P物质与大鼠结肠动力紊乱的相关研究

发布时间：2018-05-24 04:53

本文选题：应激 + 大鼠　；参考：《武汉大学》2016年博士论文

【摘要】：第一部分慢性应激大鼠模型的建立及P物质与其受体表达的变化目的：建立慢性应激模型,探讨慢性应激对P物质(Substance P)及其受体表达的影响。方法：将实验大鼠随机分为模型组和对照组,采用连续10天的反复避水应激(water-avoidance stress, WAS)建立动物模型；腹腔麻醉处死大鼠后取近端结肠,制备纵行肌(longitudinal muscle, LM)和环形肌条(circular muscle, CM),采用浴槽实验观察模型组(water-avoidance stress, WAS)和对照组(shamwater-avoidance stress, SWAS)结肠平滑肌细胞自发性的收缩活动并比较两组收缩的强度；采用酶联免疫吸附实验(ELISA)检测模型组和对照组血清及结肠黏膜SP水平；采用免疫组织化学法检测神经激肽1型受体(neurokinin-1 receptor, NK1R)和神经激肽2型受体(neurokinin-2 receptor, NK2R)在结肠的分布；采用蛋白免疫印迹法(Western blotting)测定NK1R和NK2R在模型组和对照组中结肠肌层的表达。结果：反复WAS使大鼠结肠LM和CM自发性收缩幅度增加[LM(1.14±0.05)g比.(0.94±0.04) g;CM (0.36±0.02) g比(0.25±0.02)g,P0.01],且使模型组于造模第1、3、5、7、10天的大鼠排便增加；与对照组相比,WAS组大鼠血清SP浓度显著升高[WAS (212.1±10.6)pg/mL比SWAS (105.5±4.4) pg/mL, P0.01];反复避水应激使大鼠结肠黏膜SP水平增加[WAS组(661.5±20.6) pg/mg蛋白比SWAS组(250.1±12.1) pg/mg蛋白,P0.01]; NK1R主要分布于WAS和SWAS大鼠结肠黏膜上皮、固有层平滑肌细胞及肠神经元；NK2R主要分布于WAS和SWAS大鼠结肠黏膜上皮及肠神经元,固有层平滑肌细胞散在分布；反复WAS使大鼠结肠肌层NK1R表达上调而NK2R表达无明显改变。结论：慢性应激诱导大鼠结肠动力增强,SP-NK1R信号系统可能参与该病理生理过程的发生。第二部分P物质及其受体拮抗剂对慢性应激大鼠结肠高动力的调节作用目的：探讨慢性应激对P物质(Substance P)诱导的大鼠结肠收缩活动的影响。方法：将实验动物随机分为模型组和对照组,采用连续10天的反复避水应激(water-avoidance stress, WAS)建立动物模型；腹腔麻醉处死大鼠后取近端结肠,制备纵行肌(longitudinal muscle,LM)和环形肌条(circular muscle, CM);张力换能器及多通道生理信号采集处理系统观察并记录模型组和对照组(sham water-avoidance stress, SWAS)结肠平滑肌自发性收缩活动,比较SP诱导的两组大鼠结肠平滑肌收缩强度的改变及经SP受体拮抗剂孵育后SP诱导的WAS组和SWAS组结肠收缩强度。结果：SP (0.1 μM)使模型组和对照组LM收缩增强,但两组间R值比较无显著差异(P0.05); SP(1 μM)使模型组和对照组R值分别从1增加至1.39±0.12和1.67±0.08,与基线值差异显著(P0.01),且WAS组R值显著高于SWAS组(P0.05)；SP(0.1μM)使模型组和对照组CM收缩增强,R值分别由1增加至9.65+0.85和5.35±0.29,与基线值相比,P0.01且模型组R值显著高于SWAS组(P0.01)；SP(1μM)使模型组和对照组CM的R值分别从1增加至14.49±2.75和7.31±1.45与基线值差异显著(P0.01),且WAS组R值显著高于SWAS组(P0.05); NK1R拮抗剂L-703(1μM)孵育肌条后,SP(1μM)对模型组LM和CM的增强作用逆转,对照组高于模型组。结论：慢性应激使SP诱导的大鼠结肠平滑肌收缩增强,且该作用被NK1R受体拮抗剂阻断,说明SP-NK1R系统可能参与WAS诱导的结肠高动力的发生。第三部分慢性应激大鼠近端结肠L型钙电流的变化及P物质对钙电流的影响目的：探讨慢性应激对SP诱导的大鼠结肠平滑肌L型钙电流的影响。方法：将实验动物随机分为模型组和对照组,采用连续10天的反复避水应激(water-avoidance stress, WAS)建立动物模型；腹腔麻醉处死大鼠后取近端结肠,采用酶消化法分离平滑肌细胞；运用全细胞膜片钳技术记录WAS组和对照组(sham water-avoidance stress, SWAS)组平滑肌细胞L型钙通道电流及SP对两组动物钙电流的影响。结果：SWAS组和WAS组L型钙通道峰电流密度分别为(-3.93±0.38)pA/pF和(-3.49±0.17) pA/pF,组间无显著差异(P0.05)；SP对平滑肌细胞L型钙通道有促进作用,SP (1 μM)使WAS组峰电流密度由(-3.4±0.17) pA/pF增加至(-4.8±0.31) pA/pF (P0.05),使SWAS组峰电流密度由(-3.93±0.38) pA/pF增加至(-4.54±0.39) pA/pF (P0.05)。且SP使模型组峰电流较基础值增加129±3%,使对照组较自身基础值增加116±2.7%,WAS组百分比显著高于对照组(P0.05)。结论：慢性应激不改变大鼠结肠平滑肌细胞L型钙通道电流的大小；SP通过L型钙通道促进平滑肌收缩,且慢性应激使SP对钙电流的作用增强。
[Abstract]:Objective : To establish a chronic stress model and to explore the effect of chronic stress on substance P and its receptor expression . Methods : The experimental rats were randomly divided into model group and control group , and the animal model was established with water - stress stress ( WAS ) for 10 days .
The proximal colon , longitudinal muscle ( LM ) and circular muscle ( CM ) were prepared by intraperitoneal anesthesia . The spontaneous contraction of colonic smooth muscle cells was observed in the model group ( water - induced stress , WAS ) and control group ( SWAS ) , and the intensity of contraction was compared between the two groups .
