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Study on the Induction of α-1,4-N-acetylglucosaminyltransferase by Helicobacter Pylori and TNF-α in Gastric Cancer Cell

Author: WangNa
Tutor: ZhangMuXia
School: Hebei Medical University
Course: Clinical Laboratory Diagnostics
Keywords: α4GnT Helicobacter pylori TNF - alpha Gastric cancer cells Induced by
CLC: R735.2
Type: Master's thesis
Year: 2010
Downloads: 43
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Abstract

Helicobacter pylori (Helicobacter pylori, Hp) is a gram-negative micro-aerobic, 1983 Marshal and Warren first isolated from patients with chronic gastritis, an infection rate of about 50% in the population. Closely related to Helicobacter pylori and gastritis, gastric ulcer and gastric carcinogenesis induced host inflammatory factors, transcription regulation factors, apoptosis-related factors, signal transduction factor expression of many genes and disease. In 1994 the World Health Organization be classified as a class I carcinogen. Tumor necrosis factor (Tumor necrosis factor-α, TNF-α) is important in vivo inflammatory cytokines, and its expression is increased there is a close relationship between Helicobacter pylori infection and promote the development of gastritis. Although H. pylori infection rate is very high, but the majority of infected people have no clinical symptoms, suggesting that the natural defenses of the body of Helicobacter pylori. Recent studies show that, and cardia gastric mucosa glands, pyloric glands, mucous neck cells and other parts of the presence of alpha-1 ,4-N-acetyl glucosamine transferase (alpha-1 ,4-the N-acetyglucosaminyltransferase α4GnT). It is a glycosyltransferase, to transfer N-acetylglucosamine to the end of the α-1 ,4-N-acetylglucosamine is formed having a core 2 branched O-glycans of beta galactose residues. The study found that the end of the gastric mucus is connected to the α-1 ,4-N-acetylglucosamine of mucin O-glycan natural antibiotic function. It through the influence of H. pylori cell wall components cholesterol-alpha-D-glucopyranoside biosynthesis to inhibit the proliferation of Helicobacter pylori, swimming and deforming. Speculated that gastric gland mucous cells α4GnT generated by the O-glycans of the gastric gland mucus protein α-1 ,4-N-acetylglucosamine, to invasive deep gastric mucosal barrier Helicobacter pylori, and thus has a natural protective effect on the gastric mucosa . Therefore, we apply the gastric cultured cells from proven Helicobacter pylori and TNF-of alpha on α4GnT gene regulation of gene transcription and protein levels. Objective: the application expression α4GnT gene gastric cancer cells, to explore the Helicobacter pylori cytokines TNF-the alpha on α4GnT gene expression induced mRNA and protein levels. : 1 cell culture: the recovery of the gastric cancer cell line MGC803 culture containing 10? S, 100U/ml penicillin, 100μg/ml streptomycin in RPMI 1640 medium at 37 ° C, 5% CO2 humidified incubation inside the culture, the number of 3 × 105 cells per ml were seeded in new flasks passaged The inoculated After 48h the cells were used for experiments. 2. Bacterial culture: the recovery of the Helicobacter pylori standard strain J99, inoculated on Columbia agar medium (containing 5% sheep blood, the 11μg/ml vancomycin, 11μg/ml amphotericin B, 0.47μg/ml polymyxin b) placed in culture in 37 ° C Microaerophilic conditions. 3 experimental groups: group of Helicobacter pylori: a density of 1 × 105-1 × 106CFU/ml Helicobacter pylori and MGC803 of cells co-cultured 6h, the culture medium was changed and continue to 48h. TNF-alpha group: the final concentration of 10ng/ml TNF-alpha added to the cells 48 hours role. Control group: cells was added an equal amount of Helicobacter pylori and TNF-alpha of medium and cultured 48h. 4 using RT-PCR detection of each group α4GnT gene mRNA expression level of 8% agarose gel electrophoresis, the results of electrophoresis with an inner reference GAPDH Analyzing amplified fragment. Western Blot detection the group α4GnT protein expression levels compared with GAPDH as an internal reference to determine the results of near-infrared laser imaging system scan. 6 immunohistochemical staining method detection groups α4GnT protein expression levels, DAB color light microscope cells per coverslip count of 5000 cells, the positive rate was calculated in each treatment group compared with the control group. RT-PCR and Western Blot test results by gel - protein analysis system relative quantitative analysis, as mean ± standard deviation (x ± s), application SPSS17.0 statistical software analysis, the two groups were compared using t-test . The positive rate of cells in each group, the results of immunohistochemistry application SPSS17.0 statistical analysis software, the two groups were compared using chi-square test. Significant significance level of p <0.05. Results: 1.RT-PCR detection α4GnTmRNA of expression results: Helicobacter pylori in human gastric cancer cell MGC803 α4GnT gene mRNA expression (0.1160 ± 0.011) and control group (0.1258 ± 0.00531) no significant differences, statistics showed that There was no significant difference, p> 0.05; TNF-alpha role in human gastric cancer cell MGC803 α4GnT gene mRNA expression (0.474 ± ??0.072) compared with the control group (0.1520 ± 0.001) was significantly increased, p <0.05. 2.Western Blot expression of detection α4GnT protein Results: of Helicobacter pylori group α4GnT protein expression levels (0.2200 ± 0.59) compared with the control group (0.2120 ± 0.3271) compared with no significant differences, also not statistically significant (p> 0.05); protein expression of TNF-alpha group α4GnT (0.6180 ± 0.0672) compared with the control group (0.2080 ± 0.0148) compared with an increase of 3 times, statistically significant, p <0.05. 3 the immunohistochemical detection α4GnT protein expression results: immunohistochemical staining, light microscopy under observation the Helicobacter pylori group α4GnT of positive cells (2.8%) with the control group α4GnT number of positive cells (3.4%), there is no obvious increase statistically significant difference (p> 0.05); positive cells of TNF-alpha group number (20.9%) compared with the control group α4GnT number of positive cells (5.4%) have increased significantly between the two groups difference was statistically significant (p <0.05). Conclusion: The human gastric cancer MGC803 expression α4GnT gene of Helicobacter pylori and gastric cancer cells MGC803 co-culture, did not affect the mRNA and protein expression levels of the α4GnT changed significantly. TNF-alpha role in gastric cancer cells MGC803 its α4GnT gene mRNA and protein expression levels increased, indicating that induced by TNF-alpha gene expression of MGC803 cells α4GnT. 4 MGC803 cell lines can be used as cell lines to explore α4GnT gene expression is induced factors.

Study on the Induction of α-1,4-N-acetylglucosaminyltransferase by Helicobacter Pylori and TNF-α in Gastric Cancer Cell

Abstract

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