【摘要】：目的 以培養(yǎng)的大鼠初級(jí)感覺(jué)神經(jīng)元及大鼠背根神經(jīng)節(jié)（dorsal root ganglion，DRG）為研究模型，研究白介素-1β（Interleukin-1β，IL-1β）對(duì)蛋白激酶活化受體4（protease-activated receptor-4，PAR4）在DRG初級(jí)感覺(jué)神經(jīng)元表達(dá)的影響。觀(guān)察PAR4受體及其活化的細(xì)胞內(nèi)機(jī)制，從而為進(jìn)一步闡明PAR4參與外周疼痛信號(hào)的調(diào)節(jié)作用，為外周疼痛的治療提供實(shí)驗(yàn)依據(jù)。 方法 1.初級(jí)感覺(jué)神經(jīng)元的分離和培養(yǎng) 雄性Wistar大鼠，體重約200g，應(yīng)用0.4%戊巴比妥鈉，2ml，腹腔麻醉后，急性分離大鼠背根神經(jīng)節(jié)（DRG），取雙側(cè)DRG，胰酶消化、均勻吹打，制成細(xì)胞懸液，種于六孔培養(yǎng)板，進(jìn)行初級(jí)感覺(jué)神經(jīng)元培養(yǎng)。 2. IL-1β的孵育 本實(shí)驗(yàn)分為四組：對(duì)照組（IL-1β，0ng/ml），IL-1β（25ng/ml）組，IL-1β（50ng/ml）組，IL-1β（100ng/ml）組。DRG神經(jīng)元培養(yǎng)24小時(shí)后，加入不同濃度的IL-1β（IL-1β終濃度分別為0ng/ml、25ng/ml、50ng/ml、100ng/ml），孵育、培養(yǎng)大鼠的DRG感覺(jué)神經(jīng)元，作用4小時(shí)。應(yīng)用Trizol裂解法提取培養(yǎng)的DRG感覺(jué)神經(jīng)元總RNA，用反轉(zhuǎn)錄-聚合酶鏈反應(yīng)（RT-PCR）核酸擴(kuò)增技術(shù)檢測(cè)PAR4基因的表達(dá)，及IL-1β誘導(dǎo)下對(duì)PAR4的基因表達(dá)的影響。 3．雄性Wistar大鼠，體重約200g，應(yīng)用0.4%戊巴比妥鈉，2ml，腹腔麻醉后，在大鼠的椎旁肌、椎旁皮下及L5/6椎間盤(pán)，注射IL-1β（0.5ml，50ng/ml）4小時(shí)后，急性分離L4、L5、L6的神經(jīng)節(jié)，4%多聚甲醛液固定，冰凍切片8μm，利用免疫熒光方法結(jié)合熒光顯微鏡技術(shù)，觀(guān)察PAR4在大鼠DRG感覺(jué)神經(jīng)元的表達(dá)。 4. PKC激動(dòng)劑（phorbol12-myristate13-acetate,PMA325nm）,PKC抑制劑（chelerythrine chloride,5nm),預(yù)先孵育1小時(shí)，再應(yīng)用IL-1β（50ng/ml）作用于DRG感覺(jué)神經(jīng)元，培養(yǎng)4小時(shí)， Trizol裂解法提取培養(yǎng)的DRG感覺(jué)神經(jīng)元總RNA，用反轉(zhuǎn)錄-聚合酶鏈反應(yīng)（RT-PCR）核酸擴(kuò)增技術(shù)檢測(cè)PAR4基因的表達(dá)，及PKC激動(dòng)劑及抑制劑對(duì)IL-1β調(diào)控PAR4基因表達(dá)的影響。 5.分別注射PKC激動(dòng)劑（0.5ml,20ng/mL）和抑制劑（0.5ml,20ng/mL），到大鼠的椎旁肌、椎旁皮下及L5/6椎間盤(pán)，作用1小時(shí)后，再注射IL-1β（0.5ml，50ng/ml）到椎旁肌、椎旁皮下及L5/6椎間盤(pán)，4小時(shí)。