本文選題：蛋白酶活化受體4 + 疼痛��； 參考：《泰山醫(yī)學(xué)院》2012年碩士論文

【摘要】：目的 蛋白酶活化受體4（PAR4）為G蛋白偶聯(lián)受體，參與多種病理生理的過(guò)程。最近研究發(fā)現(xiàn)PAR4在外周炎癥和疼痛調(diào)節(jié)中具有重要作用。以培養(yǎng)的大鼠背根神經(jīng)節(jié)（DRG）感覺(jué)神經(jīng)元為研究模型，研究PAR4在初級(jí)感覺(jué)神經(jīng)元的表達(dá)。觀察PAR4受體及其活化的細(xì)胞內(nèi)機(jī)制，以及與傷害性感覺(jué)受體瞬時(shí)電位香草酸受體亞型1（TRPV1）之間的相互關(guān)系，從而為進(jìn)一步闡明PAR4參與外周痛信號(hào)的調(diào)節(jié)作用提供實(shí)驗(yàn)依據(jù)。 方法 1.以急性分離、培養(yǎng)大鼠的DRG感覺(jué)神經(jīng)元為研究模型，利用免疫熒光的方法結(jié)合激光共聚焦顯微鏡技術(shù)，研究PAR4和TRPV1在體外培養(yǎng)的大鼠DRG感覺(jué)神經(jīng)元的表達(dá)以及兩者的共存。 2. Trizol裂解法提取培養(yǎng)的DRG感覺(jué)神經(jīng)元總RNA，用RT-PCR檢測(cè)PAR4基因的表達(dá)以及PAR4激動(dòng)劑對(duì)PAR4和TRPV1基因表達(dá)的影響，及PKA信號(hào)通道對(duì)PAR4表達(dá)和PAR4介導(dǎo)TRPV1基因表達(dá)的調(diào)節(jié)作用。 3. RIPA細(xì)胞裂解液提取培養(yǎng)的DRG神經(jīng)元總蛋白，檢測(cè)PAR4激動(dòng)劑對(duì)PAR4和TRPV1蛋白表達(dá)的影響以及PKA信號(hào)通道對(duì)PAR4表達(dá)和PAR4影響TRPV1蛋白表達(dá)的調(diào)節(jié)作用。 結(jié)果 1.DRG感覺(jué)神經(jīng)元的分離與培養(yǎng)： 年輕雄性Wistar大鼠，體重約100g，急性分離DRG，快速取雙側(cè)DRG，消化、吹打，制成細(xì)胞懸液，種置于六孔培養(yǎng)板觀察不同時(shí)間段細(xì)胞的數(shù)量，大小以及突起的長(zhǎng)度。接種于六孔培養(yǎng)板的神經(jīng)細(xì)胞培養(yǎng)4小時(shí)作用，倒置顯微鏡下觀察，可見(jiàn)部分神經(jīng)細(xì)胞已貼壁，貼壁的神經(jīng)細(xì)胞為圓形或橢圓形，胞體周圍有一圈光暈，尚無(wú)突起。神經(jīng)元的細(xì)胞核位于細(xì)胞中央或胞體一側(cè)，核仁明顯。接種24小時(shí)后，大部分細(xì)胞已貼壁，貼壁細(xì)胞長(zhǎng)出小突起，除單個(gè)散布的神經(jīng)細(xì)胞外，�？梢�(jiàn)幾個(gè)或多個(gè)神經(jīng)細(xì)胞聚在一起，它們向四周發(fā)出樹(shù)枝狀突起。培養(yǎng)3-4天的神經(jīng)細(xì)胞的突起逐漸延長(zhǎng)增粗，可看到神經(jīng)細(xì)胞的突觸交織在一起形成稀疏的網(wǎng)狀結(jié)構(gòu)。隨著培養(yǎng)時(shí)間的延長(zhǎng),神經(jīng)細(xì)胞突起的主干和分枝都明顯延長(zhǎng)并增粗,神經(jīng)細(xì)胞突起形成的網(wǎng)絡(luò)結(jié)構(gòu)變得更加稠密,神經(jīng)細(xì)胞胞體逐漸增大。隨著培養(yǎng)時(shí)間的延長(zhǎng)，細(xì)胞數(shù)量會(huì)減少。 2.PAR4在DRG神經(jīng)元的表達(dá)： 免疫熒光組織化學(xué)結(jié)合激光共聚焦結(jié)果顯示，在體外培養(yǎng)的大鼠DRG神經(jīng)細(xì)胞，可見(jiàn)大量的感覺(jué)神經(jīng)元表達(dá)PAR4，PAR4陽(yáng)性神經(jīng)元數(shù)量和形態(tài)變化與上述體外培養(yǎng)的DRG神經(jīng)元類似。培養(yǎng)1天的陽(yáng)性細(xì)胞多為一些中小神經(jīng)元，直徑為11-28微米，也偶可見(jiàn)一些大型神經(jīng)元表達(dá)PAR4，形態(tài)呈圓形或橢圓形。培養(yǎng)24小時(shí)后，PAR4陽(yáng)性神經(jīng)元生長(zhǎng)突起出現(xiàn)，培養(yǎng)3-4天DRG神經(jīng)元可出現(xiàn)PAR4陽(yáng)性軸突。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)表達(dá)。 3.PAR4與TRPV1在DRG初級(jí)感覺(jué)神經(jīng)元共存： 在體外培養(yǎng)的大鼠DRG的神經(jīng)細(xì)胞，培養(yǎng)1天的TRPV1陽(yáng)性細(xì)胞與PAR4陽(yáng)性細(xì)胞相似，可見(jiàn)大量的中、小型TRPV1陽(yáng)性神經(jīng)元胞體，形態(tài)呈圓形或卵圓形。TRPV1的陽(yáng)性物分布于細(xì)胞膜、細(xì)胞漿，細(xì)胞核未見(jiàn)標(biāo)記。