發(fā)布時(shí)間：2018-06-20 22:23

  本文選題：蛋白酶激活受體4 + 疼痛　； 參考：《泰山醫(yī)學(xué)院》2015年碩士論文

【摘要】：目的蛋白酶激活受體4(protease-activated receptors 4,PAR_4)為G蛋白偶聯(lián)受體,參與多種病理生理的過程。研究發(fā)現(xiàn)蛋白酶活化受體4(PAR_4)具有抑制內(nèi)臟痛和內(nèi)臟高敏感作用。由于PAR_4通過G蛋白偶聯(lián)受體發(fā)揮作用,應(yīng)該能夠找到其在內(nèi)臟痛覺信號(hào)轉(zhuǎn)導(dǎo)中的下游效應(yīng)細(xì)胞。最近研究發(fā)現(xiàn)肥大細(xì)胞(mast cells,MC)在腸易激綜合癥(IBS)內(nèi)臟高敏感的發(fā)生與肥大細(xì)胞活化以及PAR_4表達(dá)下調(diào)密切相關(guān),推測(cè)肥大細(xì)胞可能是PAR_4參與內(nèi)臟鎮(zhèn)痛的下游效應(yīng)靶點(diǎn),PAR_4可能通過作用于肥大細(xì)胞介導(dǎo)內(nèi)臟痛覺信號(hào)的轉(zhuǎn)導(dǎo)。然而其內(nèi)臟鎮(zhèn)痛作用是否直接與肥大細(xì)胞活化還不清楚。本研究以內(nèi)臟高敏感大鼠為研究模型,觀察PAR_4活化對(duì)腸肥大細(xì)胞活化的調(diào)節(jié)作用;以探討PAR_4活化對(duì)胃腸道內(nèi)臟高敏感誘發(fā)的痛覺信號(hào)轉(zhuǎn)導(dǎo)的影響,從而為IBS病人內(nèi)臟高敏感性疼痛的治療提供一個(gè)新的靶點(diǎn)。材料與方法1、采用結(jié)直腸擴(kuò)張(colorectal distension,CRD)的方法建立IBS內(nèi)臟高敏感動(dòng)物模型,并通過腹部撤回反射(abdominal withdrawal reflex,AWR)評(píng)分標(biāo)準(zhǔn),進(jìn)行半定量評(píng)分、篩選建立模型成功的動(dòng)物。對(duì)達(dá)標(biāo)模型利用BL420-F生物機(jī)能實(shí)驗(yàn)系統(tǒng)進(jìn)行肌電圖(electromyography,EMG)檢測(cè)。2、IBS模型給予PAR_4激動(dòng)劑(AYPGKF-NH2,PAR_4-AP)(100μM,200μl)直腸內(nèi)注射,并通過AWR評(píng)分標(biāo)準(zhǔn),進(jìn)行半定量評(píng)分。利用BL420-F生物機(jī)能實(shí)驗(yàn)系統(tǒng)再次進(jìn)行EMG檢測(cè)。3、應(yīng)用免疫組織化學(xué)方法結(jié)合甲苯胺藍(lán)染色技術(shù)觀察正常大鼠、IBS大鼠模型和IBS大鼠模型結(jié)腸內(nèi)注射PAR_4-AP后觀察大鼠結(jié)腸和盲腸內(nèi)PAR_4活化對(duì)NOS和P2X7在肥大細(xì)胞的表達(dá)的影響,比較正常組、IBS組、PAR_4-AP組之間的表達(dá)差異。4、免疫細(xì)胞化學(xué)技術(shù)結(jié)合結(jié)合激光共聚焦顯微鏡觀察PAR_4、NOS、P2X7和類胰蛋白酶(Tryptase,AA4)在大鼠結(jié)腸和盲腸組織內(nèi)和肥大細(xì)胞或肓腸組織內(nèi)的共表達(dá),觀察PAR_4活化對(duì)NOS、P2X7陽性標(biāo)記肥大細(xì)胞的影響。5、急性分離SD大鼠雙側(cè)股骨,骨髓干細(xì)胞定向誘導(dǎo)分化的方法培養(yǎng)骨髓源性肥大細(xì)胞(bone-marrow-derived mast cell,BMMC)。PAR_4-AP孵育培養(yǎng)的肥大細(xì)胞24h,流式細(xì)胞術(shù)和激光共聚焦顯微鏡觀察PAR_4活化對(duì)肥大細(xì)胞表達(dá)PAR_4、NOS和P2X7的影響。6、大鼠IBS模型直腸內(nèi)注射PAR_4-AP,TRIZOL提取正常組、IBS組、PAR_4-AP組直腸、結(jié)腸和盲腸組織的總RNA,用實(shí)時(shí)熒光定量PCR法檢測(cè)PAR_4活化對(duì)IL-1的基因表達(dá)變化的影響。7、大鼠IBS模型直腸內(nèi)注射PAR_4-AP,RIPA細(xì)胞裂解液提取正常組、IBS組、PAR_4-AP組直腸、結(jié)腸和盲腸組織的總蛋白,Western blot法檢測(cè)NOS、P2X7和PAR_4的表達(dá)變化。