胰腺癌甲基化异化基因的检测、芯片筛查和诊断研究
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摘要
目的:研究胰腺癌SPARC、SARP2基因CpG岛的甲基化分布特征及其与临床生物学特点的关系,以及定量检测方法的建立,并用甲基化基因芯片筛查胰腺癌新的高甲基化异化基因。
方法:首先收集23例胰腺及癌旁组织、6例慢性胰腺炎和7例正常胰腺组织作为研究对象,健康成人外周血液标本6例作为对照组。抽提上述标本DNA进行重亚硫酸盐修饰,然后进行PCR扩增SPARC、SARP2基因第一外显子区CpG岛区域,测序明确该区域CpG位点甲基化情况,并与相应的临床生物学特征的关系进行分析。另收集原发性胰腺癌10例、慢性胰腺炎10例和健康志愿者6例外周静脉血10ml,取血清提取所有研究对象的血清DNA,重亚硫酸盐修饰后,行SARP2基因BSP扩增测序。其后制备SPARC和ACTB基因检测标准品,同时设计其定量检测引物和探针,建立SPARC基因定量检测方法。最后用甲基化基因芯片筛查胰腺癌高甲基化异化基因。
结果:1.健康人白细胞、正常胰腺、慢性胰腺炎、胰腺癌及癌旁组织SPARC基因第一外显子区CpG位点甲基化率分别0%、12.4%、25.1%、56.8%、37.8%。胰腺癌SPARC基因甲基化率与正常、慢性胰腺炎比较均差别非常显著(P＜0.001),与癌旁比较差别不显著。2.健康人白细胞、正常胰腺、慢性胰腺炎、胰腺癌及癌旁组织DNA中SARP2基因第一外显子区CpG位点甲基化率分别为5.7%、0%、2.5%、37.9%、14.1%;胰腺癌组织与慢性胰腺炎、白细胞及正常胰腺胰组织比较CpG位点甲基化率差别非常显著(P＜0.01),而与癌旁比较差别显著(P＜0.05)。并且这两个基因均有部分CpG位点具有较高的甲基化率。3.在健康对照者、慢性胰腺炎及胰腺癌患者外周循环核酸中也能检测到SARP2基因甲基化,其CpG位点平均甲基化化率分别为:2.2%、10.4%、16.0%,胰腺癌与健康对照组比较差别非常显著(P＜0.01),与慢性胰腺炎比较差别不显著(P＞0.05)。4.制备了SPARC基因甲基化标准品和内参ACTB标准品,初步建立了SPARC基因实时定量检测方法(QMT)。5.初步筛查出了部分胰腺癌高甲基化基因。
结论:1.胰腺癌SPARC、SPAR2基因第一外显子区CpG位点具有较高的甲基化率,并且CpG位点甲基化的分布具有不均衡性,部分位点具有胰腺癌特异性,可作为胰腺癌基因诊断的靶点。2.在胰腺癌外周血循环核酸中可以检测到甲基化异化的SARP2基因,因此循环核酸中甲基化异化基因的检测可以用于胰腺癌的基因诊断。3.SPARC基因的甲基化实时定量检测方法稳定、敏感,可用于胰腺癌的基因诊断。4.甲基化基因芯片筛查到了部分胰腺癌高甲基化基因,对于胰腺癌的诊断和防治具有较大意义。
Objective:To study CpG island's methylated character of SPARC and SARP2 gene in pancreatic cancer and the relationship between it and clinical biology feature,establish a stable method about quantitatve detecting methylated abbrrent genes,and screen methylated aberrant gene by microarray.
Method:To collect 23 pancreatic cancer and tissues by the side of the cancer、6 chronic pancreatitis and 7 normal pancreatic tissues as research object,6 health adult peripheral blood preparations as control group.The specimen's DNA was extracted and modified by bisulfite,then SPARC,SARP2 gene extron 1 region's CpG island was amplified by PCR.The PCR products were sequenced to identify CpG site methylation situation.The relationship between it and corresponding clinical biology features was analyzed respectively.On the other hand,10 primarily pancreatic cancers、10 chronic pancreatitis and 6 health volunteers were taken 10ml peripheral vein blood respectively. Circulating nucleic acid was extracted from blood serum,modified by bisulfite.Then BSP and sequence was finished for detecting SARP2 gene methylation.While,The quantitative primer and probe of SPARC and inner conferrence gene were designed on the base of the former research,we prepared the standard preparation of target's gene and inner conferrence,and established a method about quantitatve detecting methylated abbrrent genes by quantitative MethyLight technolgy(QMT).Finally,the hyper-methylated aberrant gene was screening by microarray.
