本文選題：蛋白酶活化受體　切入點(diǎn)：4（PAR4）　出處：《泰山醫(yī)學(xué)院》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：目的 蛋白酶活化受體4（protease-activated receptor，PAR4），是蛋白酶活化受體家族成員之一，屬于G蛋白偶聯(lián)受體。研究發(fā)現(xiàn)PAR4在正常和炎癥條件下參與調(diào)節(jié)疼痛反應(yīng)，目前PAR4在痛覺(jué)調(diào)節(jié)中的作用機(jī)制尚不清楚，可能是經(jīng)過(guò)細(xì)胞內(nèi)的信號(hào)轉(zhuǎn)導(dǎo)機(jī)制作用于周圍感覺(jué)神經(jīng)元，其中部分可能是通過(guò)致敏瞬時(shí)受體電位香草酸亞型1（Transient receptor potential vanilloid1TRPV1）參與外周傷害性刺激的調(diào)節(jié)。為了進(jìn)一步了解PAR4在背根神經(jīng)節(jié)（dorsal root ganglion，DRG）初級(jí)感覺(jué)神經(jīng)元的表達(dá)，及與傷害性感覺(jué)受體TRPV1的共存，探究PAR4在外周傷害性感覺(jué)信號(hào)傳導(dǎo)中的作用提供形態(tài)學(xué)依據(jù)。 方法 1.應(yīng)用免疫熒光組織化學(xué)雙標(biāo)法結(jié)合激光共聚焦顯微鏡技術(shù)，觀察PAR4在大鼠和小鼠DRG初級(jí)感覺(jué)神經(jīng)元的表達(dá)及與TRPV1的共存。 2.腹腔內(nèi)注射醋酸建立內(nèi)臟疼痛大鼠動(dòng)物模型，應(yīng)用免疫熒光組織化學(xué)雙標(biāo)法結(jié)合激光共聚焦顯微鏡技術(shù)，觀察PAR4在DRG初級(jí)感覺(jué)神經(jīng)元的表達(dá)變化。結(jié)果 1. PAR4在小鼠DRG初級(jí)感覺(jué)神經(jīng)元的表達(dá)及與TRPV1的共存：免疫熒光顯示小鼠DRG內(nèi)見(jiàn)有大量的感覺(jué)神經(jīng)元表達(dá)PAR4，其形態(tài)多為中、小型的圓形、卵圓形，以及少量的大型神經(jīng)元，可見(jiàn)少部分陽(yáng)性神經(jīng)元纖維。有80.5%±3.1%（3672/4558）神經(jīng)元表達(dá)PAR4。免疫熒光雙標(biāo)法顯示DRG內(nèi)可見(jiàn)較多的TRPV1陽(yáng)性神經(jīng)元，，均為中、小型感覺(jué)神經(jīng)元胞體，許多DRG的PAR4陽(yáng)性神經(jīng)元的表達(dá)均與TRPV1的共存，有76.9%±3.4%(2826/3672)的PAR4陽(yáng)性神經(jīng)元表達(dá)TRPV1，有77.4%±3.1%(2826/3647)的TRPV1陽(yáng)性神經(jīng)元表達(dá)PAR4。 2. PAR4在大鼠DRG初級(jí)感覺(jué)神經(jīng)元的表達(dá)及與TRPV1的共存：PAR4在大鼠的DRG內(nèi)的表達(dá)與小鼠類似，同樣見(jiàn)有大量的感覺(jué)神經(jīng)元表達(dá)PAR4，其形態(tài)多為中小型的圓形、卵圓形，以及少量的大型神經(jīng)元，細(xì)胞計(jì)數(shù)顯示：有85.4%±4.1%（4337/5078）神經(jīng)元表達(dá)PAR4。免疫熒光雙標(biāo)法顯示有83.5%±3.6%(3623/4337)的PAR4陽(yáng)性神經(jīng)元表達(dá)TRPV1，有88.3%±3.5%(3623/4102)的TRPV1陽(yáng)性神經(jīng)元表達(dá)PAR4。 3.PAR4與CGRP在大鼠DRG初級(jí)感覺(jué)神經(jīng)元的共存：免疫熒光顯示大鼠DRG內(nèi)見(jiàn)有大量的感覺(jué)神經(jīng)元呈CGRP陽(yáng)性，其形態(tài)多為中、小型的圓形、卵圓形，以及少量的大型神經(jīng)元，并觀察到部分CGRP陽(yáng)性神經(jīng)纖維，有75.6%±4.1%（3387/4478）陽(yáng)性神經(jīng)元表達(dá)CGRP。免疫熒光雙標(biāo)法顯示有82.3%±4.6%（2788/3387）PAR4陽(yáng)性神經(jīng)元表達(dá)CGRP，有64.2%±3.7%（2788/4337）CGRP陽(yáng)性細(xì)胞均表達(dá)PAR4。 