DRG培養(yǎng)24小時(shí)后，可見(jiàn)TRPV1陽(yáng)性的樹(shù)突和軸突。熒光雙標(biāo)結(jié)合激光共聚焦結(jié)果顯示：體外培養(yǎng)的DRG神經(jīng)元均可見(jiàn)PAR4/TRPV1雙標(biāo)記。對(duì)30個(gè)培養(yǎng)孔蓋玻片上的DRG細(xì)胞計(jì)數(shù)顯示：有65.3%±3.2%(162/248)PAR4陽(yáng)性神經(jīng)元表達(dá)TRPV1，同樣，TRPV1陽(yáng)性神經(jīng)元也表達(dá)PAR4，細(xì)胞計(jì)數(shù)有95%±2.4%（162/171）TRPV1陽(yáng)性神經(jīng)元表達(dá)PAR4。 4. AYPGKF-NH2對(duì)PAR4在初級(jí)感覺(jué)神經(jīng)元表達(dá)的影響及細(xì)胞內(nèi)機(jī)制 (1)PAR4激動(dòng)劑對(duì)PAR4在初級(jí)感覺(jué)神經(jīng)元表達(dá)的影響：將PAR4激動(dòng)劑AYPGKF-NH2（10μM）加入培養(yǎng)的DRG神經(jīng)細(xì)胞內(nèi)作用1小時(shí)，Western blot結(jié)果顯示：PAR4激動(dòng)劑使PAR4蛋白的表達(dá)量增加，加PAR4激動(dòng)劑組與空白對(duì)照組比較有統(tǒng)計(jì)學(xué)意義（P0.05）。RT-PCR結(jié)果顯示：加PAR4激動(dòng)劑組與空白對(duì)照組相比PAR4mRNA的表達(dá)量增加1.2倍，加PAR4激動(dòng)劑組與空白對(duì)照組比較有統(tǒng)計(jì)學(xué)意義（P0.05）。 (2) PKA激動(dòng)劑和抑制劑對(duì)PAR4表達(dá)的影響：將PKA激動(dòng)劑（forskolin1μM）和抑制劑(PHKI14-223μM)分別孵育培養(yǎng)的神經(jīng)細(xì)胞，，作用30min后，再加入PAR4激動(dòng)劑孵育1小時(shí)，Western blot結(jié)果顯示：加PKA激動(dòng)劑和抑制劑組與加入PAR4激動(dòng)劑組相比都使PAR4蛋白表達(dá)量減少，但加PKA激動(dòng)劑組的PAR4蛋白的表達(dá)量相對(duì)于加入PKA抑制劑的DRG神經(jīng)元PAR4表達(dá)減少，加入PKA抑制劑的PAR4蛋白的表達(dá)量與加入PKA激動(dòng)劑組DRG神經(jīng)元PAR4表達(dá)增加。加入PKA激動(dòng)劑和抑制劑組和空白對(duì)照組比較均有統(tǒng)計(jì)學(xué)意義（P0.05）。RT-PCR結(jié)果顯示：PKA激動(dòng)劑組和PKA抑制劑組相比，加入PKA激動(dòng)劑的PAR4mRNA的表達(dá)量減少，加入PKA抑制劑的PAR4mRNA的表達(dá)量增加。加入PKA激動(dòng)劑組與空白對(duì)照組相比PAR4mRNA的表達(dá)量減少，加入PKA抑制劑組與空白對(duì)照組相比PAR4mRNA的表達(dá)量增加1.17倍。加入PKA抑制劑組和空白對(duì)照組比較均有統(tǒng)計(jì)學(xué)意義（P0.05）。 5. PAR4對(duì)初級(jí)感覺(jué)神經(jīng)元TRPV1表達(dá)的影響及細(xì)胞內(nèi)機(jī)制： PAR4激動(dòng)劑對(duì)TRPV1表達(dá)的影響，將PAR4激動(dòng)劑AYPGKF-NH2（10μM）加入培養(yǎng)的神經(jīng)細(xì)胞內(nèi)作用1小時(shí)，Western blot結(jié)果顯示：DRG神經(jīng)元TRPV1蛋白的表達(dá)量與空白對(duì)照組相比下降58%（n=3），加PAR4激動(dòng)劑組與空白對(duì)照組比較有統(tǒng)計(jì)學(xué)意義（P0.05）。RT-PCR結(jié)果顯示：PAR4激動(dòng)劑使TRPV1mRNA的表達(dá)量減少與空白對(duì)照組相比下降20%（n=3），加PAR4激動(dòng)劑組與空白對(duì)照組比較有統(tǒng)計(jì)學(xué)意義（P0.05）。 結(jié)論 1.體外培養(yǎng)的大鼠DRG初級(jí)感覺(jué)神經(jīng)元表達(dá)PAR4。許多PAR4陽(yáng)性神經(jīng)元與TRPV1共存。 2. AYPGKF-NH2可上調(diào)PAR4蛋白和mRNA在DRG初級(jí)感覺(jué)神經(jīng)元的表達(dá)。PKA激動(dòng)劑可抑制AYPGKF-NH2對(duì)PAR4蛋白和mRNA表達(dá)的調(diào)控作用，而PKA抑制劑可增強(qiáng)AYPGKF-NH2對(duì)PAR4蛋白和mRNA表達(dá)的調(diào)控和作用。 3.PAR4活化可下調(diào)TRPV1蛋白和mRNA在DRG初級(jí)感覺(jué)神經(jīng)元的表達(dá)。PKA激動(dòng)劑可增強(qiáng)AYPGKF-NH2對(duì)TRPV1蛋白和mRNA表達(dá)的調(diào)控作用，而PKA抑制劑可抑制AYPGKF-NH2對(duì)TRPV1蛋白和mRNA表達(dá)的調(diào)控和作用。
[Abstract]:Purpose

