301A/G位點(diǎn)的多態(tài)性改變與肝細(xì)胞肝癌的發(fā)生相關(guān)。
[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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.7
本文編號(hào)：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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.7
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
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 孫玉蘭;胡義德;;線粒體DNA與腫瘤關(guān)系的研究進(jìn)展[J];中華腫瘤防治雜志;2006年12期
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