【作者】 孙阳；

【导师】 戈应滨； 顾洛；

【作者基本信息】 南京医科大学 ， 生理学， 2004， 硕士

【摘要】 我们以往的研究表明，大鼠饮用3-14天6％酒精后，其胃粘膜会产生抵抗100％纯酒精伤害的能力，这种能力和胃粘膜细胞的细胞更新有关。胃粘膜适应性细胞保护作用的产生是多种因素共同作用的结果，其机制并不能用一种现象能够完全解释。众所周知，酒精通过各种途径损伤胃粘膜，首先作为一种攻击因子使胃粘膜直接受到损伤，其次释放氧自由基等损伤胃粘膜，同时也可以使胃粘膜血流量减少引起胃粘膜损伤。但是饮用酒精3-14天引起的适应性细胞保护作用是否和酒精浓度有关，目前还不十分明确。为进一步探讨低浓度酒精引起的胃粘膜适应性细胞保护作用的确切机制，我们设计并实施了以下实验。 首先，我们检验了饮用1％，3％，6％，12％和24％等不同浓度酒精3天后的大鼠胃粘膜对100％酒精损伤的抵抗能力和胃粘膜的细胞更新情况。结果发现，饮用6％和12％酒精3天的大鼠胃粘膜具备适应性细胞保护作用，而饮用其他浓度酒精的大鼠胃粘膜则没有。同时也只有饮用6％和12％酒精的大鼠胃粘膜的细胞增殖加快。这说明，饮用低浓度酒精确实会引起胃粘膜适应性细胞保护作用，而且这种保护作用和胃粘膜的细胞更新有关。这种现象不但和饮酒时程有关，而且和饮酒浓度有关。 其次，我们检测了大鼠饮用6％酒精3天后，对其他胃粘膜损伤因子0.6N盐酸和盐酸酒精的抵抗作用，同时检测了大鼠胃粘膜产生适南京医科大学硕士学位论文应性细胞保护作用后抵抗100%酒精损伤的能力的持续时间。结果发现，大鼠胃粘膜抵抗100%酒精损伤的能力能够持续3天，而饮用6%酒精3天对0.6N盐酸和盐酸酒精的损伤没有保护作用。大鼠胃粘膜细胞的更新周期为3一5天，本实验结果说明，胃粘膜适应性细胞保护作用确实和细胞更新加快有关，证明了我们以前的研究结果。本实验同时表明，6%酒精并没有直接增强细胞对伤害刺激的抵抗能力，而且也没有使胃粘膜的血流量和微循环得到改善。 活性氧基团是指超氧阴离子、过氧化氢和羚基等一类化学性质较02更为活泼的02的衍生物，酒精可以产生大量氧自由基，并且是引起胃粘膜损伤的重要原因之一。除氧自由基外，N0也是一种重要的自由基，大量N0不但直接损伤胃粘膜，而且还可以和超氧阴离子结合产生氧化损伤作用更强的过氧亚稍基(ONOO一)，加重细胞或胃粘膜的损伤。另一方面，也有文献报道N0具有保护胃粘膜的作用。我们以前的研究发现，大鼠饮用6%酒精后，胃粘膜清除氧自由基的能力并未增强。因此我们设想，慢性饮用低浓度酒精可能会影响胃粘膜内N0的产生，进而影响其适应性细胞保护作用的产生。为验证这一假说，我们用免疫组化和RT一PCR法检测了饮用不同浓度酒精3天后大鼠胃粘膜内三种N0产生的限速酶iNOS、nNOS和eNOS的表达。结果发现，三种合酶的免疫组化阳性细胞无论在对照组还是饮酒组均有表达;1 NOS的mRNA在对照组、饮用1%、3%和6%酒精组没有表达，在饮用12%酒精组有少量表达，而在饮用24%酒精组有高度表达;nNOS的mRNA在对照组、饮用1%、3%和24%酒精组强表达，而在饮用6%和12%酒精组的胃粘膜内却无表达;eNOS的mRNA在各组胃粘膜内无规律表达。这些结果表明，饮用6%和12%酒精3天的大鼠胃粘膜适应性细胞保护作用的产生和胃粘膜内nNOS的活性下降有关，饮用24%酒精3天的大鼠胃粘膜适应性细胞保护作用的消失和胃粘膜内iNOS的活性增强有关。 通过以上研究，我们得出下列结论: 1.慢性饮用低浓度酒精产生的胃粘膜适应性细胞保护作用不但与 饮酒时程有关，而且与饮酒浓度有关。子南京医科大学硕士学位论文2.这种适应性细胞保护作用和胃粘膜的细胞更新密切相关。3.慢性饮用低浓度酒精产生的胃粘膜适应性细胞保护作用，和胃 粘膜抵杭损伤能力的增强与否、胃粘膜血流量是否改善无关。4.慢性饮用低浓度酒精能抑制胃粘膜神经型一氧化氮合酶的活 性，使N0产生减少，进而使胃粘膜抵杭1 00%酒精伤害的能 力增强。更多还原