本實(shí)驗(yàn)分為四組：對(duì)照組（IL-1β，0ng/ml；PKC激動(dòng)劑及抑制劑，0ng/ml），IL-1β+PKC激動(dòng)劑組，IL-1β組，IL-1β+PKC抑制劑組。急性分離L4、L5、L6的神經(jīng)節(jié)，4%多聚甲醛液固定，冰凍切片8μm，利用免疫熒光方法結(jié)合熒光顯微鏡技術(shù)，觀(guān)察PAR4在大鼠DRG感覺(jué)神經(jīng)元的表達(dá)，分析應(yīng)用PKC激動(dòng)劑和抑制劑干預(yù)后的白介素誘導(dǎo)下的PAR4的表達(dá)。 結(jié)果 1. DRG內(nèi)神經(jīng)元的PAR4的分布 免疫熒光組織化學(xué)結(jié)果顯示，未應(yīng)用IL-1β作用的大鼠背根神經(jīng)節(jié)的神經(jīng)元表達(dá)PAR4，陽(yáng)性細(xì)胞多為中小型細(xì)胞,部分大型神經(jīng)元也呈PAR4陽(yáng)性表達(dá)�？梢�(jiàn)有27.31±0.49%神經(jīng)元呈PAR4陽(yáng)性，形態(tài)呈圓形或卵圓形，陽(yáng)性標(biāo)記物主要出現(xiàn)在細(xì)胞膜、細(xì)胞漿，細(xì)胞核未見(jiàn)標(biāo)記。 2. IL-1β注射后DRG內(nèi)神經(jīng)元的表達(dá)變化 應(yīng)用IL-1β干預(yù)4小時(shí)后，急性分離L4、L5、L6背根神經(jīng)節(jié)的神經(jīng)細(xì)胞，應(yīng)用免疫組化方法，可見(jiàn)大量的感覺(jué)神經(jīng)元表達(dá)PAR4陽(yáng)性�？梢�(jiàn)有63.39±0.54%神經(jīng)元呈PAR4陽(yáng)性（陽(yáng)性細(xì)胞計(jì)數(shù)均采用藥物注射椎間盤(pán)組的切片觀(guān)察分析），PAR4陽(yáng)性細(xì)胞數(shù)較正常的背根神經(jīng)細(xì)胞明顯增多，與對(duì)照組相比有統(tǒng)計(jì)學(xué)意義（P0.05）。部分細(xì)胞PAR4熒光標(biāo)記增強(qiáng)，陽(yáng)性細(xì)胞多為中小型細(xì)胞,大型神經(jīng)元也呈PAR4陽(yáng)性表達(dá)，形態(tài)呈圓形或卵圓形，陽(yáng)性標(biāo)記物主要出現(xiàn)在細(xì)胞膜、細(xì)胞漿，細(xì)胞核未見(jiàn)標(biāo)記。在神經(jīng)節(jié)部分神經(jīng)元纖維也成呈PAR4陽(yáng)性表達(dá)。 3. IL-1β對(duì)PAR4mRNA在初級(jí)感覺(jué)神經(jīng)元表達(dá)的影響 RT-PCR結(jié)果顯示：不同濃度的IL-1β使PAR4mRNA的表達(dá)量明顯增加，并隨著IL-1β濃度增加，表達(dá)量增加，呈正相關(guān)系增加,分別為對(duì)照組的1.04倍、1.18倍、1.24倍。不同濃度IL-1β組與空白對(duì)照組比較，均有統(tǒng)計(jì)學(xué)意義（P0.05），不同濃度的IL-1β組比較，也有統(tǒng)計(jì)學(xué)意義（P0.05）。 4. PKC激動(dòng)劑和抑制劑對(duì)IL-1β誘導(dǎo)PAR4mRNA表達(dá)時(shí)的調(diào)節(jié)作用 RT-PCR結(jié)果顯示：IL-1β及PKC激動(dòng)劑組使PAR4mRNA的表達(dá)量明顯，且PKC激動(dòng)劑組表達(dá)較IL-1β組表達(dá)增加，PKC激動(dòng)劑+IL-1β組是單純IL-1β組的1.11倍，有統(tǒng)計(jì)學(xué)意義（P0.05）；而PKC抑制劑使IL-1β干預(yù)后的PAR4mRNA的表達(dá)量明顯降低，與單純IL-1β組相比PAR4mRNA的表達(dá)量明顯下降，是其0.88倍，有統(tǒng)計(jì)學(xué)意義（P0.05）。 