Results:1.The methylation rate of SPARC in health adult peripheral blood WBC, pancreatic normal tissues,chronic pancreatitis,pancreatic cancer and tissues surrounding cancer were 0%、12.4%、25.1%、56.8%、37.8%respectively.The difference of CpG methylation rate between pancreatic cancer and chronic pancreatitis,as well as normal pancreatic tissues is very obvious(P＜0.001),while compared with the surrounding's of cancer,the difference is not obvious(P＞0.05).2.The methylation rate of SARP2 in health adult peripheral blood WBC,pancreatic normal tissues,chronic pancreatitis, pancreatic cancer and tissues surrounding cancer were 5.7%、0%、2.5%、37.9%、14.1% respectively.The difference of CpG sites methylation rate between pancreatic cancer and chronic pancreatitis,leukocyte as well as normal pancreatic tissues is very obvious (P＜0.01),while compared with tissues surrounding cancer,it's difference is also obvious (P＜0.05).Some CpG sites in the two genes have higher methylation rate.3.The methylation of SARP2 gene was detected in peripheral blood circulating nucleic acid of health adult,chronic pancreatitis and pancreatic cancer.Their methylation rate was 2.2%、10.4%、16.0%,respectively.There was a obvious difference between pancreatic cancer and healthy contrast(P＜0.01),but between pancreatic cancer and chronic pancreatitis(P＞0.05).4.We had prepared SPARC and inner reference(ACTB) gene's standard preparation.The quantitative detection of standard preparation is stable and sensitive.5. Some hyper-methylation gene in pancreatic cancer were found through microarray hybridization.
Conclusion:1.Hypermethylation of a part of SPARC and SARP2 extronl region's CpG sites in pancreatic cancer has tumor specific,and can be used as the target of gene diagnosis of pancreatic cancer.2.Abbrrent methylated of SARP2 gene can be detected in circulating nucleic acid of peripheral blood of pancreatic cancer,the methylation detection of SARP2 gene can be used for diagnosis and differential diagnosis of pancreatic cancer.3. The QMT is stable and reliable.The methylated detective sensitivity in tissue is up to 2.0 copy/μl,this method can be used for gene diagnosis of pancreatic cancer.4.Some hypermethylated aberrant CpG regions were found by methylated gene microarray screening.This will establish the foundation for looking for specific hypermethylated aberrant gene in pancreatic cancer.
方法:首先收集23例胰腺及癌旁组织、6例慢性胰腺炎和7例正常胰腺组织作为研究对象,健康成人外周血液标本6例作为对照组。抽提上述标本DNA进行重亚硫酸盐修饰,然后进行PCR扩增SPARC、SARP2基因第一外显子区CpG岛区域,测序明确该区域CpG位点甲基化情况,并与相应的临床生物学特征的关系进行分析。另收集原发性胰腺癌10例、慢性胰腺炎10例和健康志愿者6例外周静脉血10ml,取血清提取所有研究对象的血清DNA,重亚硫酸盐修饰后,行SARP2基因BSP扩增测序。其后制备SPARC和ACTB基因检测标准品,同时设计其定量检测引物和探针,建立SPARC基因定量检测方法。最后用甲基化基因芯片筛查胰腺癌高甲基化异化基因。
结果:1.健康人白细胞、正常胰腺、慢性胰腺炎、胰腺癌及癌旁组织SPARC基因第一外显子区CpG位点甲基化率分别0%、12.4%、25.1%、56.8%、37.8%。胰腺癌SPARC基因甲基化率与正常、慢性胰腺炎比较均差别非常显著(P＜0.001),与癌旁比较差别不显著。2.健康人白细胞、正常胰腺、慢性胰腺炎、胰腺癌及癌旁组织DNA中SARP2基因第一外显子区CpG位点甲基化率分别为5.7%、0%、2.5%、37.9%、14.1%;胰腺癌组织与慢性胰腺炎、白细胞及正常胰腺胰组织比较CpG位点甲基化率差别非常显著(P＜0.01),而与癌旁比较差别显著(P＜0.05)。并且这两个基因均有部分CpG位点具有较高的甲基化率。3.在健康对照者、慢性胰腺炎及胰腺癌患者外周循环核酸中也能检测到SARP2基因甲基化,其CpG位点平均甲基化化率分别为:2.2%、10.4%、16.0%,胰腺癌与健康对照组比较差别非常显著(P＜0.01),与慢性胰腺炎比较差别不显著(P＞0.05)。4.制备了SPARC基因甲基化标准品和内参ACTB标准品,初步建立了SPARC基因实时定量检测方法(QMT)。5.初步筛查出了部分胰腺癌高甲基化基因。
结论:1.胰腺癌SPARC、SPAR2基因第一外显子区CpG位点具有较高的甲基化率,并且CpG位点甲基化的分布具有不均衡性,部分位点具有胰腺癌特异性,可作为胰腺癌基因诊断的靶点。2.在胰腺癌外周血循环核酸中可以检测到甲基化异化的SARP2基因,因此循环核酸中甲基化异化基因的检测可以用于胰腺癌的基因诊断。3.SPARC基因的甲基化实时定量检测方法稳定、敏感,可用于胰腺癌的基因诊断。4.甲基化基因芯片筛查到了部分胰腺癌高甲基化基因,对于胰腺癌的诊断和防治具有较大意义。
Objective:To study CpG island's methylated character of SPARC and SARP2 gene in pancreatic cancer and the relationship between it and clinical biology feature,establish a stable method about quantitatve detecting methylated abbrrent genes,and screen methylated aberrant gene by microarray.
