IL-1β对蛋白激酶活化受体4在初级感觉神经元表达的影响

发布时间：2018-09-10 12:44

【摘要】：目的以培养的大鼠初级感觉神经元及大鼠背根神经节（dorsal root ganglion，DRG）为研究模型，研究白介素-1β（Interleukin-1β，IL-1β）对蛋白激酶活化受体4（protease-activated receptor-4，PAR4）在DRG初级感觉神经元表达的影响。观察PAR4受体及其活化的细胞内机制，从而为进一步阐明PAR4参与外周疼痛信号的调节作用，为外周疼痛的治疗提供实验依据。方法 1.初级感觉神经元的分离和培养雄性Wistar大鼠，体重约200g，应用0.4%戊巴比妥钠，2ml，腹腔麻醉后，急性分离大鼠背根神经节（DRG），取双侧DRG，胰酶消化、均匀吹打，制成细胞悬液，种于六孔培养板，进行初级感觉神经元培养。 2. IL-1β的孵育本实验分为四组：对照组（IL-1β，0ng/ml），IL-1β（25ng/ml）组，IL-1β（50ng/ml）组，IL-1β（100ng/ml）组。DRG神经元培养24小时后，加入不同浓度的IL-1β（IL-1β终浓度分别为0ng/ml、25ng/ml、50ng/ml、100ng/ml），孵育、培养大鼠的DRG感觉神经元，作用4小时。应用Trizol裂解法提取培养的DRG感觉神经元总RNA，用反转录-聚合酶链反应（RT-PCR）核酸扩增技术检测PAR4基因的表达，及IL-1β诱导下对PAR4的基因表达的影响。 3．雄性Wistar大鼠，体重约200g，应用0.4%戊巴比妥钠，2ml，腹腔麻醉后，在大鼠的椎旁肌、椎旁皮下及L5/6椎间盘，注射IL-1β（0.5ml，50ng/ml）4小时后，急性分离L4、L5、L6的神经节，4%多聚甲醛液固定，冰冻切片8μm，利用免疫荧光方法结合荧光显微镜技术，观察PAR4在大鼠DRG感觉神经元的表达。 4. PKC激动剂（phorbol12-myristate13-acetate,PMA325nm）,PKC抑制剂（chelerythrine chloride,5nm),预先孵育1小时，再应用IL-1β（50ng/ml）作用于DRG感觉神经元，培养4小时， Trizol裂解法提取培养的DRG感觉神经元总RNA，用反转录-聚合酶链反应（RT-PCR）核酸扩增技术检测PAR4基因的表达，及PKC激动剂及抑制剂对IL-1β调控PAR4基因表达的影响。 5.分别注射PKC激动剂（0.5ml,20ng/mL）和抑制剂（0.5ml,20ng/mL），到大鼠的椎旁肌、椎旁皮下及L5/6椎间盘，作用1小时后，再注射IL-1β（0.5ml，50ng/ml）到椎旁肌、椎旁皮下及L5/6椎间盘，4小时。本实验分为四组：对照组（IL-1β，0ng/ml；PKC激动剂及抑制剂，0ng/ml），IL-1β+PKC激动剂组，IL-1β组，IL-1β+PKC抑制剂组。急性分离L4、L5、L6的神经节，4%多聚甲醛液固定，冰冻切片8μm，利用免疫荧光方法结合荧光显微镜技术，观察PAR4在大鼠DRG感觉神经元的表达，分析应用PKC激动剂和抑制剂干预后的白介素诱导下的PAR4的表达。结果 1. DRG内神经元的PAR4的分布免疫荧光组织化学结果显示，未应用IL-1β作用的大鼠背根神经节的神经元表达PAR4，阳性细胞多为中小型细胞,部分大型神经元也呈PAR4阳性表达。可见有27.31±0.49%神经元呈PAR4阳性，形态呈圆形或卵圆形，阳性标记物主要出现在细胞膜、细胞浆，细胞核未见标记。 2. IL-1β注射后DRG内神经元的表达变化应用IL-1β干预4小时后，急性分离L4、L5、L6背根神经节的神经细胞，应用免疫组化方法，可见大量的感觉神经元表达PAR4阳性。可见有63.39±0.54%神经元呈PAR4阳性（阳性细胞计数均采用药物注射椎间盘组的切片观察分析），PAR4阳性细胞数较正常的背根神经细胞明显增多，与对照组相比有统计学意义（P0.05）。部分细胞PAR4荧光标记增强，阳性细胞多为中小型细胞,大型神经元也呈PAR4阳性表达，形态呈圆形或卵圆形，阳性标记物主要出现在细胞膜、细胞浆，细胞核未见标记。