蛋白酶激活受体4在肥大细胞介导的内脏高敏感性疼痛的作用和调节机制的研究

发布时间：2018-06-20 22:23

本文选题：蛋白酶激活受体4 + 疼痛　；参考：《泰山医学院》2015年硕士论文

【摘要】：目的蛋白酶激活受体4(protease-activated receptors 4,PAR_4)为G蛋白偶联受体,参与多种病理生理的过程。研究发现蛋白酶活化受体4(PAR_4)具有抑制内脏痛和内脏高敏感作用。由于PAR_4通过G蛋白偶联受体发挥作用,应该能够找到其在内脏痛觉信号转导中的下游效应细胞。最近研究发现肥大细胞(mast cells,MC)在肠易激综合症(IBS)内脏高敏感的发生与肥大细胞活化以及PAR_4表达下调密切相关,推测肥大细胞可能是PAR_4参与内脏镇痛的下游效应靶点,PAR_4可能通过作用于肥大细胞介导内脏痛觉信号的转导。然而其内脏镇痛作用是否直接与肥大细胞活化还不清楚。本研究以内脏高敏感大鼠为研究模型,观察PAR_4活化对肠肥大细胞活化的调节作用;以探讨PAR_4活化对胃肠道内脏高敏感诱发的痛觉信号转导的影响,从而为IBS病人内脏高敏感性疼痛的治疗提供一个新的靶点。材料与方法1、采用结直肠扩张(colorectal distension,CRD)的方法建立IBS内脏高敏感动物模型,并通过腹部撤回反射(abdominal withdrawal reflex,AWR)评分标准,进行半定量评分、筛选建立模型成功的动物。对达标模型利用BL420-F生物机能实验系统进行肌电图(electromyography,EMG)检测。2、IBS模型给予PAR_4激动剂(AYPGKF-NH2,PAR_4-AP)(100μM,200μl)直肠内注射,并通过AWR评分标准,进行半定量评分。利用BL420-F生物机能实验系统再次进行EMG检测。3、应用免疫组织化学方法结合甲苯胺蓝染色技术观察正常大鼠、IBS大鼠模型和IBS大鼠模型结肠内注射PAR_4-AP后观察大鼠结肠和盲肠内PAR_4活化对NOS和P2X7在肥大细胞的表达的影响,比较正常组、IBS组、PAR_4-AP组之间的表达差异。4、免疫细胞化学技术结合结合激光共聚焦显微镜观察PAR_4、NOS、P2X7和类胰蛋白酶(Tryptase,AA4)在大鼠结肠和盲肠组织内和肥大细胞或肓肠组织内的共表达,观察PAR_4活化对NOS、P2X7阳性标记肥大细胞的影响。5、急性分离SD大鼠双侧股骨,骨髓干细胞定向诱导分化的方法培养骨髓源性肥大细胞(bone-marrow-derived mast cell,BMMC)。PAR_4-AP孵育培养的肥大细胞24h,流式细胞术和激光共聚焦显微镜观察PAR_4活化对肥大细胞表达PAR_4、NOS和P2X7的影响。6、大鼠IBS模型直肠内注射PAR_4-AP,TRIZOL提取正常组、IBS组、PAR_4-AP组直肠、结肠和盲肠组织的总RNA,用实时荧光定量PCR法检测PAR_4活化对IL-1的基因表达变化的影响。7、大鼠IBS模型直肠内注射PAR_4-AP,RIPA细胞裂解液提取正常组、IBS组、PAR_4-AP组直肠、结肠和盲肠组织的总蛋白,Western blot法检测NOS、P2X7和PAR_4的表达变化。结果1、PAR_4活化对IBS大鼠内脏高敏感性疼痛的作用(1)直结肠扩张后AWR评分。与正常组相比,IBS组SD大鼠的AWR评分升高,内脏疼痛敏感性升高;PAR_4激动组的AWR评分较IBS组降低,说明PAR_4激动剂可以降低内脏高敏感性。(2)腹直肌肌电检测。结果显示大鼠IBS模型直肠内注射PAR_4-AP能明显降低直肠扩张诱发的肌电图和平均峰值的曲线面积(area under the curve,AUC)。直肠扩张在20mmHg的压力下IBS组与PAR_4激动组之间有差异,IBS组与正常组之间有差异;在40mmHg的压力下各组间差异均有统计学意义;在60mmHg的压力IBS组与PAR_4激动组之间差异有统计学意义。2、PAR_4活化对IBS大鼠结肠和盲肠内NOS和P2X7阳性肥大细胞的影响(1)PAR_4阳性细胞的变化。IBS组盲肠中PAR_4阳性细胞数高于正常组和PAR_4激动组;PAR_4激动组高于正常组。(2)肥大细胞的变化。IBS组盲肠中Tryptase阳性肥大细胞数量明显高于正常组和PAR_4激动组。(3)NOS阳性细胞的变化。IBS组盲肠中NOS阳性细胞数高于正常组;PAR_4激动组高于正常组;PAR_4激动组与IBS组盲肠中NOS阳性细胞数没有明显差异。(4)P2X7阳性细胞的变化。IBS组盲肠中P2X7阳性细胞数高于正常组和PAR_4激动组;PAR_4激动组高于正常组。3、PAR_4、NOS和P2X7在IBS大鼠肠粘膜或培养肥大细胞的表达免疫细胞化学技术结合激光共聚焦显微镜显示在IBS大鼠模型肠粘膜或骨髓干细胞定向诱导分化法培养的肥大细胞表达PAR_4、NOS和P2X7。可见AA4分别与PAR_4、NOS和P2X7共存。4、PAR_4活化对肥大细胞的影响(1)PAR_4激动剂对PAR_4在肥大细胞内表达的影响。Western blotting和流式细胞技术显示,PAR_4激动剂使培养的肥大细胞PAR_4蛋白的表达明显增加。(2)PAR_4激动剂对NOS在肥大细胞表达的影响。Western blotting和流式细胞技术显示,PAR_4激动剂使使培养的肥大细胞NOS表达明显增加。5、PAR_4活化下调IL-1βmRNA表达。实时定量PCR技术检测显示大鼠IBS模型直肠内注射PAR_4-AP能明显降低IL-1βmRNA水平。IBS组IL-1β基因表达量明显高于对照组正常组,PAR_4-AP组大鼠肠组织中IL-1β基因表达量介于IBS组与正常对照组之间。结论1、IBS组大鼠的AWR评分升高,内脏疼痛敏感性升高;PAR_4激动剂可以降低内脏高敏感性。2、IBS中MC细胞数高于正常组和PAR_4激动组,PAR_4激动组高于正常组。3、PAR_4通过肥大细胞来介导NOS、P2X7、IL-1β表达变化,参与内脏高敏感性疼痛的调节作用。
[Abstract]:The purpose of protease activated receptor 4 (protease-activated receptors 4, PAR_4) for G protein coupled receptors, is involved in many physiological and pathological findings. The protease activated receptor 4 (PAR_4) can inhibit the visceral pain and visceral hypersensitivity. Due to the PAR_4 through G protein coupled receptors play a role, should be able to find the letter in visceral pain The downstream effects of signal transduction in cells. Recent studies have found that mast cells (mast, cells, MC) in irritable bowel syndrome (IBS) and high sensitive visceral mast cell activation and expression of PAR_4 is closely related, it