乌司他丁预处理对氧糖剥夺诱导的GES-1细胞损伤的影响

发布时间：2018-02-02 08:21

本文关键词： 乌司他丁预处理 GES-1细胞氧糖剥夺 TRPV1　出处：《南方医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景急性胃粘膜损伤(AGML)是临床上常见的围术期并发症,其具体的发病机制至今仍未完全明确。目前普遍认为胃粘膜缺血是AGML发病的主要环节。我们课题组前期已通过大鼠的浸水束缚应激模型,分别从损伤性应激和非损伤的心理应激等层面探讨了 AGML的发病进程。然而却一直未从缺血的角度探讨AGML的发生机制及可能的防护措施。乌司他丁(UTI)作为一种广谱尿胰蛋白酶抑制剂,具有减少氧自由基、稳定溶酶体膜、减轻炎症、改善循环等作用。研究表明UTI对危重患者肠粘膜具有保护作用,但其对胃粘膜是否也有保护作用尚无报道。目的本研究采用人胃粘膜上皮细胞株(GES-1),运用氧糖剥夺(OGD)方法来构建细胞的缺氧缺糖模型,单纯从“缺血”角度研究胃粘膜损伤的机制,并观察UTI预处理是否对损伤的细胞有保护作用。内容与方法1.构建GES-1细胞株的OGD模型:采用无糖培养基和三气培养箱对GES-1细胞给予不同的OGD时间(0、2、4、6、8h),用CCK-8法检测细胞活力,Hoechst染色法和流式细胞仪检测细胞凋亡,免疫印迹法检测Bcl-2,Bax蛋白的表达;2.摸索UTI合适的作用浓度:将GES-1细胞按顺序分别加入浓度为0、100、200、400、800、1000U/mL 的 UTI,作用 12h 后进行 CCK-8 法检测,发现 1000 U/ml组细胞存活率较OU/ml组明显下降(P0.01),而其余组细胞存活率无明显变化。故在OGD损伤6h前,去掉1000U/ml浓度组,将细胞随机分为正常对照组、氧糖剥夺组、不同浓度UTI预处理组(UTI浓度分别为100、200、400、800U/mL),处理后用CCK-8法检测;3.探索UTI减轻GES-1细胞OGD损伤的机制:GES-1细胞随机分为正常对照组(N组)、氧糖剥夺组(O组)、UTI预处理组(U组)。N组不做任何处理,O组和U组细胞均进行OGD处理6h,且U组细胞在OGD前先用800U/ml UTI提前作用12h。处理后,用CCK-8法测定细胞活力,流式细胞术检测细胞凋亡,免疫印迹法检测Caspase-3和Cleaved Caspase-3的蛋白表达水平,实时荧光定量PCR法检测细胞内TRPV1 mRNA的表达水平。结果1.检测结果显示,随着OGD时间的延长,细胞活力明显下降,凋亡细胞明显增多,凋亡率显著上升,凋亡相关蛋白Bax/Bcl-2比值逐渐增加(P0.01或P0.05)。2.与氧糖剥夺组比较,细胞存活率在UTI浓度提高到400U/ml和800U/ml时明显增加,且在UTI浓度为800U/ml时细胞存活率最高(P0.01)。3.结果显示,与N组比较,O组细胞存活率显著降低,凋亡率增加,Caspase-3 和 Cleaved Caspase-3 表达增加,TRPV1 mRNA 表达降低(P0.05);而 UTI预处理能显著抑制上述O组的变化,差异具有统计学意义。结论利用无糖培养基和三气培养箱可成功构建GES-1细胞的OGD模型,能较好地观察缺氧缺糖对胃粘膜上皮细胞的损伤情况;UTI预处理可减轻GES-1细胞OGD损伤,其保护机制可能与减少细胞凋亡以及对TRPV1的上调相关。
[Abstract]:Background AGMLs with acute gastric mucosal injury is a common perioperative complication. At present, it is generally believed that gastric mucosal ischemia is the main link in the pathogenesis of AGML. Our group has passed the rat model of restraint stress in the early stage. From the aspects of injurious stress and non-injurious psychological stress, respectively, this paper discusses the problem of psychological stress. The pathogenesis of AGML. However, the pathogenesis and possible protective measures of AGML have not been discussed from the point of view of ischemia. UTI) as a broad-spectrum urinary trypsin inhibitor. It can reduce oxygen free radical, stabilize lysosomal membrane, reduce inflammation, improve circulation and so on. Studies have shown that UTI has protective effect on intestinal mucosa in critically ill patients. But whether it can protect gastric mucosa has not been reported. Objective to construct the model of hypoxia and glucose deficiency by using the method of oxygen glucose deprivation (OGDD) and human gastric mucosal epithelial cell line GES-1. To study the mechanism of gastric mucosal injury from the angle of "ischemia". The effects of UTI pretreatment on the injured cells were observed. 1. The OGD model of GES-1 cell line was established. The GES-1 cells were treated with different OGD time in glucose free medium and three gas incubator. 0. (2) CCK-8 assay was used to detect cell viability and apoptosis was detected by flow cytometry, and the expression of Bcl-2P Bax protein was detected by Western blot. 2. To find out the appropriate concentration of UTI: the GES-1 cells were added in the order of the concentration of 0 ~ 100U ~ (200) O ~ (400) ~ (400) ~ (-1) U / mL of UTI. The cell survival rate of 1 000 U / ml group was significantly lower than that of OU/ml group (P 0.01). However, the survival rate of the other groups did not change significantly, so before 6 hours of OGD injury, the cells were randomly divided into normal control group and oxygen glucose deprivation group. The concentration of UTI in different concentrations of UTI pretreatment group was 100U / mLX / m ~ (-1), respectively, and was detected by CCK-8 method after treatment. 3. To explore the mechanism of UTI in alleviating OGD damage in GES-1 cells. The cells were randomly divided into normal control group (n group) and oxygen glucose deprivation group (group O). The cells of UTI preconditioning group were treated with OGD for 6 h without any treatment. U group cells were treated with 800U / ml UTI for 12h before OGD, then the cell viability was measured by CCK-8 assay and apoptosis was detected by flow cytometry. The protein expression of Caspase-3 and Cleaved Caspase-3 was detected by Western blot. The expression of TRPV1 mRNA in cells was detected by real-time fluorescence quantitative PCR. Results 1. The results showed that with the prolongation of OGD time, cell viability decreased significantly. 2. The apoptotic cells increased significantly, the apoptosis rate increased significantly, and the ratio of apoptosis-related protein Bax/Bcl-2 increased gradually (P0.01 or P0.050.2.Compared with the oxygen glucose deprivation group. Cell viability was significantly increased when UTI concentration increased to 400U / ml and 800U / ml. When the concentration of UTI was 800U / ml, the cell survival rate was the highest (P 0.01). The results showed that compared with N group, the cell survival rate was significantly lower and the apoptosis rate was increased. The expression of Caspase-3 and Cleaved Caspase-3 increased and the expression of TRPV1 mRNA decreased. UTI pretreatment can significantly inhibit the changes of the above O group, the difference is statistically significant. Conclusion the OGD model of GES-1 cells can be successfully constructed by using sugar-free culture medium and three-air incubator. The injury of gastric mucosal epithelial cells induced by hypoxia and glucose deficiency was observed. UTI pretreatment can attenuate the OGD damage of GES-1 cells, and its protective mechanism may be related to the decrease of apoptosis and the up-regulation of TRPV1.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R614

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