在神经节部分神经元纤维也成呈PAR4阳性表达。 3. IL-1β对PAR4mRNA在初级感觉神经元表达的影响 RT-PCR结果显示：不同浓度的IL-1β使PAR4mRNA的表达量明显增加，并随着IL-1β浓度增加，表达量增加，呈正相关系增加,分别为对照组的1.04倍、1.18倍、1.24倍。不同浓度IL-1β组与空白对照组比较，均有统计学意义（P0.05），不同浓度的IL-1β组比较，也有统计学意义（P0.05）。 4. PKC激动剂和抑制剂对IL-1β诱导PAR4mRNA表达时的调节作用 RT-PCR结果显示：IL-1β及PKC激动剂组使PAR4mRNA的表达量明显，且PKC激动剂组表达较IL-1β组表达增加，PKC激动剂+IL-1β组是单纯IL-1β组的1.11倍，有统计学意义（P0.05）；而PKC抑制剂使IL-1β干预后的PAR4mRNA的表达量明显降低，与单纯IL-1β组相比PAR4mRNA的表达量明显下降，是其0.88倍，有统计学意义（P0.05）。 5. PKC对IL-1β注射后PAR4免疫阳性细胞的表达变化的影响分别在大鼠皮下注射、椎旁肌及椎间盘中，三个部位注射PKC激动剂(0.5ml，20ng/mL)及抑制剂(0.5ml，20ng/mL),1小时，再注射IL-1β（0.5ml，，50ng/mL），4小时后，通过免疫荧光组织化学结果显示，L4-L6DRG内的PAR4阳性细胞数明显发生变化，PKC激动剂使得PAR4的表达的阳性细胞数明显增多，与IL-1β组相比，PKC激动剂+IL-1β组PAR4阳性细胞计数是71.21±0.57%（实验细胞计数均采用药物注射腰椎间盘的组织切片观察分析），有统计学意义（P0.05），其中仍以中小型细胞为主,部分大型神经元表达；PKC抑制剂使得PAR4的表达的阳性细胞数明显减少，与IL-1β组相比，PKC抑制剂+IL-1β组PAR4阳性细胞计数是21.93±0.33%（实验细胞计数均采用药物注射腰椎间盘的组织切片观察分析），有统计学意义（P0.05），部分阳性细胞荧光显示的亮度减弱，大、中、小细胞数量减少上没有明显差异。结论 1.免疫荧光显示，大鼠DRG部分中小型初级感觉神经元呈PAR4免疫细胞化学标记阳性。 2.在大鼠的椎旁肌、皮下及椎间盘注射IL-1β，4小时后， PAR4阳性神经元表达较未行IL-1β注射组均增加，主要是一些中小型细胞，部分大细胞也存在表达 3.不同浓度的IL-1β作用于体外培养的大鼠DRG初级感觉神经元，PAR4mRNA表达量明显增加，且IL-1β浓度与PAR4mRNA表达呈正相关系。说明IL-1β对PAR4mRNA表达的起到一定的调控作用 4. PKC激动剂增强IL-1β对PAR4mRNA调节作用，相反，PKC抑制剂降低IL-1β对PAR4mRNA调节作用；同样预先在大鼠的椎旁肌、皮下及椎间盘注射PKC激动剂或抑制剂,能够增加或减少DRG内PAR4阳性神经元的数量。表明PKC信号参与IL-1β对DRG初级感觉神经元PAR4的调节作用。
[Abstract]:objective
The effects of interleukin-1 beta (IL-1 beta) on the expression of protein kinase-activated receptor-4 (PAR4) in primary sensory neurons and dorsal root ganglion (DRG) of rats were studied. The intracellular mechanisms of PAR4 receptor and its activation were observed. Therefore, PAR4 may be involved in the regulation of peripheral pain signal and provide experimental basis for the treatment of peripheral pain.
