| 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.
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