本文關(guān)鍵詞： 鈉尿肽 肝郁脾虛 胃腸運(yùn)動(dòng) Cajal間質(zhì)細(xì)胞　出處：《大連醫(yī)科大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:探討鈉尿肽信號(hào)通路在肝郁脾虛胃腸運(yùn)動(dòng)障礙大鼠胃ICC丟失中的作用,為肝郁脾虛胃腸運(yùn)動(dòng)障礙的臨床治療提供新思路。方法:1.首先用慢性輕度不可預(yù)知刺激聯(lián)合單籠喂養(yǎng)建立肝郁脾虛大鼠模型。然后檢測(cè)造模前后兩組大鼠的體重變化,用曠場(chǎng)實(shí)驗(yàn)、糖水偏好實(shí)驗(yàn)檢測(cè)造模前后大鼠行為學(xué)變化,檢測(cè)胃殘留率間接檢測(cè)胃排空功能,檢測(cè)尿D-木糖排泄率觀察小腸的吸收功能,用以上方法進(jìn)行模型鑒定。2.用Western-blotting方法檢測(cè)肝郁脾虛大鼠胃中三種鈉尿肽受體(Natriuretic peptide receptor A,B,C)和c-kit及其配體干細(xì)胞因子(Stem cell factor,SCF)蛋白表達(dá)情況。用Real-Time PCR方法檢測(cè)肝郁脾虛大鼠胃中三種鈉尿肽受體(NPR-A,B,C)和c-kit及其配體SCF基因表達(dá)的變化情況。3.提取正常SD大鼠的胃平滑肌細(xì)胞,用免疫細(xì)胞化學(xué)方法進(jìn)行細(xì)胞鑒定并培養(yǎng)。用c ANF(一種特異性的NPR-C激動(dòng)劑)、8-Brom-cGMP(8-Br-cGMP,一種膜通透性cGMP類似物)和不同濃度的C型鈉尿肽(C-type natriuretic peptide,CNP)處理培養(yǎng)的正常胃平滑肌細(xì)胞,然后用Western-blotting方法檢測(cè)細(xì)胞中SCF表達(dá)的差異。用MTT比色法檢測(cè)不同濃度的c ANF處理正常培養(yǎng)的胃平滑肌細(xì)胞后其增殖情況的變化。結(jié)果:1.肝郁脾虛模型組大鼠一般狀態(tài)差;體重增長(zhǎng)緩慢;曠場(chǎng)實(shí)驗(yàn)中的修飾次數(shù)、站立次數(shù)、穿格次數(shù)均顯著減少,糖水偏好率下降,說(shuō)明大鼠自主探索能力下降,自主活動(dòng)減少;胃殘留率高于正常組,說(shuō)明胃排空功能下降;尿D-木糖排泄率下降說(shuō)明小腸吸收功能差。2.Western-blotting結(jié)果顯示,與正常組相比,肝郁脾虛模型組大鼠胃中鈉尿肽受體(NPR-A,B,C)蛋白表達(dá)上調(diào),c-kit及其配體SCF蛋白表達(dá)下調(diào)。RTPCR結(jié)果顯示,肝郁脾虛大鼠胃中NPR-A,B,C基因表達(dá)均上調(diào),c-kit及SCF基因表達(dá)下調(diào),變化趨勢(shì)與蛋白表達(dá)情況一致。3.免疫細(xì)胞化學(xué)結(jié)果顯示,陽(yáng)性細(xì)胞胞漿呈黃棕色,細(xì)胞核呈藍(lán)色,陰性細(xì)胞胞漿無(wú)色透明,細(xì)胞核同樣染成藍(lán)色。用不同濃度的CNP(10~(-8),10~(-7),10~(-6)mol/L)處理培養(yǎng)的胃平滑肌細(xì)胞后,高濃度的CNP(10~(-7),10~(-6) mol/L)使細(xì)胞中SCF表達(dá)下調(diào)。用相同濃度(10~(-6) mol/L)的CNP、8-Br-cGMP、c ANF處理細(xì)胞后,結(jié)果顯示CNP及8-Br-cGMP使細(xì)胞中SCF表達(dá)下調(diào)。MTT結(jié)果顯示,用不同濃度的c ANF(10~(-8),10~(-7),10~(-6) mol/L)處理正常細(xì)胞后,高濃度的c ANF(10~(-7),10~(-6) mol/L)會(huì)抑制細(xì)胞的增殖。結(jié)論:1.用慢性輕度不可預(yù)知刺激聯(lián)合單籠喂養(yǎng)可以成功制備肝郁脾虛證動(dòng)物模型;2.在肝郁脾虛大鼠胃中鈉尿肽信號(hào)通路表達(dá)上調(diào),同時(shí)SCF/c-kit信號(hào)通路下調(diào);3.CNP/NPR-A,B/cGMP信號(hào)通路使SCF表達(dá)下調(diào);4.c ANF/NPR-C信號(hào)通路抑制胃平滑肌細(xì)胞的增殖。鈉尿肽信號(hào)通路上調(diào)可能通過(guò)降低SCF的產(chǎn)生間接參與Cajal細(xì)胞的丟失。
[Abstract]:Objective: to investigate the role of natriuretic peptide signaling pathway in gastric ICC loss in rats with gastrointestinal motility disorder due to liver depression and spleen deficiency. To provide a new idea for the clinical treatment of gastrointestinal motility disorder due to liver stagnation and spleen deficiency. Methods 1. First, the rat model of liver depression and spleen deficiency was established by chronic mild unpredictable stimulation combined with single cage feeding. Then, the body weight changes of the two groups were measured before and after the establishment of the model. The behavioral changes, gastric emptying function, urine D-xylose excretion rate and intestinal absorption function were detected by open-field test and sugar water preference test before and after model making in rats. Western-blotting method was used to detect the expression of three natriuretic peptide receptor Agna (C), c-kit and its ligand stem cell factor Stem cell factor-SCFs in the stomach of rats with liver stagnation and spleen deficiency. Real-Time PCR method was used to detect the expression of liver depression and spleen deficiency. The changes of SCF gene expression of three kinds of natriuretic peptide receptors (NPR-AZB), c-kit and their ligands in rat stomach. 