PAR4在初级感觉神经元的表达及对TRPV1调控机制的研究

发布时间：2018-05-16 07:11

本文选题：蛋白酶活化受体4 + 疼痛　；参考：《泰山医学院》2012年硕士论文

【摘要】：目的蛋白酶活化受体4（PAR4）为G蛋白偶联受体，参与多种病理生理的过程。最近研究发现PAR4在外周炎症和疼痛调节中具有重要作用。以培养的大鼠背根神经节（DRG）感觉神经元为研究模型，研究PAR4在初级感觉神经元的表达。观察PAR4受体及其活化的细胞内机制，以及与伤害性感觉受体瞬时电位香草酸受体亚型1（TRPV1）之间的相互关系，从而为进一步阐明PAR4参与外周痛信号的调节作用提供实验依据。方法 1.以急性分离、培养大鼠的DRG感觉神经元为研究模型，利用免疫荧光的方法结合激光共聚焦显微镜技术，研究PAR4和TRPV1在体外培养的大鼠DRG感觉神经元的表达以及两者的共存。 2. Trizol裂解法提取培养的DRG感觉神经元总RNA，用RT-PCR检测PAR4基因的表达以及PAR4激动剂对PAR4和TRPV1基因表达的影响，及PKA信号通道对PAR4表达和PAR4介导TRPV1基因表达的调节作用。 3. RIPA细胞裂解液提取培养的DRG神经元总蛋白，检测PAR4激动剂对PAR4和TRPV1蛋白表达的影响以及PKA信号通道对PAR4表达和PAR4影响TRPV1蛋白表达的调节作用。结果 1.DRG感觉神经元的分离与培养：年轻雄性Wistar大鼠，体重约100g，急性分离DRG，快速取双侧DRG，消化、吹打，制成细胞悬液，种置于六孔培养板观察不同时间段细胞的数量，大小以及突起的长度。接种于六孔培养板的神经细胞培养4小时作用，倒置显微镜下观察，可见部分神经细胞已贴壁，贴壁的神经细胞为圆形或椭圆形，胞体周围有一圈光晕，尚无突起。神经元的细胞核位于细胞中央或胞体一侧，核仁明显。接种24小时后，大部分细胞已贴壁，贴壁细胞长出小突起，除单个散布的神经细胞外，常可见几个或多个神经细胞聚在一起，它们向四周发出树枝状突起。培养3-4天的神经细胞的突起逐渐延长增粗，可看到神经细胞的突触交织在一起形成稀疏的网状结构。随着培养时间的延长,神经细胞突起的主干和分枝都明显延长并增粗,神经细胞突起形成的网络结构变得更加稠密,神经细胞胞体逐渐增大。随着培养时间的延长，细胞数量会减少。 2.PAR4在DRG神经元的表达：免疫荧光组织化学结合激光共聚焦结果显示，在体外培养的大鼠DRG神经细胞，可见大量的感觉神经元表达PAR4，PAR4阳性神经元数量和形态变化与上述体外培养的DRG神经元类似。培养1天的阳性细胞多为一些中小神经元，直径为11-28微米，也偶可见一些大型神经元表达PAR4，形态呈圆形或椭圆形。培养24小时后，PAR4阳性神经元生长突起出现，培养3-4天DRG神经元可出现PAR4阳性轴突。阳性标记物主要出现在细胞膜、细胞浆，细胞核未见表达。 3.PAR4与TRPV1在DRG初级感觉神经元共存：在体外培养的大鼠DRG的神经细胞，培养1天的TRPV1阳性细胞与PAR4阳性细胞相似，可见大量的中、小型TRPV1阳性神经元胞体，形态呈圆形或卵圆形。TRPV1的阳性物分布于细胞膜、细胞浆，细胞核未见标记。DRG培养24小时后，可见TRPV1阳性的树突和轴突。荧光双标结合激光共聚焦结果显示：体外培养的DRG神经元均可见PAR4/TRPV1双标记。对30个培养孔盖玻片上的DRG细胞计数显示：有65.3%±3.2%(162/248)PAR4阳性神经元表达TRPV1，同样，TRPV1阳性神经元也表达PAR4，细胞计数有95%±2.4%（162/171）TRPV1阳性神经元表达PAR4。 4. AYPGKF-NH2对PAR4在初级感觉神经元表达的影响及细胞内机制 (1)PAR4激动剂对PAR4在初级感觉神经元表达的影响：将PAR4激动剂AYPGKF-NH2（10μM）加入培养的DRG神经细胞内作用1小时，Western blot结果显示：PAR4激动剂使PAR4蛋白的表达量增加，加PAR4激动剂组与空白对照组比较有统计学意义（P0.05）。RT-PCR结果显示：加PAR4激动剂组与空白对照组相比PAR4mRNA的表达量增加1.2倍，加PAR4激动剂组与空白对照组比较有统计学意义（P0.05）。 (2) PKA激动剂和抑制剂对PAR4表达的影响：将PKA激动剂（forskolin1μM）和抑制剂(PHKI14-223μM)分别孵育培养的神经细胞，，作用30min后，再加入PAR4激动剂孵育1小时，Western blot结果显示：加PKA激动剂和抑制剂组与加入PAR4激动剂组相比都使PAR4蛋白表达量减少，但加PKA激动剂组的PAR4蛋白的表达量相对于加入PKA抑制剂的DRG神经元PAR4表达减少，加入PKA抑制剂的PAR4蛋白的表达量与加入PKA激动剂组DRG神经元PAR4表达增加。加入PKA激动剂和抑制剂组和空白对照组比较均有统计学意义（P0.05）。RT-PCR结果显示：PKA激动剂组和PKA抑制剂组相比，加入PKA激动剂的PAR4mRNA的表达量减少，加入PKA抑制剂的PAR4mRNA的表达量增加。加入PKA激动剂组与空白对照组相比PAR4mRNA的表达量减少，加入PKA抑制剂组与空白对照组相比PAR4mRNA的表达量增加1.17倍。加入PKA抑制剂组和空白对照组比较均有统计学意义（P0.05）。 5. PAR4对初级感觉神经元TRPV1表达的影响及细胞内机制： PAR4激动剂对TRPV1表达的影响，将PAR4激动剂AYPGKF-NH2（10μM）加入培养的神经细胞内作用1小时，Western blot结果显示：DRG神经元TRPV1蛋白的表达量与空白对照组相比下降58%（n=3），加PAR4激动剂组与空白对照组比较有统计学意义（P0.05）。RT-PCR结果显示：PAR4激动剂使TRPV1mRNA的表达量减少与空白对照组相比下降20%（n=3），加PAR4激动剂组与空白对照组比较有统计学意义（P0.05）。结论 1.体外培养的大鼠DRG初级感觉神经元表达PAR4。许多PAR4阳性神经元与TRPV1共存。 2. AYPGKF-NH2可上调PAR4蛋白和mRNA在DRG初级感觉神经元的表达。PKA激动剂可抑制AYPGKF-NH2对PAR4蛋白和mRNA表达的调控作用，而PKA抑制剂可增强AYPGKF-NH2对PAR4蛋白和mRNA表达的调控和作用。 3.PAR4活化可下调TRPV1蛋白和mRNA在DRG初级感觉神经元的表达。PKA激动剂可增强AYPGKF-NH2对TRPV1蛋白和mRNA表达的调控作用，而PKA抑制剂可抑制AYPGKF-NH2对TRPV1蛋白和mRNA表达的调控和作用。
[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 .

【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】：R363

【参考文献】