4. TRPV1與CGRP在大鼠DRG初級(jí)感覺(jué)神經(jīng)元的共存：DRG內(nèi)TRPV1陽(yáng)性神經(jīng)元與CGRP陽(yáng)性細(xì)胞類似，均為中、小型感覺(jué)神經(jīng)元胞體和一些彌散神經(jīng)纖維。免疫熒光雙標(biāo)顯示TRPV1/CGRP在DRG感覺(jué)神經(jīng)元內(nèi)廣泛的共存，有83.9%±3.6%（2842/3387）CGRP陽(yáng)性細(xì)胞均表達(dá)TRPV1，有69.2%±3.2%（2842/4102）TRPV1陽(yáng)性細(xì)胞表達(dá)CGRP，也可觀察到少量的TRPV1單標(biāo)神經(jīng)元或CGRP單標(biāo)神經(jīng)元。 5. PAR4在內(nèi)臟疼痛大鼠動(dòng)物模型中DRG感覺(jué)神經(jīng)元的表達(dá)變化，在內(nèi)臟疼痛大鼠動(dòng)物模型DRG內(nèi)PAR4陽(yáng)性感覺(jué)神經(jīng)元的數(shù)量明顯增加，細(xì)胞計(jì)數(shù)顯示PAR4陽(yáng)性感覺(jué)神經(jīng)元的數(shù)量與正常大鼠相比，增長(zhǎng)了11.1%±2.3%，有96.5%±4.3%（5336/5528）神經(jīng)元表達(dá)PAR4。同時(shí)DRG內(nèi)TRPV1陽(yáng)性神經(jīng)元的數(shù)量也明顯增加，有95.0%±4.4%（5110/5378）神經(jīng)元表達(dá)TRPV1。免疫熒光雙標(biāo)顯示有較多的PAR4/TRPV1雙標(biāo)細(xì)胞,分別占PAR4陽(yáng)性和TRPV1陽(yáng)性細(xì)胞的88.8%±3.7%(4742/5336)和92.7%±3.4%(4742/5110)。實(shí)驗(yàn)結(jié)果顯示在大鼠DRG內(nèi)見(jiàn)有許多感覺(jué)神經(jīng)元表達(dá)PAR4，并與TRPV1廣泛的共存。結(jié)論 1.PAR4在大鼠和小鼠的DRG初級(jí)感覺(jué)神經(jīng)元有廣泛的表達(dá)，主要為中、小型神經(jīng)元。 2.PAR4與TRPV1在DRG初級(jí)感覺(jué)神經(jīng)元存在廣泛的共存，說(shuō)明PAR4參與外周傷害性刺激可能與TRPV1的功能調(diào)節(jié)有關(guān)。 3.在內(nèi)臟疼痛大鼠動(dòng)物模型中，PAR4在DRG的表達(dá)明顯增加，說(shuō)明PAR4在內(nèi)臟傷害性刺激的傳導(dǎo)過(guò)程具有重要的作用。
[Abstract]:objective
Protease activated receptor 4 (protease-activated, receptor, PAR4) is one of the protease activated receptor family member, belongs to the G protein coupled receptor. The study found that PAR4 is involved in the regulation of pain response in normal and inflammatory conditions, the mechanism of PAR4 in the regulation of pain is unclear, probably through signal transduction in cells for peripheral making machine some sensory neurons, possibly through sensitization of transient receptor potential vanilloid 1 (Transient receptor potential vanilloid1TRPV1) is involved in the regulation of peripheral noxious stimulation. In order to further understand the PAR4 in dorsal root ganglion (dorsal root, ganglion, DRG) expression in primary sensory neurons, and nociceptive sensory receptor TRPV1 coexist, provide a morphological basis for exploring PAR4 in peripheral nociceptive sensory signal transduction.