3. The gastric smooth muscle cells of normal SD rats were extracted. The cultured normal gastric smooth muscle cells were treated with c-ANF8-Brom-cGMP8-Br-cGMP8-Br-cGMP8 and C-type natriuretic peptidea of different concentrations of C-type natriuretic peptide (C-type natriuretic peptidea) by immunocytochemistry, and the normal gastric smooth muscle cells were treated with c-ANF-8-Brom-cGMP8-Br-cGMP8-Br-cGMP8-Br-cGMP8-Br-cGMP8. Then the difference of SCF expression in the cells was detected by Western-blotting method. The proliferation of normal cultured gastric smooth muscle cells was detected by MTT colorimetry after treated with different concentrations of c ANF. Results 1. The normal state of the rats in the model group of liver stagnation and spleen deficiency was poor. In the open field experiment, the number of modifications, standing times, and the number of lattice piercing decreased significantly, and the preference rate of sugar water decreased, which indicated that the ability of autonomous exploration and autonomous activity were decreased, and the gastric residual rate was higher than that in the normal group, and the rate of gastric remnant was higher than that of the normal group. The decrease of urine D-xylose excretion rate indicates that the intestinal absorption function is poor. 2. Western-blotting results show that compared with the normal group, The down-regulated expression of c-kit and its ligand SCF protein in the stomach of the rats with liver stagnation and spleen deficiency. The results showed that the expression of NPR-A- BPG-C gene in stomach of rats with liver-stagnation and spleen deficiency was up-regulated, and the expression of c-kit and SCF genes were down-regulated. The result of immunocytochemistry showed that the cytoplasm of positive cells was yellowish brown, the nucleus was blue, and the cytoplasm of negative cells was colorless and transparent. The nuclei were also stained blue. After treated with different concentrations of CNP10 ~ (-10) ~ (-8) ~ 10 ~ (-10) ~ (-6) mol 路L ~ (-1), the high concentration of CNP ~ (10) -10 ~ (-6) mol / L) down-regulated the expression of SCF in the cells. After the cells were treated with the same concentration of 10 ~ (-6) mol / L ANF, the cells were treated with the same concentration of CNPS-8-Br-cGMPc / L, and the cells were treated with the same concentration of CNP ~ (10 ~ (10) ~ (-6)) mol 路L ~ (-1). The results showed that CNP and 8-Br-cGMP could down-regulate the expression of SCF. The results showed that normal cells were treated with different concentrations of c-ANF-10 ~ (-10) -7 ~ (-7) ~ (10) mol ~ (-6) mol 路L ~ (-1). Conclusion: 1. Chronic mild unpredictable stimulation combined with single cage feeding can successfully establish the animal model of liver stagnation and spleen deficiency. The expression of natriuretic peptide signal pathway in the stomach of rats with liver depression and spleen deficiency can be upregulated. At the same time, the SCF/c-kit signaling pathway down-regulated the SCF expression and inhibited the proliferation of gastric smooth muscle cells. The up-regulation of natriuretic peptide signaling pathway may be indirectly involved in the loss of Cajal cells by reducing the production of SCF.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R259

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