結(jié)果1、PAR_4活化對(duì)IBS大鼠內(nèi)臟高敏感性疼痛的作用(1)直結(jié)腸擴(kuò)張后AWR評(píng)分。與正常組相比,IBS組SD大鼠的AWR評(píng)分升高,內(nèi)臟疼痛敏感性升高;PAR_4激動(dòng)組的AWR評(píng)分較IBS組降低,說明PAR_4激動(dòng)劑可以降低內(nèi)臟高敏感性。(2)腹直肌肌電檢測(cè)。結(jié)果顯示大鼠IBS模型直腸內(nèi)注射PAR_4-AP能明顯降低直腸擴(kuò)張誘發(fā)的肌電圖和平均峰值的曲線面積(area under the curve,AUC)。直腸擴(kuò)張?jiān)?0mmHg的壓力下IBS組與PAR_4激動(dòng)組之間有差異,IBS組與正常組之間有差異;在40mmHg的壓力下各組間差異均有統(tǒng)計(jì)學(xué)意義;在60mmHg的壓力IBS組與PAR_4激動(dòng)組之間差異有統(tǒng)計(jì)學(xué)意義。2、PAR_4活化對(duì)IBS大鼠結(jié)腸和盲腸內(nèi)NOS和P2X7陽性肥大細(xì)胞的影響(1)PAR_4陽性細(xì)胞的變化。IBS組盲腸中PAR_4陽性細(xì)胞數(shù)高于正常組和PAR_4激動(dòng)組;PAR_4激動(dòng)組高于正常組。(2)肥大細(xì)胞的變化。IBS組盲腸中Tryptase陽性肥大細(xì)胞數(shù)量明顯高于正常組和PAR_4激動(dòng)組。(3)NOS陽性細(xì)胞的變化。IBS組盲腸中NOS陽性細(xì)胞數(shù)高于正常組;PAR_4激動(dòng)組高于正常組;PAR_4激動(dòng)組與IBS組盲腸中NOS陽性細(xì)胞數(shù)沒有明顯差異。(4)P2X7陽性細(xì)胞的變化。IBS組盲腸中P2X7陽性細(xì)胞數(shù)高于正常組和PAR_4激動(dòng)組;PAR_4激動(dòng)組高于正常組。3、PAR_4、NOS和P2X7在IBS大鼠腸粘膜或培養(yǎng)肥大細(xì)胞的表達(dá)免疫細(xì)胞化學(xué)技術(shù)結(jié)合激光共聚焦顯微鏡顯示在IBS大鼠模型腸粘膜或骨髓干細(xì)胞定向誘導(dǎo)分化法培養(yǎng)的肥大細(xì)胞表達(dá)PAR_4、NOS和P2X7�？梢夾A4分別與PAR_4、NOS和P2X7共存。4、PAR_4活化對(duì)肥大細(xì)胞的影響(1)PAR_4激動(dòng)劑對(duì)PAR_4在肥大細(xì)胞內(nèi)表達(dá)的影響。Western blotting和流式細(xì)胞技術(shù)顯示,PAR_4激動(dòng)劑使培養(yǎng)的肥大細(xì)胞PAR_4蛋白的表達(dá)明顯增加。(2)PAR_4激動(dòng)劑對(duì)NOS在肥大細(xì)胞表達(dá)的影響。Western blotting和流式細(xì)胞技術(shù)顯示,PAR_4激動(dòng)劑使使培養(yǎng)的肥大細(xì)胞NOS表達(dá)明顯增加。5、PAR_4活化下調(diào)IL-1βmRNA表達(dá)。實(shí)時(shí)定量PCR技術(shù)檢測(cè)顯示大鼠IBS模型直腸內(nèi)注射PAR_4-AP能明顯降低IL-1βmRNA水平。IBS組IL-1β基因表達(dá)量明顯高于對(duì)照組正常組,PAR_4-AP組大鼠腸組織中IL-1β基因表達(dá)量介于IBS組與正常對(duì)照組之間。結(jié)論1、IBS組大鼠的AWR評(píng)分升高,內(nèi)臟疼痛敏感性升高;PAR_4激動(dòng)劑可以降低內(nèi)臟高敏感性。2、IBS中MC細(xì)胞數(shù)高于正常組和PAR_4激動(dòng)組,PAR_4激動(dòng)組高于正常組。3、PAR_4通過肥大細(xì)胞來介導(dǎo)NOS、P2X7、IL-1β表達(dá)變化,參與內(nèi)臟高敏感性疼痛的調(diào)節(jié)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.
【學(xué)位授予單位】：泰山醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R402

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