Razi Journal of Medical Sciences

fa بررسی بیان ژن VPR2 در سلولهای AGS ترانسفکت شده با وکتور نوترکیب حامل ژن tagD هلیکوباکتر پیلوری Investigation of VPR2 gene expression in AGS cells transfected with recombinant vector carrier of tagD gene of Helicobacter pyloriExpression of VPR2 Gene in AGS ژنتیک Genetic پژوهشي Research زمینه و هدف: هلیکوباکتر پیلوری با پیشرفت سرطان معده ارتباط دارد. آنزیم تیول پروکسیداز که در این باکتری توسط ژن tagD رمزگذاری می‌شود، نقش مهمی را در اتصال باکتری و کلونیزاسیون آن در معده انسان بازی می‌کند. هدف از این تحقیق بررسی بیان ژن VPR2 در سلول‌های AGS ترانسفکت شده با وکتور نوترکیب حامل ژن tagD هلیکوباکتر پیلوری است. روش کار: در این پژوهش، سلول‌های AGS در محیط RPMI-1640 دارای 10% سرم جنین گاوی و در شرایط استاندارد کشت داده شدند. این سلول‌ها با وکتور نوترکیب PFLAG-CMV-3-tagD یا وکتور خالی PFLAG-CMV-3 به عنوان کنترل، ترانسفکت شدند. RNA کامل سلول استخراج شد و سنتز cDNA صورت پذیرفت. سپس با روش real time RT-PCR  میزان بیان ژن VPR2 در هر دو گروه سلول‌های AGS ترانسفکت شده تست و کنترل، بررسی شد. در نهایت با استفاده از نرم افزار  SPSS و آزمون‌های آماری t-test Indepent  بیان هر یک از ژن‌ها مورد بررسی قرار گرفت. یافته‌ها: بیان یوکاریوتی ژن tagD در سلول‌های AGS دریافت کننده ژن tagD با انجام آزمایش RT-PCR تایید شد. بیان ژنهای VPR2  در سلول‌های AGS دریافت کننده ژن tagD نسبت به سلول‌های کنترل به صورت معنی‌داری افزایش نشان داد (0203/0p= ). نتیجه‌گیری: براساس یافته‌های حاصل، می‌توان نتیجه گرفت که ژن tagD هلیکوباکتر پیلوری، با اثر بر سلول‌های AGS، می‌تواند سبب کاهش یا افزایش بیان در این سلول‌ها گردد. ژن tagD یکی از ژن‌های مهم هلیکوباکتر پیلوری است که می‌تواند بر روی سلول‌های میزبان تأثیر بگذارد.   <div dir="RTL" style="text-align: left;">  Background: 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.</div> باکتری هلیکوباکتر پیلوری, tagD,VPR2 Helicobacter pylori, tagD, VPR2 40 49 http://rjms.iums.ac.ir/browse.php?a_code=A-10-4905-2&slc_lang=fa&sid=1 Shahrzad Afsharipoor شهرزاد افشاری پور shariiiiiii7272@yahoo.com 3900319475328460054296 3900319475328460054296 No Shahrekord Branch, Islamic Azad University, Shahrekord, Iran واحد شهرکرد، دانشگاه آزاد اسلامی، شهرکرد، ایران Abbas Doosti عباس دوستی abbasdoosti@yahoo.com 3900319475328460054297 3900319475328460054297 Yes Shahrekord Branch Islamic Azad University, Shahrekord, Iran واحد شهرکرد، دانشگاه آزاد اسلامی، شهرکرد، ایران