發(fā)布時(shí)間：2018-02-02 08:21

  本文關(guān)鍵詞： 烏司他丁 預(yù)處理 GES-1細(xì)胞 氧糖剝奪 TRPV1　出處：《南方醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：背景急性胃粘膜損傷(AGML)是臨床上常見的圍術(shù)期并發(fā)癥,其具體的發(fā)病機(jī)制至今仍未完全明確。目前普遍認(rèn)為胃粘膜缺血是AGML發(fā)病的主要環(huán)節(jié)。我們課題組前期已通過大鼠的浸水束縛應(yīng)激模型,分別從損傷性應(yīng)激和非損傷的心理應(yīng)激等層面探討了 AGML的發(fā)病進(jìn)程。然而卻一直未從缺血的角度探討AGML的發(fā)生機(jī)制及可能的防護(hù)措施。烏司他丁(UTI)作為一種廣譜尿胰蛋白酶抑制劑,具有減少氧自由基、穩(wěn)定溶酶體膜、減輕炎癥、改善循環(huán)等作用。研究表明UTI對(duì)危重患者腸粘膜具有保護(hù)作用,但其對(duì)胃粘膜是否也有保護(hù)作用尚無報(bào)道。目的本研究采用人胃粘膜上皮細(xì)胞株(GES-1),運(yùn)用氧糖剝奪(OGD)方法來構(gòu)建細(xì)胞的缺氧缺糖模型,單純從“缺血”角度研究胃粘膜損傷的機(jī)制,并觀察UTI預(yù)處理是否對(duì)損傷的細(xì)胞有保護(hù)作用。內(nèi)容與方法1.構(gòu)建GES-1細(xì)胞株的OGD模型:采用無糖培養(yǎng)基和三氣培養(yǎng)箱對(duì)GES-1細(xì)胞給予不同的OGD時(shí)間(0、2、4、6、8h),用CCK-8法檢測細(xì)胞活力,Hoechst染色法和流式細(xì)胞儀檢測細(xì)胞凋亡,免疫印跡法檢測Bcl-2,Bax蛋白的表達(dá);2.摸索UTI合適的作用濃度:將GES-1細(xì)胞按順序分別加入濃度為0、100、200、400、800、1000U/mL 的 UTI,作用 12h 后進(jìn)行 CCK-8 法檢測,發(fā)現(xiàn) 1000 U/ml組細(xì)胞存活率較OU/ml組明顯下降(P0.01),而其余組細(xì)胞存活率無明顯變化。故在OGD損傷6h前,去掉1000U/ml濃度組,將細(xì)胞隨機(jī)分為正常對(duì)照組、氧糖剝奪組、不同濃度UTI預(yù)處理組(UTI濃度分別為100、200、400、800U/mL),處理后用CCK-8法檢測;3.探索UTI減輕GES-1細(xì)胞OGD損傷的機(jī)制:GES-1細(xì)胞隨機(jī)分為正常對(duì)照組(N組)、氧糖剝奪組(O組)、UTI預(yù)處理組(U組)。N組不做任何處理,O組和U組細(xì)胞均進(jìn)行OGD處理6h,且U組細(xì)胞在OGD前先用800U/ml UTI提前作用12h。處理后,用CCK-8法測定細(xì)胞活力,流式細(xì)胞術(shù)檢測細(xì)胞凋亡,免疫印跡法檢測Caspase-3和Cleaved Caspase-3的蛋白表達(dá)水平,實(shí)時(shí)熒光定量PCR法檢測細(xì)胞內(nèi)TRPV1 mRNA的表達(dá)水平。結(jié)果1.檢測結(jié)果顯示,隨著OGD時(shí)間的延長,細(xì)胞活力明顯下降,凋亡細(xì)胞明顯增多,凋亡率顯著上升,凋亡相關(guān)蛋白Bax/Bcl-2比值逐漸增加(P0.01或P0.05)。2.與氧糖剝奪組比較,細(xì)胞存活率在UTI濃度提高到400U/ml和800U/ml時(shí)明顯增加,且在UTI濃度為800U/ml時(shí)細(xì)胞存活率最高(P0.01)。3.結(jié)果顯示,與N組比較,O組細(xì)胞存活率顯著降低,凋亡率增加,Caspase-3 和 Cleaved Caspase-3 表達(dá)增加,TRPV1 mRNA 表達(dá)降低(P0.05);而 UTI預(yù)處理能顯著抑制上述O組的變化,差異具有統(tǒng)計(jì)學(xué)意義。結(jié)論利用無糖培養(yǎng)基和三氣培養(yǎng)箱可成功構(gòu)建GES-1細(xì)胞的OGD模型,能較好地觀察缺氧缺糖對(duì)胃粘膜上皮細(xì)胞的損傷情況;UTI預(yù)處理可減輕GES-1細(xì)胞OGD損傷,其保護(hù)機(jī)制可能與減少細(xì)胞凋亡以及對(duì)TRPV1的上調(diào)相關(guān)。
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R614

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