本文選題：應激 + 大鼠��； 參考：《武漢大學》2016年博士論文

【摘要】：第一部分慢性應激大鼠模型的建立及P物質(zhì)與其受體表達的變化目的：建立慢性應激模型,探討慢性應激對P物質(zhì)(Substance P)及其受體表達的影響。方法：將實驗大鼠隨機分為模型組和對照組,采用連續(xù)10天的反復避水應激(water-avoidance stress, WAS)建立動物模型；腹腔麻醉處死大鼠后取近端結腸,制備縱行肌(longitudinal muscle, LM)和環(huán)形肌條(circular muscle, CM),采用浴槽實驗觀察模型組(water-avoidance stress, WAS)和對照組(shamwater-avoidance stress, SWAS)結腸平滑肌細胞自發(fā)性的收縮活動并比較兩組收縮的強度；采用酶聯(lián)免疫吸附實驗(ELISA)檢測模型組和對照組血清及結腸黏膜SP水平；采用免疫組織化學法檢測神經(jīng)激肽1型受體(neurokinin-1 receptor, NK1R)和神經(jīng)激肽2型受體(neurokinin-2 receptor, NK2R)在結腸的分布；采用蛋白免疫印跡法(Western blotting)測定NK1R和NK2R在模型組和對照組中結腸肌層的表達。結果：反復WAS使大鼠結腸LM和CM自發(fā)性收縮幅度增加[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],且使模型組于造模第1、3、5、7、10天的大鼠排便增加；與對照組相比,WAS組大鼠血清SP濃度顯著升高[WAS (212.1±10.6)pg/mL比SWAS (105.5±4.4) pg/mL, P0.01];反復避水應激使大鼠結腸黏膜SP水平增加[WAS組(661.5±20.6) pg/mg蛋白比SWAS組(250.1±12.1) pg/mg蛋白,P0.01]; NK1R主要分布于WAS和SWAS大鼠結腸黏膜上皮、固有層平滑肌細胞及腸神經(jīng)元；NK2R主要分布于WAS和SWAS大鼠結腸黏膜上皮及腸神經(jīng)元,固有層平滑肌細胞散在分布；反復WAS使大鼠結腸肌層NK1R表達上調(diào)而NK2R表達無明顯改變。結論：慢性應激誘導大鼠結腸動力增強,SP-NK1R信號系統(tǒng)可能參與該病理生理過程的發(fā)生。第二部分P物質(zhì)及其受體拮抗劑對慢性應激大鼠結腸高動力的調(diào)節(jié)作用目的：探討慢性應激對P物質(zhì)(Substance P)誘導的大鼠結腸收縮活動的影響。方法：將實驗動物隨機分為模型組和對照組,采用連續(xù)10天的反復避水應激(water-avoidance stress, WAS)建立動物模型；腹腔麻醉處死大鼠后取近端結腸,制備縱行肌(longitudinal muscle,LM)和環(huán)形肌條(circular muscle, CM);張力換能器及多通道生理信號采集處理系統(tǒng)觀察并記錄模型組和對照組(sham water-avoidance stress, SWAS)結腸平滑肌自發(fā)性收縮活動,比較SP誘導的兩組大鼠結腸平滑肌收縮強度的改變及經(jīng)SP受體拮抗劑孵育后SP誘導的WAS組和SWAS組結腸收縮強度。結果：SP (0.1 μM)使模型組和對照組LM收縮增強,但兩組間R值比較無顯著差異(P0.05); SP(1 μM)使模型組和對照組R值分別從1增加至1.39±0.12和1.67±0.08,與基線值差異顯著(P0.01),且WAS組R值顯著高于SWAS組(P0.05)；SP(0.1μM)使模型組和對照組CM收縮增強,R值分別由1增加至9.65+0.85和5.35±0.29,與基線值相比,P0.01且模型組R值顯著高于SWAS組(P0.01)；SP(1μM)使模型組和對照組CM的R值分別從1增加至14.49±2.75和7.31±1.45與基線值差異顯著(P0.01),且WAS組R值顯著高于SWAS組(P0.05); NK1R拮抗劑L-703(1μM)孵育肌條后,SP(1μM)對模型組LM和CM的增強作用逆轉,對照組高于模型組。結論：慢性應激使SP誘導的大鼠結腸平滑肌收縮增強,且該作用被NK1R受體拮抗劑阻斷,說明SP-NK1R系統(tǒng)可能參與WAS誘導的結腸高動力的發(fā)生。第三部分慢性應激大鼠近端結腸L型鈣電流的變化及P物質(zhì)對鈣電流的影響目的：探討慢性應激對SP誘導的大鼠結腸平滑肌L型鈣電流的影響。方法：將實驗動物隨機分為模型組和對照組,采用連續(xù)10天的反復避水應激(water-avoidance stress, WAS)建立動物模型；腹腔麻醉處死大鼠后取近端結腸,采用酶消化法分離平滑肌細胞；運用全細胞膜片鉗技術記錄WAS組和對照組(sham water-avoidance stress, SWAS)組平滑肌細胞L型鈣通道電流及SP對兩組動物鈣電流的影響。結果：SWAS組和WAS組L型鈣通道峰電流密度分別為(-3.93±0.38)pA/pF和(-3.49±0.17) pA/pF,組間無顯著差異(P0.05)；SP對平滑肌細胞L型鈣通道有促進作用,SP (1 μM)使WAS組峰電流密度由(-3.4±0.17) pA/pF增加至(-4.8±0.31) pA/pF (P0.05),使SWAS組峰電流密度由(-3.93±0.38) pA/pF增加至(-4.54±0.39) pA/pF (P0.05)。且SP使模型組峰電流較基礎值增加129±3%,使對照組較自身基礎值增加116±2.7%,WAS組百分比顯著高于對照組(P0.05)。結論：慢性應激不改變大鼠結腸平滑肌細胞L型鈣通道電流的大�。籗P通過L型鈣通道促進平滑肌收縮,且慢性應激使SP對鈣電流的作用增強。
[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 鹵 0.05 ) g , ( 0.94 鹵 0.04 ) g , CM ( 0.36 鹵 0.02 ) g ( 0.25 鹵 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 鹵 10.6 ) pg / mL , P0.01 ) . The level of SP in colonic mucosa of rats was increased by 661.5 鹵 20.6 pg / mg protein ( 250.1 鹵 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 鹵 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 鹵 0.38 ) pA / pF and ( - 3.49 鹵 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 鹵 0.38 ) pA / pF to ( - 4.54 鹵 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 .
【學位授予單位】：武漢大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R574.62

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