Method:To collect 23 pancreatic cancer and tissues by the side of the cancer、6 chronic pancreatitis and 7 normal pancreatic tissues as research object,6 health adult peripheral blood preparations as control group.The specimen's DNA was extracted and modified by bisulfite,then SPARC,SARP2 gene extron 1 region's CpG island was amplified by PCR.The PCR products were sequenced to identify CpG site methylation situation.The relationship between it and corresponding clinical biology features was analyzed respectively.On the other hand,10 primarily pancreatic cancers、10 chronic pancreatitis and 6 health volunteers were taken 10ml peripheral vein blood respectively. Circulating nucleic acid was extracted from blood serum,modified by bisulfite.Then BSP and sequence was finished for detecting SARP2 gene methylation.While,The quantitative primer and probe of SPARC and inner conferrence gene were designed on the base of the former research,we prepared the standard preparation of target's gene and inner conferrence,and established a method about quantitatve detecting methylated abbrrent genes by quantitative MethyLight technolgy(QMT).Finally,the hyper-methylated aberrant gene was screening by microarray.
Results:1.The methylation rate of SPARC in health adult peripheral blood WBC, pancreatic normal tissues,chronic pancreatitis,pancreatic cancer and tissues surrounding cancer were 0%、12.4%、25.1%、56.8%、37.8%respectively.The difference of CpG methylation rate between pancreatic cancer and chronic pancreatitis,as well as normal pancreatic tissues is very obvious(P＜0.001),while compared with the surrounding's of cancer,the difference is not obvious(P＞0.05).2.The methylation rate of SARP2 in health adult peripheral blood WBC,pancreatic normal tissues,chronic pancreatitis, pancreatic cancer and tissues surrounding cancer were 5.7%、0%、2.5%、37.9%、14.1% respectively.The difference of CpG sites methylation rate between pancreatic cancer and chronic pancreatitis,leukocyte as well as normal pancreatic tissues is very obvious (P＜0.01),while compared with tissues surrounding cancer,it's difference is also obvious (P＜0.05).Some CpG sites in the two genes have higher methylation rate.3.The methylation of SARP2 gene was detected in peripheral blood circulating nucleic acid of health adult,chronic pancreatitis and pancreatic cancer.Their methylation rate was 2.2%、10.4%、16.0%,respectively.There was a obvious difference between pancreatic cancer and healthy contrast(P＜0.01),but between pancreatic cancer and chronic pancreatitis(P＞0.05).4.We had prepared SPARC and inner reference(ACTB) gene's standard preparation.The quantitative detection of standard preparation is stable and sensitive.5. Some hyper-methylation gene in pancreatic cancer were found through microarray hybridization.
Conclusion:1.Hypermethylation of a part of SPARC and SARP2 extronl region's CpG sites in pancreatic cancer has tumor specific,and can be used as the target of gene diagnosis of pancreatic cancer.2.Abbrrent methylated of SARP2 gene can be detected in circulating nucleic acid of peripheral blood of pancreatic cancer,the methylation detection of SARP2 gene can be used for diagnosis and differential diagnosis of pancreatic cancer.3. The QMT is stable and reliable.The methylated detective sensitivity in tissue is up to 2.0 copy/μl,this method can be used for gene diagnosis of pancreatic cancer.4.Some hypermethylated aberrant CpG regions were found by methylated gene microarray screening.This will establish the foundation for looking for specific hypermethylated aberrant gene in pancreatic cancer.
引文
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