【作者】 崔晶；

【导师】 来茂德；

【作者基本信息】 浙江大学 ， 病理学与病理生理学， 2006， 博士

【摘要】 结直肠癌是严重危害人类健康的常见恶性肿瘤之一。大量的研究证明,大多数结直肠癌是从腺瘤癌变而来,这个过程约5—20年。切除腺瘤能有效降低结直肠癌的发病率。大约30%—46%的腺瘤患者治疗后2.5—7年就会复发。目前缺乏有效的指标来预测腺瘤的进一步演进或复发。因此研究腺瘤的发生机制及进展为癌的机制对于结直肠癌的防治意义重大。虽然结直肠癌发生的分子机理相对于其它肿瘤研究较为深入,但细胞的恶性转化毕竟是一个复杂的多步骤的过程,具体的细节还不清楚。除已知基因的作用,还有许多未知基因的作用有待进一步鉴定。因此,通过一定的方法识别、分离和鉴定结直肠腺瘤、结直肠癌发生相关的差异表达基因,寻找结直肠癌发生不同阶段转变过程中的关键基因,有助于更深入地认识结直肠腺瘤及结直肠癌发生的机理,并为早期诊断和治疗提供依据。 我们实验室于1999年应用抑制性差减杂交法(SSH)利用一个同时患有结肠腺瘤和腺癌的病例构建了三个cDNA差减文库,即腺瘤相对于正常粘膜的差异片段文库(A-N),腺癌相对于正常粘膜的差异片段文库(T-N)和腺癌相对于腺瘤的差异片段文库(T-A)。在分析A—N文库(以腺瘤为检测样本,正常粘更多还原

