P38MAPK与caspase-3在大鼠胃缺血再灌注中的表达及其相关性研究
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摘要
实验目的
胃肠缺血再灌注损伤是胃肠手术、全身炎症反应综合症和机体应激后导致胃肠溃疡、出血或功能障碍等的一种基本病理生理变化。本课题采用免疫组织化学等检测方法,观察大鼠胃缺血30min后不同再灌注时间段胃黏膜表达p38MAPK (mitogen-activated protein kinase, MAPK)和半胱天冬氨酸蛋白酶-3(caspase-3)的变化趋势；结合相应时间段胃黏膜损伤指数及其组织病理学变化,探索胃缺血再灌注损伤(gastric ischemia-reperfusion injury, GI-RI)中p38MAPK和caspase-3表达的规律；最后用医学统计学方法分析p38MAPK. caspase-3的表达与胃粘膜损伤程度的关系,探讨两者在GI-RI中表达的相关关系。
实验材料与方法
健康成年SD大鼠30只,雌雄各半,体重220±10g,随机分为假手术组和缺血再灌注组。10%水合氯醛0.3ml/100g腹腔注射,麻醉成功后取腹部正中切口,分离并阻断胃主要动脉(胃左动脉、胃右动脉、胃网膜左动脉、胃网膜右动脉以及胃后、胃短动脉)30min后去除动脉夹再灌注0.5h、1h、3h、6h和24h,将大鼠断颈椎处死,开腹取胃。首先以两把血管钳分别夹闭食管和十二指肠胃端,而后胃内注射10%中性甲醛溶液1ml,取胃：沿胃大弯纵行切开,以冰盐水清洗后置入10%中性甲醛溶液测量胃黏膜出血情况,标记后置入10%中性甲醛溶液固定约12h；取材,主要取腺胃组织约0.5×1cm2,石蜡包埋,制作5μm连续切片(每个样本做出3张组织切片分别用于p38MAPK、caspase-3检测和HE染色)。应用免疫组织化学方法检测30例大鼠胃的p38MAPK和caspase-3的表达情况,以假手术组作对照,显微镜400×下,每个切片在胃黏膜区域随机选取5个视野。应用IPP6.0图像分析软件检测积分光密度(IOD)和面积(Area)值,计算求得各组平均光密度值(mean density),测得数据以均数±标准差表示。应用SPSS13.0软件对相关数据进行分析,取p=0.05为检验水准。
结果
假手术组胃黏膜表达p38MAPK和caspase-3水平较低,GI-R后胃黏膜表达p38MAPK和caspase-3水平迅速上升；再灌注0.5h时caspase-3表达呈高峰,随后开始下降,至再灌注后3h降至最低,而后又缓慢升高,至再灌注24h达第二个高峰；p38MAPK在再灌注1h时达到峰值,随后下降,但仍维持在较高水平至再灌注：24h。GI-R后胃黏膜损伤指数显著上升,至再灌注1h达峰值；随着再灌注时间延长,胃黏膜在多种因素作用下进行修复或适应,损伤指数开始下降,至再灌注3h到低谷；此后损伤指数又开始缓慢上升,胃黏膜损伤又有所加重但低于前者,再灌注后6h至24h没有明显变化。
结论
GI-RI是由多因素参与的损伤与修复过程,其中caspase-3表达是促进GI-RI进展的重要因素之一；GI-RI在GI-R 1h内进展最快、表现最严重,该时间段可能是防治GI-RI的关键；p38MAPK调控的炎性反应、细胞凋亡等,参与了GI-RI的形成、发展和转归；在GI-RI中,p38MAPK和caspase-3的表达在统计学上不表现为相关性。
Objective
Gastrointestinal ischemia-reperfusion is a basic pathological change for gastrointestinal ulceration, bleeding and dysfunction after the gastrointestinal surgery, systemic inflammatory response and so on. The expression and application of p38 MAPK in gastrointestinal ischemia-reperfusion has little reported home and abroad. This study uses immunohistochemistry methods to detect the expression of p38MAPK and caspase-3 factors in different time after reperfusion, integrating the gastric mucosal damage index and the pathological change with the corresponding time, to export the expression law of p38MAPK and caspase-3 factor. Moreover, this text will discuss their relationship with gastric mucosal injury degree and analysis the expression pertinence between p38MAPK and caspase-3 factors by SPSS 13.0 software.