Method
1. the expression of PAR4 in primary sensory neurons and coexistence with TRPV1 in rat and mouse DRG were observed by immunofluorescence histochemical double labeling combined with confocal laser scanning microscopy.
2. an animal model of visceral pain was established by intraperitoneal injection of acetic acid. The expression of PAR4 in primary sensory neurons of DRG was observed by immunofluorescence histochemical double labeling combined with confocal laser scanning microscopy.
1. PAR4 expression in mouse DRG primary sensory neurons and coexist with TRPV1. Immunofluorescence showed a large number of sensory neurons expressing PAR4 mouse DRG see, its shape is small, round, oval, large and small neurons, visible a few positive neuron fibers. 80.5% + 3.1% (3672/4558) neurons expression of PAR4. double immunofluorescence assay showed that TRPV1 DRG could be seen in the more positive neurons were in small sensory neurons, the expression of PAR4 DRG positive neurons and TRPV1 coexistence, 76.9% + 3.4% (2826/3672) PAR4 positive neurons express TRPV1, 77.4% + 3.1% (2826/3647). The expression of TRPV1 in PAR4.
2. PAR4 expression in DRG rat primary sensory neurons and coexist with TRPV1: PAR4 in rat DRG expression in mice with similar, also see a large number of sensory neurons express PAR4, its shape is small and round, oval, large and small neurons, cell count: 85.4% + 4.1% (4337/5078) neurons expression of PAR4. immunofluorescence staining showed that 83.5% + 3.6% (3623/4337) PAR4 positive neurons express TRPV1, 88.3% + 3.5% (3623/4102) TRPV1 positive neurons and expression of PAR4.
The coexistence of 3.PAR4 and CGRP in DRG rat primary sensory neurons: immunofluorescence showed a large number of sensory neurons were positive for CGRP see DRG in rat, its shape is small, round, oval, large and small neurons, and observed that CGRP positive nerve fiber, 75.6% + 4.1% (3387/4478) positive neurons express CGRP. double immunofluorescence method showed that 82.3% + 4.6% (2788/3387) PAR4 positive neurons express CGRP, 64.2% + 3.7% (2788/4337) PAR4. expression of CGRP positive cells
The coexistence of 4. TRPV1 and CGRP DRG in rat primary sensory neurons: DRG TRPV1 positive neurons and CGRP positive cells were similar in small sensory neurons and some diffuse nerve fibers. Double immunofluorescent staining showed that TRPV1/CGRP in DRG sensory neurons within the broad coexist, there are 83.9% + 3.6% (2842/3387) TRPV1 expression CGRP positive cells, 69.2% + 3.2% (2842/4102) expression of TRPV1 CGRP, can also be observed in a small number of TRPV1 labeled neurons and CGRP labeled neurons.
Expression of PAR4 in 5. animal models of pain in rats with visceral sensory neurons in DRG, the number of visceral pain animal model of rat DRG PAR4 positive sensory neurons increased significantly, the number of PAR4 positive cells show that sensory neurons compared with normal rats, growth of 11.1% + 2.3%, 96.5% + 4.3% (5336/5528) neurons the expression of PAR4. and DRG in the number of TRPV1 positive neurons was significantly increased, there are 95% + 4.4% (5110/5378) neurons expression of TRPV1. double immunofluorescence showed more PAR4/TRPV1 positive cells were PAR4 positive and TRPV1 positive cells (4742/5336) 88.8% + 3.7% and 92.7% + 3.4% (4742/5110). Experimental results show that in rats DRG seen in many sensory neurons express PAR4 and TRPV1 coexist and widely. Conclusion
1.PAR4 is widely expressed in DRG primary sensory neurons in rats and mice, mainly medium and small neurons.
2.PAR4 and TRPV1 exist widely in DRG primary sensory neurons, indicating that the involvement of PAR4 in peripheral nociceptive stimulation may be related to the function regulation of TRPV1.
3. in the animal model of visceral pain, the expression of PAR4 in DRG increased significantly, indicating that PAR4 plays an important role in the conduction of visceral nociceptive stimuli.

【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】：2012
【分類號(hào)】：R338

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