【Abstract】 Colorectal Cancer （CRC） is a common gastrointestinal malignancy which threatens the health of human. The adenoma-carcinoma sequence is now widely accepted as the major pathway for the development of colorectal cancer in the general population. Colorectal adenomas are recognized as the precursor lesions for most cases of colorectal cancer. Removal of these adenomas resulted in a markedly lower colorectal cancer occurence than without polypectomy. The progression from adenoma to cancer takes various time, range from 5 years to more than 20 years. A 30% -46% recurrence rate of adenomas has been reported in studies with a follow-up period of 2.5-7 years. There is no effective criterion in evaluating whether adenoma will develop cancer or recur. A better understanding of the mechanism of adenoma development is important for prevention and diagnosis of colorectal cancer. Although many genes have been reported to be closely related to the progression of colorectal carcinogenesis, they are far from enough to explain the transformation. A lot of other genes, known or unknown, remain to be discovered. The identification and characterization of humangenes expressed exclusively or preferentially in tumor tissue will, hopefully, shed light on the mechanisms of colorectal carcinogenesis and provide useful genetic markers for early diagnosis and therapy.Three subtracted cDNA libraries were constructed by SSH in our lab in 1999. They were colonic adenocarcinoma-normal mucosa cDNA subtracted library （subtracted T-N library）, colonic adenoma-normal mucosa cDNA subtracted library （subtracted A-N library）, colonic adenocarcinoma-adenoma cDNA subtracted library （subtracted T-A library）. Four differentially expressed sequences located in the 3’UTR of C6orf37 gene were found in A-N library. C6orf37 had been shown a novel candidate gene associated with human retinal disease, but the function of C6orf37 is still unknown. In this research we planed to confirm the relationship between the 4 differentially expressed sequences and C6orf37, and then study its expression pattern in colorectal tumor and explore the possible functional role of this gene in colorectal carcinogenesis.To obtain the full length of differentially expressed sequences and confirm their identity, 3’RACE was used to amplify 3’ end sequence of differentially expressed cDNA and long PCR was used to amplify the upstream sequence 5’RACE was used to amplify the 5’ end. The full length of cDNA was obtained by assembling the results of RACE and long PCR. The full length of differentially cDNA was 5547 nucleotides. It contains an open reading frame of 1314bp that encodes a 437-amino acid protein. The similarity between the full length and C6orf37 sequence （AF₃50451） is 98%. The differentially expressed gene that we screened by SSH is C6orf37 gene. Manybioinformatics softwares and internet programmes were used to analyze the characters of C6orf37 mRNA and protein to find hints indicating its function. Results of bioinformatics analysis indicate C6orf37 protein is a globule cytoplasm protein, whose sequence is highly conservative. A number of conserved potential phosphorylation sites exist in C6orf37 protein. C6orf37 gene is expressed in a wide range of human tissues. Differential expression is observed among fetal, juvenile and adult tissues. C6orf37 gene is also screened in many cancer SAGE libraries.A novel VNTR sequence was found in C6orf37 second exon. We tried to find the association of the VNTR polymorphism with colorectal cancer risk. DHPLC was applied in detecting the VNTR genetypes in 166 Chinese healthy people and 122 colorectal cancer patients. The VNTR was composed of 15 base pairs which consensus sequence encoding 5-amino-acid （G-G-D-F-G）. The repeat times ranged from 3 to 5. There were three common alleles containing three repeats （a）, four repeats （b） and five repeats （c） respectively, which produced three homozygotes （a/a, b/b and c/c） and three heterozygotes （a/b, a/c and b/c）. a, b, c alleles frequency were 0.143, 0.332,0.548 respectively in Chinese population. Heterozygosity （H） was 0.583. Polymorphism information content （PIC） was 0.510. The distribution of genotypes and allele frequencies of the VNTR reached no significant difference between patients and healthy controls. There was no correlation between VNTR polymorphism and colorectal cancer clinicopathological features.The expression of C6orf37 gene at mRNA level in colorectal mucosa, adenoma and carcinoma tissues was detected to confirm the result of SSH subtracted A-N library.Semi-quantitative RT-PCR was performed to study the expression of C6orf37 gene at mRNA level in 13 matched normal colorectal mucosa, adenoma and carcinoma tissues. Results of RT-PCR showed that the mRNA expression level of C6orf37 gene in colorectal adenoma was significantly higher than those in paired normal mucosa. Although the expression level of carcinoma was also higher, the difference between carcinoma and normal mucosa did not reach statistic significance. There was no difference between adenoma and carcinoma.ISH was used to further testify the overexpression of C6orf37 mRNA in 24 adenomas and carcinoma tissues. Positive stain of ISH was located in cytoplasm of epithelial tissue. mRNA expression level of C6orf37 in colorectal adenoma and carcinoma was higher than in normal mucosa. There was no difference between adenoma and carcinoma. No relationship was observed between C6orf37 mRNA expression and clinicopathological characters of colorectal tumor.We generated two rabbit anti-C6orf37 polyclonal antibodies by synthesized immunogenic peptides combined with carrier proteins. The titer and speciality of immune serum were confirmed. These polyclonal antibodies were successfully used in immunohistochemistry to detect the expression of C6orf37 protein in 81 colorectal carcinoma and adenoma tissue. C6orf37 protein was highly expressed in adenoma than in normal mucosa. No difference was detected between normal mucosa and carcinoma as well as between adenoma and carcinoma. The cancer cells located in the invasive front of cancer showed stronger staining of C6orf37 protein than those in the center. There was no relationship between C6orO7 protein expression andclincopathological parameters. The distribution of C6orf37 protein stained by IHC was consistent with the results of ISH. C6orf37 expressed in a wide rage of human tissue such as stomach, pancreatic island, lymphoid node and nerve tissue.In conclusion, the differential expressed gene of SSH subtracted A-N library is C6orf37 gene. C6orf37 is a globule cytoplasm protein and may play an important role in human growth;A novel VNTR polymorphism in C6orf37 exists in Chinese population and is not associated with colorectal cancer risk;C6orf37 is a novel gene up-regulated in colorectal adenoma and is associated with colorectal tumorgenesis.更多还原