Method
1. isolation and culture of primary sensory neurons
Male Wistar rats, weighing about 200 g, were anesthetized with 0.4% sodium pentobarbital, 2 ml. After intraperitoneal anesthesia, the dorsal root ganglion (DRG) of the rats was isolated and cultured. Bilateral DRG, digested with trypsin, and blown evenly, were made into cell suspension. The primary sensory neurons were cultured on a six-well plate.
Incubation of 2. IL-1 beta
The experiment was divided into four groups: control group (IL-1 beta, 0ng/ml), IL-1 beta (25ng/ml), IL-1 beta (50ng/ml) and IL-1 beta (100ng/ml). After 24 hours of culture, DRG neurons were incubated with different concentrations of IL-1 beta (the final concentration of IL-1 beta was 0ng/ml, 25ng/ml, 50ng/ml, 100ng/ml) for 4 hours. Total RNA of DRG sensory neurons was extracted and the expression of PAR4 gene was detected by reverse transcription-polymerase chain reaction (RT-PCR) and the effect of IL-1beta on the expression of PAR4 gene was studied.
3. Male Wistar rats, weighing about 200 g, were anesthetized with 0.4% sodium pentobarbital, 2 ml. After intraperitoneal anesthesia, the paravertebral muscles, paravertebral subcutaneous and L5/6 intervertebral discs were injected with IL-1 beta (0.5 ml, 50 ng/ml) for 4 hours, the ganglia of L4, L5, L 6 were separated, fixed with 4% paraformaldehyde solution and frozen section for 8 microns. The expression of PAR4 in the DRG sensory neurons of rats was observed.
4. PKC agonists (phorbol 12-myristate 13-acetate, PMA325nm), PKC inhibitors (chelerythrine chloride, 5nm) were incubated for 1 hour, then the DRG sensory neurons were treated with IL-1beta (50ng/ml) for 4 hours. Total RNA of DRG sensory neurons was extracted by Trizol lysis and detected by RT-PCR. The expression of PAR4 gene and the effects of PKC agonists and inhibitors on the regulation of PAR4 gene expression by IL-1 beta were measured.
5. PKC agonists (0.5ml, 20ng/mL) and inhibitors (0.5ml, 20ng/mL) were injected into paravertebral muscles, paravertebral subcutaneous and L5/6 intervertebral discs respectively. After 1 hour of treatment, IL-1beta (0.5ml, 50ng/ml) was injected into paravertebral muscles, paravertebral subcutaneous and L5/6 intervertebral discs for 4 hours. The experiment was divided into four groups: control group (IL-1beta, 0ng/ml; PKC agonists and inhibitors, 0ng/ml). L4, L5, L6 ganglia were isolated, fixed with 4% paraformaldehyde solution, and frozen sections were cut into 8 microns. The expression of PAR4 in DRG sensory neurons was observed by immunofluorescence combined with fluorescence microscopy. The expression of PAR4 in DRG sensory neurons was analyzed after the intervention of PKC agonists and inhibitors. The expression of PAR4.
Result
PAR4 distribution of neurons in 1. DRG
Immunofluorescence histochemistry showed that PAR4 was expressed in the dorsal root ganglion neurons of rats without IL-1 beta. Most of the positive cells were small and medium sized, and some of the large neurons were PAR4 positive. 27.31 [0.49]% of the neurons were PAR4 positive. The morphology of the neurons was round or oval, and the positive markers mainly appeared in the cell membrane. Cytoplasm and nuclei were not marked.
