چكيده فارسي :
زمينه و هدف
هليكوباكتر پيلوري با پيشرفت سرطان معده ارتباط دارد. آنزيم تيول پروكسيداز كه در اين باكتري توسط ژن tagD رمزگذاري مي شود، نقش مهمي را در اتصال باكتري و كلونيزاسيون آن در معده انسان بازي مي كند. هدف از اين تحقيق بررسي بيان ژن VPR2 در سلول هاي AGS ترانسفكت شده با وكتور نوتركيب حامل ژن tagD هليكوباكتر پيلوري است.
روش كار
در اين پژوهش، سلول هاي AGS در محيط RPMI-1640 داراي 10% سرم جنين گاوي و در شرايط استاندارد كشت داده شدند. اين سلول ها با وكتور نوتركيب PFLAG-CMV-3-tagD يا وكتور خالي PFLAG-CMV-3 به عنوان كنترل، ترانسفكت شدند. RNA كامل سلول استخراج شد و سنتز cDNA صورت پذيرفت. سپس با روش real time RT-PCR ميزان بيان ژن VPR2 در هر دو گروه سلول هاي AGS ترانسفكت شده تست و كنترل، بررسي شد. در نهايت با استفاده از نرم افزار SPSS و آزمون هاي آماري t-test Indepent بيان هر يك از ژن ها مورد بررسي قرار گرفت.
يافته ها
بيان يوكاريوتي ژن tagD در سلول هاي AGS دريافت كننده ژن tagD با انجام آزمايش RT-PCR تاييد شد. بيان ژنهاي VPR2 در سلول هاي AGS دريافت كننده ژن tagD نسبت به سلول هاي كنترل به صورت معني داري افزايش نشان داد (0/0203=p).
نتيجه گيري
براساس يافته هاي حاصل، مي توان نتيجه گرفت كه ژن tagD هليكوباكتر پيلوري، با اثر بر سلول هاي AGS، مي تواند سبب كاهش يا افزايش بيان در اين سلول ها گردد. ژن tagD يكي از ژن هاي مهم هليكوباكتر پيلوري است كه مي تواند بر روي سلول هاي ميزبان تاثير بگذارد.
چكيده لاتين :
Cancer is the first and second leading cause of death in developed and developing countries and gastric cancer is one of the most common and dangerous types of cancer in Iran, where 10,000 people are infected with this disease every year and it is the most common type. Cancer is also among men The Ministry of Health has announced that Iran has the most stomach cancer in the world. The main etiology for gastric cancer is Helicobacter pylori infection. Many studies have shown that most people with gastric cancer have a previous history of Helicobacter pylori infection, while less than one in 100 people with Helicobacter pylori infection are at risk for gastric cancer. Take; Therefore, Helicobacter pylori infection is an important factor in the development of gastric cancer, but there must be many environmental and possibly genetic factors in order to lead to gastric cancer. Helicobacter pylori is a small, highly motile gram-negative rod-shaped bacterium that infects the mucous membrane of the human stomach. Infection of this bacterium can occur in childhood, but its prevalence is directly related to aging and most cases are asymptomatic, but if the infection continues, 10 to 15% of people will develop gastric ulcer or gastric cancer. The thiol peroxidase-binding tagD gene in Helicobacter pylori is one of the most important adhesion molecules in H. pylori, which encodes triglyceride Citidylyltransferase. HpTpx protein is an antioxidant protein that plays an important role in empowering Helicobacter pylori to survive in gastric oxidative stress. HpTpr is present in the form of a hydrodynamic monomer and is an enzyme whose peroxidase and antioxidant activities are fully preserved. H. pylori produces a set of antioxidant enzymes, including superoxide dismutase (SOD), catalase, and peroxidase, to survive in the gastric environment, which can reverse the ROS reaction. Reduction of TagD protein leads to gradual transition from cell cycle stage to cell lysis and also causes bacterial deformation from bacillus to spherical state, irregular bacterial growth, swollen spherical shapes, unequal distribution in receptors, peptidoglycan thickening and cell lysis. Gene 2 VPR2 (Vasopressin receptor type) is located in the Xq28 position in humans and consists of 5 exons. This hormone has a peptide structure and is released from the pituitary gland. Lack of secretion of this hormone from the pituitary gland causes neurogenic type 2 diabetes and its lack of effect on the kidney causes nephrogenic type 2 diabetes. VPR2 gene expression has been shown in fetal lung tissue and lung cancer. Therefore, in the present study, our aim was to investigate the expression of VPR2 gene in AGS cells transfected with the recombinant vector carrying the tagD gene of Helicobacter pylori.
Methods
The present study was performed at the Biotechnology Research Center of Islamic Azad University of Shahrekord. The duration of the research lasted from January 2016 to June 2017. Gastric cancer cells, AGS class, were obtained from the Biotechnology Research Center of Shahrekord Branch of Azad University. The recombinant plasmid was then confirmed by enzymatic digestion. To confirm the presence of tagD gene in plasmid 3-pFLAG-CMV, dual enzymatic digestion was used and BglII and EcoRV restriction enzymes were used. AGS cells were cultured in a T25 flask. When the cells reached a density of 70%, cell pellets were prepared and cell suspension per ml was prepared from the culture medium. 10 μl of the suspension along with 10 μl of trypan blue dye was placed on the neobar beam and counted after lamination and then the number of cells in 1 ml of culture medium was calculated and finally the cells of the treatment and control groups were precipitated. In the present study, Anxin V kit was used to determine the percentage of apoptotic cells for rapid analysis of different stages of apoptosis. To prove the correctness of the synthesized cDNA, PCR was performed with GAPDH gene primers. Then, to determine the exact temperature of annealing primers, PCR was performed with appropriate primers. Using SPSS software and t-test Independent statistical tests, the expression of each gene was examined and compared. If p>0.05 is, the difference in data is statistically significant.
Results
Eukaryotic expression of tagD gene in AGS cells of tagD gene was confirmed by RT-PCR. Expression of VPR2 genes in AGS cells receiving tagD gene was significantly higher than control cells (p = 0. 0203).
Conclusion
The findings of the present study indicate the effects of decreasing or increasing expression in some cell genes under the influence of the tagD gene of Helicobacter pylori. Because the tagD gene is one of the most important genes in Helicobacter pylori, it plays a vital role in its colonization in the human stomach. Therefore, this gene may be involved in cell proliferation. Overall, the results of our study showed that VPR2 gene expression can be affected by Helicobacter pylori tagD gene, and it appears that this bacterium alters tagD gene expression in host cells.
