PAR4在背根神经节感觉神经元的表达及与TRPV1的共存

发布时间：2018-03-22 04:02

本文选题：蛋白酶活化受体　切入点：4（PAR4）　出处：《泰山医学院》2012年硕士论文　论文类型：学位论文

【摘要】：目的蛋白酶活化受体4（protease-activated receptor，PAR4），是蛋白酶活化受体家族成员之一，属于G蛋白偶联受体。研究发现PAR4在正常和炎症条件下参与调节疼痛反应，目前PAR4在痛觉调节中的作用机制尚不清楚，可能是经过细胞内的信号转导机制作用于周围感觉神经元，其中部分可能是通过致敏瞬时受体电位香草酸亚型1（Transient receptor potential vanilloid1TRPV1）参与外周伤害性刺激的调节。为了进一步了解PAR4在背根神经节（dorsal root ganglion，DRG）初级感觉神经元的表达，及与伤害性感觉受体TRPV1的共存，探究PAR4在外周伤害性感觉信号传导中的作用提供形态学依据。方法 1.应用免疫荧光组织化学双标法结合激光共聚焦显微镜技术，观察PAR4在大鼠和小鼠DRG初级感觉神经元的表达及与TRPV1的共存。 2.腹腔内注射醋酸建立内脏疼痛大鼠动物模型，应用免疫荧光组织化学双标法结合激光共聚焦显微镜技术，观察PAR4在DRG初级感觉神经元的表达变化。结果 1. PAR4在小鼠DRG初级感觉神经元的表达及与TRPV1的共存：免疫荧光显示小鼠DRG内见有大量的感觉神经元表达PAR4，其形态多为中、小型的圆形、卵圆形，以及少量的大型神经元，可见少部分阳性神经元纤维。有80.5%±3.1%（3672/4558）神经元表达PAR4。免疫荧光双标法显示DRG内可见较多的TRPV1阳性神经元，，均为中、小型感觉神经元胞体，许多DRG的PAR4阳性神经元的表达均与TRPV1的共存，有76.9%±3.4%(2826/3672)的PAR4阳性神经元表达TRPV1，有77.4%±3.1%(2826/3647)的TRPV1阳性神经元表达PAR4。 2. PAR4在大鼠DRG初级感觉神经元的表达及与TRPV1的共存：PAR4在大鼠的DRG内的表达与小鼠类似，同样见有大量的感觉神经元表达PAR4，其形态多为中小型的圆形、卵圆形，以及少量的大型神经元，细胞计数显示：有85.4%±4.1%（4337/5078）神经元表达PAR4。免疫荧光双标法显示有83.5%±3.6%(3623/4337)的PAR4阳性神经元表达TRPV1，有88.3%±3.5%(3623/4102)的TRPV1阳性神经元表达PAR4。 3.PAR4与CGRP在大鼠DRG初级感觉神经元的共存：免疫荧光显示大鼠DRG内见有大量的感觉神经元呈CGRP阳性，其形态多为中、小型的圆形、卵圆形，以及少量的大型神经元，并观察到部分CGRP阳性神经纤维，有75.6%±4.1%（3387/4478）阳性神经元表达CGRP。免疫荧光双标法显示有82.3%±4.6%（2788/3387）PAR4阳性神经元表达CGRP，有64.2%±3.7%（2788/4337）CGRP阳性细胞均表达PAR4。 4. TRPV1与CGRP在大鼠DRG初级感觉神经元的共存：DRG内TRPV1阳性神经元与CGRP阳性细胞类似，均为中、小型感觉神经元胞体和一些弥散神经纤维。免疫荧光双标显示TRPV1/CGRP在DRG感觉神经元内广泛的共存，有83.9%±3.6%（2842/3387）CGRP阳性细胞均表达TRPV1，有69.2%±3.2%（2842/4102）TRPV1阳性细胞表达CGRP，也可观察到少量的TRPV1单标神经元或CGRP单标神经元。 5. PAR4在内脏疼痛大鼠动物模型中DRG感觉神经元的表达变化，在内脏疼痛大鼠动物模型DRG内PAR4阳性感觉神经元的数量明显增加，细胞计数显示PAR4阳性感觉神经元的数量与正常大鼠相比，增长了11.1%±2.3%，有96.5%±4.3%（5336/5528）神经元表达PAR4。同时DRG内TRPV1阳性神经元的数量也明显增加，有95.0%±4.4%（5110/5378）神经元表达TRPV1。免疫荧光双标显示有较多的PAR4/TRPV1双标细胞,分别占PAR4阳性和TRPV1阳性细胞的88.8%±3.7%(4742/5336)和92.7%±3.4%(4742/5110)。实验结果显示在大鼠DRG内见有许多感觉神经元表达PAR4，并与TRPV1广泛的共存。结论 1.PAR4在大鼠和小鼠的DRG初级感觉神经元有广泛的表达，主要为中、小型神经元。 2.PAR4与TRPV1在DRG初级感觉神经元存在广泛的共存，说明PAR4参与外周伤害性刺激可能与TRPV1的功能调节有关。 3.在内脏疼痛大鼠动物模型中，PAR4在DRG的表达明显增加，说明PAR4在内脏伤害性刺激的传导过程具有重要的作用。
[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.

【学位授予单位】：泰山医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R338

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