【Abstract】 Our previous studies indicated that the gastric mucosa of the rats with 6% ethanol exposure in 3rd to 14th days had the ability to resist subsequent injury of pure ethanol, which was related to the enhanced cell turnover of gastric mucosa. Various mechanisms contributed to this phenomenon, neither can explain it completely. It is well known that the ethanol can injury the gastric mucosa through multiple pathways, including direct assaulting, mediated by reactive oxygen species (ROS), and reducing gastric mucosal blood flow (GMBF). Whether this adaptive cytoprotection is related to the ethanol concentration is so far unknown. To clarify its mechanism, the following experiments were designed and performed.At first, the ability to resist the injury of pure ethanol and the cell turnover of gastric mucosa in rats treated with alcohol of 1%, 3%, 6%, 12%, and 24% concentration were examined, respectively. The adaptive cytoprotection was found only in 6% and 12% group. The enhanced cell turnover of gastric mucosa was also found in these two groups. These results indicated that this phenomenon could be induced by adequate chronic alcohol drinking, involving the role of enhanced gastric mucosal cell turnover. The gastric mucosal adaptive cytoprotection induced byethanol intake was related to both of drinking duration and ethanol concentration.Secondly, the resistance of gastric mucosa to 0.6N HC1 and HC1-ethanol were also investigated. The gastric mucosa treated with 6% ethanol for 3 days could not be able to against the injury of 0.6N HC1 and HC1-ethanol. It is well known that the cells renewing of gastric mucosa was 3 to 5 days. Our results supported the concept of the adaptive cytoprotection of gastric mucosa mediated by enhanced cell turnover. The results also indicated that stimulus with 6% ethanol did not increase the ability of cells to resist the injury of noxious agents (0.6N HC1, HCl-ethanol and so on). And the blood flow and microcirculation of gastric mucosa were not improved. We also investigated the duration of the ability against pure ethanol, and found it could last 3 days.Reactive oxygen species (ROS) were radical derivatives of molecular oxygen, including the superoxide radical (O2- ), hydrogen peroxide (H2O2), and the hydroxyl radical (OH). The gastric mucosal injury induced by ethanol was mainly mediated by ROS derived from ethanol stimulation. The nitric oxide (NO) was another important free radical. Overproduction of NO could damage gastric mucosa directly. NO also interacted with O2 to form the toxic species, peroxynitrite (ONOO-). On the other hand, it has been reported that NO could play a cytoprotection role in gastric mucosa. Our previous studies showed that the scavenging free radical ability of gastric mucosa had not been enhanced after treatment with 6% ethanol. In this case, we hypothesized that adaptive cytoprotection of chronic drinking rat gastric mucosa should be related to the production of NO. To address this issue, the expressions of neuronal nitrogen monoxide synthase (nNOS), endothelial nitrogen monoxide synthase (eNOS), and inducible nitrogen monoxide synthase (iNOS) were measured in gastric mucosa of rats treated with the ethanol in different concentration by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. Inimmunohistochemical study, positive staining cells of all three NOSs were detected in both control and ethanol groups. The results of RT-PCR showed that iNOS mRNA was overexpressed in the rat gastric mucosa of the groups in which drinking 12% and 24% ethanol. In contrast, the rat gastric mucosa in other groups such as drinking 0%, 1%, 3%, and 6% ethanol, the mRNA of iNOS was not expressed. The mRNA of nNOS was expressed markedly in the control, 1%, 3%, and 24% ethanol groups, while not expressed in 6% and 12% ethanol groups. The mRNA of eNOS was expressed random in all groups. These results suggested that the adaptive cytoprotection induced by 6% or 12% ethanol may be associated with the downregulation of the act更多还原