Materials and Methods
30 healthy adult SD rats, male and female in half, weight 220±10g, were randomly divided into six groups, sham-operated group (the same surgical procedure without clamping the celiac artery), GI-R groups (reperfusion 0.5h,1h,3h,6h,24h, respectively, after 30min of ischemia).10% chloral hydrate 0.3ml/100g inject into abdominal cavity for anaesthesia. After successful anaesthesia, cut open the abdominal cavity, the celiac artery and its adjacent tissues were carefully isolated. The celiac artery was clamped with a small non-traumatic vascular clamp for 30 min to induce ischemia and then released to allow reperfusion. After reperfusion, the rats were sacrificed to remove the stomach immediately. And then,5 pieces of tissue of 1.0mm*2.0 mm at the greater curvature from the gastric mucosa were immersed in 2.5%glutaraldehyde solution at 4℃for transmission microscopy. The paraffin tissue slices (5μm) were pasted discontinuously to glass pretreated with poly-lysine. The part of paraffin tissue slices was stained with hematoxylin-eosin. After that, use immunohistochemical assay to detect the expression of p38 and caspase-3 of this rats. Compared with the sham group, the GI-R groups were counted in 5 random hign-power (*40) or lower-power (*10) fields. Take IPP map analysis software to detect the IOD and area value, calculate the mean density value with each group, and express as mean±D. At last, use spss 13.0 software to statistic the data, selecta=0.05 as a standard.
Result and conclusion
The gastric mucosal express p38MAPK and caspase-3 factor in false operation group much less than operation group. After gastric ischemia-reperfusion (GI-R) caspase-3 expression increase rapidly, and it reach first peak in 0.5h, and then decline, to 3h it go to the bottom, and then increase slowly, to 24h it reach the maximum value. As to p38MAPK expression, lh it reach its peak, and then it decline, but its decline trend has no any statistic meaning, because it still stay in high level. Meanwhile, gastric mucosal damage index increasing when start with GI-R. and it reach its peak in 1h, with the time going on, the injury of gastric mucosal start to repair, so the damage index decline in time, going to bottom at 3h, but then it climbs slowly within 3h to 6h, that its to say, the injury aggravation,6h to 24h that is no significant change.
As we seen from above result, the expressions of p38MAPK and caspase-3 factor have association with the gastric mucosal damage index in the progress of gastric ischemia-reperfusion injury. At first they show the positive correction with each other. Gastric ischemia-reperfusion lesion and repair are caused by multiple factors. P38MAPK regulate and control the inflammatory response and cells apoptotic, promoting gastric ischemia-reperfusion injury form and development.
胃肠缺血再灌注损伤是胃肠手术、全身炎症反应综合症和机体应激后导致胃肠溃疡、出血或功能障碍等的一种基本病理生理变化。本课题采用免疫组织化学等检测方法,观察大鼠胃缺血30min后不同再灌注时间段胃黏膜表达p38MAPK (mitogen-activated protein kinase, MAPK)和半胱天冬氨酸蛋白酶-3(caspase-3)的变化趋势；结合相应时间段胃黏膜损伤指数及其组织病理学变化,探索胃缺血再灌注损伤(gastric ischemia-reperfusion injury, GI-RI)中p38MAPK和caspase-3表达的规律；最后用医学统计学方法分析p38MAPK. caspase-3的表达与胃粘膜损伤程度的关系,探讨两者在GI-RI中表达的相关关系。
实验材料与方法
健康成年SD大鼠30只,雌雄各半,体重220±10g,随机分为假手术组和缺血再灌注组。10%水合氯醛0.3ml/100g腹腔注射,麻醉成功后取腹部正中切口,分离并阻断胃主要动脉(胃左动脉、胃右动脉、胃网膜左动脉、胃网膜右动脉以及胃后、胃短动脉)30min后去除动脉夹再灌注0.5h、1h、3h、6h和24h,将大鼠断颈椎处死,开腹取胃。首先以两把血管钳分别夹闭食管和十二指肠胃端,而后胃内注射10%中性甲醛溶液1ml,取胃：沿胃大弯纵行切开,以冰盐水清洗后置入10%中性甲醛溶液测量胃黏膜出血情况,标记后置入10%中性甲醛溶液固定约12h；取材,主要取腺胃组织约0.5×1cm2,石蜡包埋,制作5μm连续切片(每个样本做出3张组织切片分别用于p38MAPK、caspase-3检测和HE染色)。应用免疫组织化学方法检测30例大鼠胃的p38MAPK和caspase-3的表达情况,以假手术组作对照,显微镜400×下,每个切片在胃黏膜区域随机选取5个视野。应用IPP6.0图像分析软件检测积分光密度(IOD)和面积(Area)值,计算求得各组平均光密度值(mean density),测得数据以均数±标准差表示。应用SPSS13.0软件对相关数据进行分析,取p=0.05为检验水准。
结果
假手术组胃黏膜表达p38MAPK和caspase-3水平较低,GI-R后胃黏膜表达p38MAPK和caspase-3水平迅速上升；再灌注0.5h时caspase-3表达呈高峰,随后开始下降,至再灌注后3h降至最低,而后又缓慢升高,至再灌注24h达第二个高峰；p38MAPK在再灌注1h时达到峰值,随后下降,但仍维持在较高水平至再灌注：24h。GI-R后胃黏膜损伤指数显著上升,至再灌注1h达峰值；随着再灌注时间延长,胃黏膜在多种因素作用下进行修复或适应,损伤指数开始下降,至再灌注3h到低谷；此后损伤指数又开始缓慢上升,胃黏膜损伤又有所加重但低于前者,再灌注后6h至24h没有明显变化。
结论
GI-RI是由多因素参与的损伤与修复过程,其中caspase-3表达是促进GI-RI进展的重要因素之一；GI-RI在GI-R 1h内进展最快、表现最严重,该时间段可能是防治GI-RI的关键；p38MAPK调控的炎性反应、细胞凋亡等,参与了GI-RI的形成、发展和转归；在GI-RI中,p38MAPK和caspase-3的表达在统计学上不表现为相关性。
Objective
Gastrointestinal ischemia-reperfusion is a basic pathological change for gastrointestinal ulceration, bleeding and dysfunction after the gastrointestinal surgery, systemic inflammatory response and so on. The expression and application of p38 MAPK in gastrointestinal ischemia-reperfusion has little reported home and abroad. This study uses immunohistochemistry methods to detect the expression of p38MAPK and caspase-3 factors in different time after reperfusion, integrating the gastric mucosal damage index and the pathological change with the corresponding time, to export the expression law of p38MAPK and caspase-3 factor. Moreover, this text will discuss their relationship with gastric mucosal injury degree and analysis the expression pertinence between p38MAPK and caspase-3 factors by SPSS 13.0 software.