is speculated that mast cells may be involved in visceral pain in the lower reaches of the PAR_4 effect target, PAR_4 may be acting on mast cell mediated A visceral pain signal transduction. However the visceral analgesic effect is directly related to the activation of mast cells is not clear. In this study, visceral hypersensitivity in rats, observe the PAR_4 activation effect on intestinal mast cells; to investigate the effect of activation of PAR_4 signal transduction on gastrointestinal pain induced visceral hypersensitivity. From The treatment for IBS patients with visceral pain sensibility Gao Min provides a new target. Materials and methods 1 with colorectal distension (colorectal distension CRD) method to establish animal model of visceral hypersensitivity in IBS, and through the Abdominal withdrawal reflex (Abdominal withdrawal, reflex, AWR) standard for evaluation, semi quantitative score, were established model The success of the animal. The standard EMG model using BL420-F biological function experimental system (electromyography, EMG) detection of.2, IBS model PAR_4 agonists (AYPGKF-NH2, PAR_4-AP) (100 M, 200 L) was injected into the rectum, and through the AWR standard for evaluation, semi quantitative score. Again EMG examination by BL420-F the experimental system of biological function .3, immunohistochemistry and toluidine blue staining were observed in normal rat colon of rats injected PAR_4-AP model and IBS IBS rats were observed after the colon and cecum in rats PAR_4 on activation of NOS and P2X7 in the expression of mast cells, compared to normal group, IBS group, the difference of.4 expression in PAR_4-AP group between the immune cells Immunocytochemical technique combined with confocal microscopy combined with PAR_4, NOS laser, P2X7 and tryptase (Tryptase, AA4) were expressed in colon and cecum tissue in rats and mast cells or cecum tissue, observe the PAR_4 activation of NOS, P2X7 positive mast cells influence.5, acute separation SD rat bilateral femur bone marrow stem cell set Bone marrow mast cells to differentiation medium (bone-marrow-derived mast cell, BMMC.PAR_4-AP) were incubated with 24h mast cell culture, flow cytometry and confocal microscopy observation of PAR_4 activation on the expression of PAR_4 in mast cells, effects of.6 and P2X7 NOS, PAR_4-AP IBS in rat model of rectal injection, normal TRIZOL extraction group, IBS group, PAR_4-AP group, RNA total rectum, colon and cecum tissue. The effect of.7 was detected by the real-time quantitative PCR PAR_4 on activation of IL-1 gene expression changes, PAR_4-AP model of internal rectal injection in rats IBS, RIPA cell lysates were extracted from normal group, IBS group, PAR_4-AP group, rectum, colon and cecum tissue total protein the detection of NOS, Western blot, P The expression of 2X7 and PAR_4. The results of 1 PAR_4 on activation of IBS rat visceral pain sensibility Gao Min effect (1) after colorectal distention score of AWR. Compared with the normal group, IBS group, SD rats AWR score increased, increased visceral pain sensitivity; reduce the activation of PAR_4 group in the AWR score compared with the IBS group, indicating PAR_4 agonist can decrease visceral sensibility. Gao