钠尿肽信号通路上调对肝郁脾虚大鼠胃Cajal细胞丢失的影响

发布时间：2018-02-27 08:29

本文关键词： 钠尿肽肝郁脾虚胃肠运动 Cajal间质细胞　出处：《大连医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的:探讨钠尿肽信号通路在肝郁脾虚胃肠运动障碍大鼠胃ICC丢失中的作用,为肝郁脾虚胃肠运动障碍的临床治疗提供新思路。方法:1.首先用慢性轻度不可预知刺激联合单笼喂养建立肝郁脾虚大鼠模型。然后检测造模前后两组大鼠的体重变化,用旷场实验、糖水偏好实验检测造模前后大鼠行为学变化,检测胃残留率间接检测胃排空功能,检测尿D-木糖排泄率观察小肠的吸收功能,用以上方法进行模型鉴定。2.用Western-blotting方法检测肝郁脾虚大鼠胃中三种钠尿肽受体(Natriuretic peptide receptor A,B,C)和c-kit及其配体干细胞因子(Stem cell factor,SCF)蛋白表达情况。用Real-Time PCR方法检测肝郁脾虚大鼠胃中三种钠尿肽受体(NPR-A,B,C)和c-kit及其配体SCF基因表达的变化情况。3.提取正常SD大鼠的胃平滑肌细胞,用免疫细胞化学方法进行细胞鉴定并培养。用c ANF(一种特异性的NPR-C激动剂)、8-Brom-cGMP(8-Br-cGMP,一种膜通透性cGMP类似物)和不同浓度的C型钠尿肽(C-type natriuretic peptide,CNP)处理培养的正常胃平滑肌细胞,然后用Western-blotting方法检测细胞中SCF表达的差异。用MTT比色法检测不同浓度的c ANF处理正常培养的胃平滑肌细胞后其增殖情况的变化。结果:1.肝郁脾虚模型组大鼠一般状态差;体重增长缓慢;旷场实验中的修饰次数、站立次数、穿格次数均显著减少,糖水偏好率下降,说明大鼠自主探索能力下降,自主活动减少;胃残留率高于正常组,说明胃排空功能下降;尿D-木糖排泄率下降说明小肠吸收功能差。2.Western-blotting结果显示,与正常组相比,肝郁脾虚模型组大鼠胃中钠尿肽受体(NPR-A,B,C)蛋白表达上调,c-kit及其配体SCF蛋白表达下调。RTPCR结果显示,肝郁脾虚大鼠胃中NPR-A,B,C基因表达均上调,c-kit及SCF基因表达下调,变化趋势与蛋白表达情况一致。3.免疫细胞化学结果显示,阳性细胞胞浆呈黄棕色,细胞核呈蓝色,阴性细胞胞浆无色透明,细胞核同样染成蓝色。用不同浓度的CNP(10~(-8),10~(-7),10~(-6)mol/L)处理培养的胃平滑肌细胞后,高浓度的CNP(10~(-7),10~(-6) mol/L)使细胞中SCF表达下调。用相同浓度(10~(-6) mol/L)的CNP、8-Br-cGMP、c ANF处理细胞后,结果显示CNP及8-Br-cGMP使细胞中SCF表达下调。MTT结果显示,用不同浓度的c ANF(10~(-8),10~(-7),10~(-6) mol/L)处理正常细胞后,高浓度的c ANF(10~(-7),10~(-6) mol/L)会抑制细胞的增殖。结论:1.用慢性轻度不可预知刺激联合单笼喂养可以成功制备肝郁脾虚证动物模型;2.在肝郁脾虚大鼠胃中钠尿肽信号通路表达上调,同时SCF/c-kit信号通路下调;3.CNP/NPR-A,B/cGMP信号通路使SCF表达下调;4.c ANF/NPR-C信号通路抑制胃平滑肌细胞的增殖。钠尿肽信号通路上调可能通过降低SCF的产生间接参与Cajal细胞的丢失。
[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.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R259

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