The levels of SP in serum and colon mucosa were determined by enzyme linked immunosorbent assay ( ELISA ) .
The distribution of neurokinin - 1 receptor ( NK1R ) and neurokinin - 2 receptor ( NK2R ) in colon was detected by immunohistochemistry .
The expression of NK1R and NK2R in model group and control group were measured by Western blotting . Results : Repeated WAS increased the spontaneous contraction amplitude of colon LM and CM in rats ( 1.14 卤 0.05 ) g , ( 0.94 卤 0.04 ) g , CM ( 0.36 卤 0.02 ) g ( 0.25 卤 0.02 ) g , P0.01 respectively .
Compared with the control group , the serum SP concentration of the rats was significantly higher than that in SWAS group ( 105.1 卤 10.6 ) pg / mL , P0.01 ) . The level of SP in colonic mucosa of rats was increased by 661.5 卤 20.6 pg / mg protein ( 250.1 卤 12.1 ) pg / mg protein ( P0.01 ) . NK1R was mainly distributed in colonic mucosa epithelium , lamina propria smooth muscle cells and intestinal neurons of WAS and SWAS rats .
NK2R was mainly distributed in the colonic mucosa and intestinal neurons of WAS and SWAS rats .
The effects of chronic stress on colonic contraction in rats with chronic stress induced by P substance and its receptor antagonists were investigated .
The results showed that SP ( 0.1 渭M ) increased the contractile intensity of colonic smooth muscle in the model group and control group . The results showed that SP ( 0.1 渭M ) increased the contractile intensity of colonic smooth muscle in the model group and control group . The results showed that SP ( 0.1 渭M ) increased the model group and the control group ( P < 0.01 ) . The R value of the group was significantly higher than that in SWAS group ( P0.05 ) .
SP ( 0.1 渭M ) increased the contraction of CM in model group and control group , and the R value increased from 1 to 9.65 + 0.85 and 5.35 卤 0.29 respectively , compared with baseline , P0.01 and model group R value was significantly higher than that in SWAS group ( P0.01 ) .
The effects of chronic stress on L - type calcium current in colonic smooth muscle of rats induced by SP were studied .
The proximal colon was removed by intraperitoneal anesthesia , and smooth muscle cells were isolated by enzymatic digestion .
The results showed that the current density of L - type calcium channel in SWAS group and WAS group was ( - 3.93 卤 0.38 ) pA / pF and ( - 3.49 卤 0.17 ) pA / pF , there was no significant difference between the groups ( P0.05 ) .
SP ( 1 渭M ) increased the peak current density from ( - 3.93 卤 0.38 ) pA / pF to ( - 4.54 卤 0.39 ) pA / pF ( P0.05 ) .
SP promotes smooth muscle contraction through L - type calcium channel , and chronic stress increases the effect of SP on calcium current .
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R574.62

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