Min (2) Detection of the rectus abdominis. The results showed that rectal IBS rat model of PAR_4-AP injection can significantly reduce rectal distension evoked EMG curve area and average peak (area under the curve, AUC). Rectal distension under 20mmHg pressure difference between IBS group and PAR_4 group have significant differences between the IBS excited, group and normal group; in the pressure of 40mmHg The sense of the difference between the groups were statistically; between IBS group and PAR_4 60mmHg pressure excited group had significant difference.2, PAR_4 activation effect on NOS and P2X7 positive mast cells in the colon and cecum of rats in IBS (1) PAR_4 positive cell number changes of.IBS positive cells in PAR_4 group was higher than that of the normal group and the PAR_4 excited PAR_4 laser group; Dynamic group is higher than the normal group. (2) the number of Tryptase positive mast cell changes of mast cells in.IBS group was significantly higher than that in normal group and PAR_4 agonist group. (3) the number of NOS positive cells in.IBS positive cells in NOS group was higher than the normal group; PAR_4 agonist group is higher than the normal group; PAR_4 group and positive cells excited IBS group was the number of NOS in no Ming Significant difference. (4) the number of P2X7 positive cells in.IBS positive cells in P2X7 group was higher than that in normal group and PAR_4 group PAR_4 excited excited group than the normal group;.3, PAR_4, NOS and P2X7 IBS in rat intestinal mucosa or cultured mast cells expressing immunocytochemistry combined with confocal laser microscopy showed in the model the intestinal mucosa of IBS rats or bone Mast cell differentiation of bone marrow stem cell directional cultivation method the expression of PAR_4, NOS and P2X7. AA4 were visible with PAR_4, NOS and P2X7 coexistence of.4, PAR_4 activation of mast cells (1) PAR_4 agonists showed the effect of blotting on the.Western expression of PAR_4 in mast cells and flow cytometry, the PAR_4 agonist mast cell PAR in vitro The expression of _4 protein increased significantly. (2) PAR_4 agonists showed the effect of blotting on the.Western expression of NOS in mast cells and flow cytometry, PAR_4 agonists make mast cells cultured in NOS increased the expression of.5 and activation of PAR_4 regulated expression of IL-1 beta mRNA. Real time quantitative PCR detection technology shows that the rectum of rat IBS model PAR_4-AP injection Can significantly reduce the IL-1 level of.IBS group IL-1 beta beta mRNA gene expression was significantly higher than the control group normal group, the expression of IL-1 gene in intestinal tissue in PAR_4-AP rats weight between IBS group and normal control group. Conclusion: 1, IBS group rats AWR score increased and increased visceral pain sensitivity; PAR_4 agonist can decrease Gao Min.2 IBS in visceral sensibility, MC The number of cells was higher than normal group and PAR_4 group PAR_4 excited, excited group is higher than the normal group.3, PAR_4 mediated NOS by mast cell P2X7, IL-1 expression changes, regulation of the visceral emotional Gao Min pain.
【学位授予单位】：泰山医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R402

【参考文献】