5. PKC對(duì)IL-1β注射后PAR4免疫陽(yáng)性細(xì)胞的表達(dá)變化的影響 分別在大鼠皮下注射、椎旁肌及椎間盤(pán)中，三個(gè)部位注射PKC激動(dòng)劑(0.5ml，20ng/mL)及抑制劑(0.5ml，20ng/mL),1小時(shí)，再注射IL-1β（0.5ml，，50ng/mL），4小時(shí)后，通過(guò)免疫熒光組織化學(xué)結(jié)果顯示，L4-L6DRG內(nèi)的PAR4陽(yáng)性細(xì)胞數(shù)明顯發(fā)生變化，PKC激動(dòng)劑使得PAR4的表達(dá)的陽(yáng)性細(xì)胞數(shù)明顯增多，與IL-1β組相比，PKC激動(dòng)劑+IL-1β組PAR4陽(yáng)性細(xì)胞計(jì)數(shù)是71.21±0.57%（實(shí)驗(yàn)細(xì)胞計(jì)數(shù)均采用藥物注射腰椎間盤(pán)的組織切片觀(guān)察分析），有統(tǒng)計(jì)學(xué)意義（P0.05），其中仍以中小型細(xì)胞為主,部分大型神經(jīng)元表達(dá)；PKC抑制劑使得PAR4的表達(dá)的陽(yáng)性細(xì)胞數(shù)明顯減少，與IL-1β組相比，PKC抑制劑+IL-1β組PAR4陽(yáng)性細(xì)胞計(jì)數(shù)是21.93±0.33%（實(shí)驗(yàn)細(xì)胞計(jì)數(shù)均采用藥物注射腰椎間盤(pán)的組織切片觀(guān)察分析），有統(tǒng)計(jì)學(xué)意義（P0.05），部分陽(yáng)性細(xì)胞熒光顯示的亮度減弱，大、中、小細(xì)胞數(shù)量減少上沒(méi)有明顯差異。 結(jié)論 1.免疫熒光顯示，大鼠DRG部分中小型初級(jí)感覺(jué)神經(jīng)元呈PAR4免疫細(xì)胞化學(xué)標(biāo)記陽(yáng)性。 2.在大鼠的椎旁肌、皮下及椎間盤(pán)注射IL-1β，4小時(shí)后， PAR4陽(yáng)性神經(jīng)元表達(dá)較未行IL-1β注射組均增加，主要是一些中小型細(xì)胞，部分大細(xì)胞也存在表達(dá) 3.不同濃度的IL-1β作用于體外培養(yǎng)的大鼠DRG初級(jí)感覺(jué)神經(jīng)元，PAR4mRNA表達(dá)量明顯增加，且IL-1β濃度與PAR4mRNA表達(dá)呈正相關(guān)系。說(shuō)明IL-1β對(duì)PAR4mRNA表達(dá)的起到一定的調(diào)控作用 4. PKC激動(dòng)劑增強(qiáng)IL-1β對(duì)PAR4mRNA調(diào)節(jié)作用，相反，PKC抑制劑降低IL-1β對(duì)PAR4mRNA調(diào)節(jié)作用；同樣預(yù)先在大鼠的椎旁肌、皮下及椎間盤(pán)注射PKC激動(dòng)劑或抑制劑,能夠增加或減少DRG內(nèi)PAR4陽(yáng)性神經(jīng)元的數(shù)量。表明PKC信號(hào)參與IL-1β對(duì)DRG初級(jí)感覺(jué)神經(jīng)元PAR4的調(diào)節(jié)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.
【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】：2013
【分類(lèi)號(hào)】：R338

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