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دراسة جزيئية، وخلوية وراثية، ومناعية لمرض سرطان القولون والمستقيم في العراق == Molecular, Cytogenetic And Immunological Study of Colorectal Cancer In Iraqi Patients
Author name:
حيدر جاسم محمد الخفاجي
Supervisor name:
علي حمود السعدي | عزام قنبر اغا
General topic:
Biology
Specific topic:
Zoology
Degree:
Doctorate
University:
University of Babylon - College Of Science - Department Of Biology
Language:
English
University location:
Babylon
First pages:
24T2729 - p.pdf
Abstract:
The study consists of three parts related with patients of CRC as in : 1 - Molecular part 2 - Cytogenetic and molecular genetics part and 3 - Immunological part. In addition of their relatives of clinical and pathological diagnosis, genus, and tumor locations in 150 of Iraqi patients confirmed with CRC collected from Teaching Hospitals of some Iraqi governorates. The percent of male to female (54.66% / 45.43%) and the aged range of study were 16 to 80 years.The first part consist a study of five types of genes related with CRC represented by MLH1, MSH2, APC, K ras, and SMAD7 genes. Some of these genes develop with germline mutations as in MLH 1 exons1 and 15 so as MSH2 genes. While the rest genes related with a sporadic of CRC. All these genes were amplified by conventional polymerase chain reaction (PCR) for study the types of the mutations and their defect of these genes on CRC. The APC exon11and K ras exon1 genes were processed by single strand conformational polymorphism (SSCP) technique. The results of PCR - SSCP technique for two genes represented by appeared three types of bands, which were (AA), (AB) and (AC) bands. The important band was (AB) which constitutes the variability region represented by 84.2% and 44.82% in patients for both genes. The results of patients with mutated bands with high significant differences(P?0.05).compared with healthy control. Patients carried these bands subjected to PCR - Sequences technique. The analysis results for APC gene sequences were substitutions mutations type of single nucleotide polymorphism (SNP) change Cytocine to Thiamin (C>T) at 1972 location of genome. While the analysis results of Kras gene appeared two types of mutations, substitution and frame shifts represented by deletion mutation and so as appeared stop codon mutation.The other genes represented by MLH1, MSH2, and SMAD detected by direct PCR - Sequences. The results analysis of MLH1 gene, exon (1and 15) represented by nucleotides change with high frequencies of substitution mutation through exon1 at different locations of nucleotides. So as frame shift mutation type deletion through exon15. While for MSH2 gene, exon 6 from 6.66% represent by deletion mutation. A spread study for SMAD7 gene, exon4 were done through 30 patients average age 52 years (male to female 56.7% : 53.3%) confirmed colorectal adenocarcinoma compared with 15 healthy. A mutation of 20% of (SNPs) were identified.The second part which represented standard conventional cytogenetic and molecular genetics were done through peripheral blood culture (PBC). The study showed increasing of mitotic index (MI) in patients with significant differences (P?0.05) compared with healthy controls indicating for increasing of proliferation of the lymphocytes division. The study showed some chromosomal aberrations with significant, the majority of aberrations were higher significant in dwarf and aneuploidy chromosomes so as elongation chromosomes revealed significant differences (P?0.05) between patients and healthy. The appeared of Polyploidy chromosomes, with significant differences (P?0.05), that’s seem to be good indicator for cancer disease. The molecular genetics alterations which dependent on cytogenetic investigation were done through deletion of long arm of 5 chromosome which APC gene is located. The test elucidates the importance of inactivation (deletion) of APC gene in elicited of colorectal cancer. The study of 20 patients, were investigated by fluorescent in situ hybridization (FISH). The results showed 60% patients which have 5q arm deletion of (+). And (5%) have two deletions (++) which was a male aged less than 60 years. The results analysis explained significant differences (p?0.01) between studied group and deletion (+) of arm 5q. A correlation represented by significant differences (p?0.01)through dysfunction of APC gene deletion of 5q were found with high percent among sporadic adenocarcinoma 58.3%, with less in attenuated familial adenomatous polyposis (AFAP) and familial adenomatous polyposis (FAP) represented by the (25%), and (16.7) respectively.The last part related with immunological test were done by detection of tumor markers represented by Carcino Embryonic Antigen (CEA), IL - 33 and IL - 31.These markers represented a confirmative indicators for colorectal adenocarcinoma which were tested by ELISA technique in (79) patients confirmed with CRC of male and female. These tumor markers in tested with patients showed high significant differences (p?0.01) compared with healthy controls. Another immunological test for detection of Human papilloma virus (HPV) type 18 E6 gene expression in CRC by Immune histochemistry technique (IHC) in 71 (FFPET) of CRC. The results of this study showed there were a correlations between patients and adenocarcinoma positive HPV18 E6 infection 43.7% of patients infected with virus, with high significant differences (p?0.01) and higher infection in female (67.7%) than male (32.3%). So as a correlations were found between HPV18 E6 expression and tumor tissue locations, explained by high percentage 45.2% in rectum.While a tumor marker was (CD8+) was used to determine its role in human immune surveillance in tumor regions of CRC so as with regions which were infected by HPV. The study revealed high significant differences between healthy control and studied groups which have (CD8+) positive infiltration in tumor origins through detection by (IHC).In conclusions diagnosis of CRC can be detected by special genes like K ras gene through professional PCR processing, compact with tumor markers, so as Fluorescent in situ hybridization (FISH) technique provides a precise method which can be used for detection of alteration of molecular and cytogenetic related with CRC.