Expression changes of neurons in DRG after 2. IL-1 injection
Immunohistochemical staining showed that PAR4 was expressed in a large number of sensory neurons. 63.39 (+0.54%) neurons were PAR4 positive (positive cell counts were observed in the intervertebral disc injection group) and the number of PAR4 positive cells was normal. PAR4 fluorescence labeling was enhanced in some cells, most of the positive cells were small and medium sized cells, and the large neurons were also PAR4 positive expression. The shape was round or oval. The positive markers mainly appeared in the cell membrane, cytoplasm and nucleus. Some nerve fibers also showed positive expression of PAR4.
Effect of 3. IL-1 beta on PAR4mRNA expression in primary sensory neurons
The results of RT-PCR showed that the expression of PAR4 mRNA was significantly increased in different concentrations of IL-1beta. The expression of PAR4 mRNA was positively correlated with the increase of IL-1beta concentration, which was 1.04, 1.18 and 1.24 times of that in the control group, respectively. There was statistical significance (P0.05).
Regulatory effects of 4. PKC agonists and inhibitors on IL-1 beta induced PAR4mRNA expression
RT-PCR results showed that the expression of PAR4 mRNA was significantly increased in the PKC agonist group compared with the IL-1 beta group, and the expression of PAR4 mRNA in the PKC agonist + IL-1 beta group was 1.11 times higher than that in the IL-1 beta group (P 0.05), while the expression of PAR4 mRNA was significantly decreased in the PKC inhibitor group compared with the IL-1 beta group. The expression of 4mRNA decreased significantly 0.88 times, with statistical significance (P0.05).
Effect of 5. PKC on the expression of PAR4 immunoreactive cells after IL-1 beta injection
PKC agonists (0.5ml, 20ng/mL) and inhibitors (0.5ml, 20ng/mL) were injected subcutaneously, paravertebral muscles and intervertebral discs in rats respectively. After 1 hour, IL-1 beta (0.5ml, 50ng/mL) was injected again. Immunofluorescence histochemistry showed that the number of PAR4 positive cells in L4-L6DRG changed significantly. PKC agonists made the expression of PAR4. The number of PAR4 positive cells in the PKC agonist + IL-1 beta group was 71.21 (+ 0.57%) compared with the IL-1 beta group (all of the experimental cells were observed and analyzed by the tissue sections of the lumbar intervertebral disc injected with drugs), and there was statistical significance (P 0.05). The expression of PAR4 positive cells in the PKC agonist + IL-1 beta group was mainly small and medium sized cells, and some large neurons were expressed. Compared with the IL-1 beta group, the number of PAR4 positive cells in the PKC inhibitor + IL-1 beta group was 21.93 (+ 0.33%) with statistical significance (P 0.05). The fluorescence intensity of some positive cells decreased, and the number of large, medium and small cells in the PKC inhibitor + IL-1 beta group was 21.93 (+ 0.33%). There is no significant difference in volume reduction.
conclusion
1. Immunofluorescence showed that PAR4 immunocytochemical staining was positive in some small and medium primary sensory neurons of DRG.
2. The expression of PAR4 positive neurons in paravertebral muscles, subcutaneous and intervertebral discs of rats was increased 4 hours after injection of IL-1beta. The expression of PAR4 positive neurons was mainly small and medium-sized cells, and some large cells were also expressed.
3. The expression of PAR4 mRNA in primary sensory neurons of DRG was significantly increased by different concentrations of IL-1beta, and the expression of PAR4 mRNA was positively correlated with the concentration of IL-1beta.
4. PKC agonists enhanced the regulation of IL-1beta on PAR4 mRNA, on the contrary, PKC inhibitors decreased the regulation of IL-1beta on PAR4 mRNA; the same pre-injection of PKC agonists or inhibitors into paravertebral muscles, subcutaneous and intervertebral discs in rats could increase or decrease the number of PAR4-positive neurons in DRG. Regulatory role of yuan PAR4.
【学位授予单位】：泰山医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R338

【参考文献】