Materials and Methods
30 healthy adult SD rats, male and female in half, weight 220±10g, were randomly divided into six groups, sham-operated group (the same surgical procedure without clamping the celiac artery), GI-R groups (reperfusion 0.5h,1h,3h,6h,24h, respectively, after 30min of ischemia).10% chloral hydrate 0.3ml/100g inject into abdominal cavity for anaesthesia. After successful anaesthesia, cut open the abdominal cavity, the celiac artery and its adjacent tissues were carefully isolated. The celiac artery was clamped with a small non-traumatic vascular clamp for 30 min to induce ischemia and then released to allow reperfusion. After reperfusion, the rats were sacrificed to remove the stomach immediately. And then,5 pieces of tissue of 1.0mm*2.0 mm at the greater curvature from the gastric mucosa were immersed in 2.5%glutaraldehyde solution at 4℃for transmission microscopy. The paraffin tissue slices (5μm) were pasted discontinuously to glass pretreated with poly-lysine. The part of paraffin tissue slices was stained with hematoxylin-eosin. After that, use immunohistochemical assay to detect the expression of p38 and caspase-3 of this rats. Compared with the sham group, the GI-R groups were counted in 5 random hign-power (*40) or lower-power (*10) fields. Take IPP map analysis software to detect the IOD and area value, calculate the mean density value with each group, and express as mean±D. At last, use spss 13.0 software to statistic the data, selecta=0.05 as a standard.
Result and conclusion
The gastric mucosal express p38MAPK and caspase-3 factor in false operation group much less than operation group. After gastric ischemia-reperfusion (GI-R) caspase-3 expression increase rapidly, and it reach first peak in 0.5h, and then decline, to 3h it go to the bottom, and then increase slowly, to 24h it reach the maximum value. As to p38MAPK expression, lh it reach its peak, and then it decline, but its decline trend has no any statistic meaning, because it still stay in high level. Meanwhile, gastric mucosal damage index increasing when start with GI-R. and it reach its peak in 1h, with the time going on, the injury of gastric mucosal start to repair, so the damage index decline in time, going to bottom at 3h, but then it climbs slowly within 3h to 6h, that its to say, the injury aggravation,6h to 24h that is no significant change.
As we seen from above result, the expressions of p38MAPK and caspase-3 factor have association with the gastric mucosal damage index in the progress of gastric ischemia-reperfusion injury. At first they show the positive correction with each other. Gastric ischemia-reperfusion lesion and repair are caused by multiple factors. P38MAPK regulate and control the inflammatory response and cells apoptotic, promoting gastric ischemia-reperfusion injury form and development.
引文
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[8]梁晚益,黄跃生,杨宗城.线粒体在缺血再灌注细胞损伤中的作用[J].国外医学生理病理科学与临床分册,2000,20：364-366.
[9]王刚石,王孟薇,吴本俨.人胃粘膜细胞的分离与羟自由基损伤模型的建立.世界华人消化杂志,1999,7：1006-1008.
[10]Liu F, Li JX, Li CM, et al. Plasma endothelin in patients with endotoxemia and dynamic comparison between vasoconstrictor and vasodilator in cirrhotic patients[J]. World J Gastroenterol,2001,7:126-127.
[11]郭津生,古永亮,王吉耀,等.结构型与诱生型一氧化氮合酶在大鼠胃溃疡模型中的表达和活性变化[J].世界华人消化杂志,2001,9：288-292.
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[14]Brzozowski T, Konturek PC, Konturek SJ, et al. Role of gastric acid secretion in progression of acute gastric erosions induced by ischemia-reperfusion into gastric ulcers[J]. Eur J Pharmacol,2000,398:147-158.
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