In order to further clarify the relationship between PAR4 receptor and its activated intracellular mechanism , and the relationship between PAR4 receptor and its activated intracellular mechanism and the transient potential vanillic acid receptor subtype 1 ( TRL 1 ) of the injured sensory receptor , the experimental basis was provided to further elucidate the role of PAR4 in the regulation of peripheral pain signal .

method

1 . The rat DRG sensory neurons were isolated and cultured as the research model , and the expression of the rat DRG sensory neurons cultured in vitro and the coexistence of both were studied by immunofluorescence .

2 . The total RNA of cultured DRG sensory neurons was extracted by Trizol lysis method , the expression of PAR4 gene was detected by RT - PCR , and the effect of PAR4 agonist on PAR4 gene expression and the effect of PAR4 agonist on PAR4 expression and PAR4 gene expression were studied .

3 . The total protein of DRG neurons cultured in RIPA cell lysate was extracted . The effect of PAR4 agonist on the expression of PAR4 and TRpv1 and the regulation of the expression of PAR4 and PAR4 on PAR4 expression and PAR4 were detected .

Results

1 . Isolation and culture of DRG sensory neurons :

In young male Wistar rats , weighing about 100g , acute isolated DRG , quickly taking bilateral DRG , digesting and blowing , making into cell suspension , putting into six - hole culture plate to observe the quantity , size and length of cells .

2 . PAR4 expression in DRG neurons :

The expression of PAR4 and PAR4 positive neurons in cultured rat DRG neurons was similar to that of DRG neurons cultured in vitro .

3 . par4 and trpv1 co - exist with the primary sensory neurons of the DRG :

In vitro cultured rat DRG neurons were cultured for 1 day , TRv1 - positive cells were similar to PAR4 - positive cells .

4 . Effect of AYPGKF - NH2 on the expression of PAR4 in primary sensory neurons and intracellular mechanism

( 1 ) The effect of PAR4 agonist on the expression of PAR4 agonist in primary sensory neurons : PAR4 agonist AYPGKF - NH2 ( 10 渭M ) was added to cultured DRG neurons for 1 hour . Western blot showed that PAR4 agonist increased PAR4 protein expression . Compared with blank control group , PAR4 agonist increased by 1.2 times compared with blank control group ( P0.05 ) .

Compared with the control group , the expression of PAR4 mRNA was decreased , and the expression of PAR4 mRNA was increased by 1.17 times . Compared with the control group , the expression of PAR4 mRNA was increased by 1.17 times compared with the control group .

5 . Effects of PAR4 on the expression of primary sensory neurons and intracellular mechanisms :

The results showed that PAR4 agonist decreased the expression of TRPV1mRNA by 20 % ( n = 3 ) compared with blank control group ( P0.05 ) .

Conclusion

1 . PAR4 was expressed in rat DRG primary sensory neurons cultured in vitro .

2 . The expression of PAR4 protein and mRNA in DRG primary sensory neurons can be increased by AYPGKF - NH2 , and the effect of AYPGKF - NH2 on PAR4 protein and mRNA expression can be inhibited and the regulation and effect of AYPGKF - NH2 on PAR4 protein and mRNA expression can be enhanced .

3 . PAR4 activation can downregulate the expression of TRpv1 protein and mRNA in DRG primary sensory neurons . The effect of AYPGKF - NH2 on the protein and mRNA expression in DRG can be enhanced by the activity of protein and protein , while the inhibitor can inhibit the regulation and effect of AYPGKF - NH2 on the protein and mRNA expression in DRG .

【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)】：R363

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