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

【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蛋白偶聯受體。研究發(fā)現PAR4在正常和炎癥條件下參與調節(jié)疼痛反應，目前PAR4在痛覺調節(jié)中的作用機制尚不清楚，可能是經過細胞內的信號轉導機制作用于周圍感覺神經元，其中部分可能是通過致敏瞬時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節(jié)。為了進一步了解PAR4在背根神經節(jié)（dorsal root ganglion，DRG）初級感覺神經元的表達，及與傷害性感覺受體TRPV1的共存，探究PAR4在外周傷害性感覺信號傳導中的作用提供形態(tài)學依據。 方法 1.應用免疫熒光組織化學雙標法結合激光共聚焦顯微鏡技術，觀察PAR4在大鼠和小鼠DRG初級感覺神經元的表達及與TRPV1的共存。 2.腹腔內注射醋酸建立內臟疼痛大鼠動物模型，應用免疫熒光組織化學雙標法結合激光共聚焦顯微鏡技術，觀察PAR4在DRG初級感覺神經元的表達變化。結果 1. PAR4在小鼠DRG初級感覺神經元的表達及與TRPV1的共存：免疫熒光顯示小鼠DRG內見有大量的感覺神經元表達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元，可見少部分陽性神經元纖維。有80.5%±3.1%（3672/4558）神經元表達PAR4。免疫熒光雙標法顯示DRG內可見較多的TRPV1陽性神經元，，均為中、小型感覺神經元胞體，許多DRG的PAR4陽性神經元的表達均與TRPV1的共存，有76.9%±3.4%(2826/3672)的PAR4陽性神經元表達TRPV1，有77.4%±3.1%(2826/3647)的TRPV1陽性神經元表達PAR4。 2. PAR4在大鼠DRG初級感覺神經元的表達及與TRPV1的共存：PAR4在大鼠的DRG內的表達與小鼠類似，同樣見有大量的感覺神經元表達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元，細胞計數顯示：有85.4%±4.1%（4337/5078）神經元表達PAR4。免疫熒光雙標法顯示有83.5%±3.6%(3623/4337)的PAR4陽性神經元表達TRPV1，有88.3%±3.5%(3623/4102)的TRPV1陽性神經元表達PAR4。 3.PAR4與CGRP在大鼠DRG初級感覺神經元的共存：免疫熒光顯示大鼠DRG內見有大量的感覺神經元呈CGRP陽性，其形態(tài)多為中、小型的圓形、卵圓形，以及少量的大型神經元，并觀察到部分CGRP陽性神經纖維，有75.6%±4.1%（3387/4478）陽性神經元表達CGRP。免疫熒光雙標法顯示有82.3%±4.6%（2788/3387）PAR4陽性神經元表達CGRP，有64.2%±3.7%（2788/4337）CGRP陽性細胞均表達PAR4。 4. TRPV1與CGRP在大鼠DRG初級感覺神經元的共存：DRG內TRPV1陽性神經元與CGRP陽性細胞類似，均為中、小型感覺神經元胞體和一些彌散神經纖維。免疫熒光雙標顯示TRPV1/CGRP在DRG感覺神經元內廣泛的共存，有83.9%±3.6%（2842/3387）CGRP陽性細胞均表達TRPV1，有69.2%±3.2%（2842/4102）TRPV1陽性細胞表達CGRP，也可觀察到少量的TRPV1單標神經元或CGRP單標神經元。 5. PAR4在內臟疼痛大鼠動物模型中DRG感覺神經元的表達變化，在內臟疼痛大鼠動物模型DRG內PAR4陽性感覺神經元的數量明顯增加，細胞計數顯示PAR4陽性感覺神經元的數量與正常大鼠相比，增長了11.1%±2.3%，有96.5%±4.3%（5336/5528）神經元表達PAR4。同時DRG內TRPV1陽性神經元的數量也明顯增加，有95.0%±4.4%（5110/5378）神經元表達TRPV1。免疫熒光雙標顯示有較多的PAR4/TRPV1雙標細胞,分別占PAR4陽性和TRPV1陽性細胞的88.8%±3.7%(4742/5336)和92.7%±3.4%(4742/5110)。實驗結果顯示在大鼠DRG內見有許多感覺神經元表達PAR4，并與TRPV1廣泛的共存。結論 1.PAR4在大鼠和小鼠的DRG初級感覺神經元有廣泛的表達，主要為中、小型神經元。 2.PAR4與TRPV1在DRG初級感覺神經元存在廣泛的共存，說明PAR4參與外周傷害性刺激可能與TRPV1的功能調節(jié)有關。 3.在內臟疼痛大鼠動物模型中，PAR4在DRG的表達明顯增加，說明PAR4在內臟傷害性刺激的傳導過程具有重要的作用。
[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.

【學位授予單位】：泰山醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R338

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