RNA干扰对P815细胞中PARs表达的影响的研究

发布时间：2018-03-11 18:32

本文选题：蛋白酶激活受体(PARs)　切入点：RNA干扰　出处：《汕头大学》2008年博士论文　论文类型：学位论文

【摘要】： 蛋白酶激活受体(protease/proteinase-activated receptors, PARs)属于与G蛋白相偶联、有七个跨膜单位的受体家族[1],目前在人和小鼠中共发现4种PARs,分别为PAR-1、PAR-2、PAR-3和PAR-4。其中PAR-1, PAR-3和PAR-4是凝血酶受体[2,3,4],PAR-1, PAR-2和PAR-4是胰蛋白酶受体,PAR-2是类胰蛋白酶受体。 RNA干扰(RNA interference,RNAi )是是指细胞产生内源性双链RNA(double stranded RNA,dsRNA )或导入外源性dsRNA后,与dsRNA同源的内源性mRNA发生特异性的降解,从而导致基因表达沉默的现象。因这种现象发生在转录后水平,故又称为转录后基因沉默(post-transcriptional gene silencing, PTGS )[5]。这种RNA水平上的基因抑制,提供了一种特异性失活功能基因的方法,成为基因表达调控和功能基因组学研究的一个重要手段。本研究通过RNA干扰技术,抑制PAR-1、PAR-2和PAR-4基因的表达,并探讨了PARs基因与IL-4、IL-6和IL-13分泌的影响。分别合成了含有21个核苷酸PAR-1, PAR-2和PAR-4的的小双链干扰RNA(siRNA),每种基因合成3对siRNA(siRNA PAR1-1、siRNA PAR1-2和siRNA PAR1-3;siRNA PAR2-1、siRNA PAR2-2和siRNA PAR2-3;siRNA PAR4-1、siRNA PAR4-2和siRNA PAR4-3)。利用RNA干扰技术,将以上9种siRNA分别导入P815细胞中。通过实时定量PCR、Western Blot免疫印记检测技术、流式细胞术和激光共聚焦技术在mRNA水平和蛋白水平分别检测PAR-1,PAR-2和PAR-4表达情况;用PARs的激动肽、胰蛋白酶、类胰蛋白酶和凝血酶分别激发干扰了PAR-1、PAR-2和PAR-4表达40小时后的P815细胞16小时,用ELISA检测P815细胞细胞培养上清液中的IL-4、IL-6和IL-13。实验结果显示:3种PAR-1的siRNA中,siRNA PAR1-1对目的基因无明显抑制作用;PAR1-2在浓度为3nm,转染48小时时的抑制效果最强,siRNA PAR1-3有较弱的抑制作用;3种PAR-2的siRNA中,PAR2-1、PAR2-2、PAR2-3对目的基因均有一定的抑制作,其中siRNA PAR2-3对PAR2的抑制作用最强,其次为siRNA PAR2-2;3种PAR-4的siRNA中,siRNA PAR4-3在转染不同的时间对PAR4均有较稳定的抑制作用,其中在转染48小时时,对PAR4的抑制作用最强,且较低浓度的siRNA抑制作用更强。siRNA PAR4-1和PAR4-2对目的基因的抑制作用较弱。 PAR-2和PAR-4不直接参与IL-4、IL-6和IL-13的释放;PAR-1也不参与IL-4和IL-13的释放;PAR-1被抑制时,IL-6的释放量增加,暗示存在某种途径可以通过PAR-1来抑制IL-6的释放。在mRNA水平上沉默PAR-1、PAR-2和PAR-4的表达后,胰蛋白酶和类胰蛋白酶将不再能促进IL-4的分泌,说明胰蛋白酶和类胰蛋白酶对肥大细胞P815的作用极可能通过激活PARs实现。而凝血酶对肥大细胞P815的作用有可能是通过激活PAR-1和PAR-4实现的。当P815细胞中的PAR-1的表达被抑制时,Il-6的释放增加,提示PAR-1可能通过某种途径参与了IL-6的释放。凝血酶、胰蛋白酶和类胰蛋白酶引起的IL-6的产生至少有部分途径是通过PAR-1和PAR-2而起作用。PAR-4参与了这些丝氨酸蛋白酶引起的IL-6的释放,并且这种作用具有放大效应,故当PAR-4被抑制时,P815细胞的IL-6的释放与对照相比会明显减少。在mRNA水平上沉默PAR-1、PAR-2和PAR-4的表达后,PAR-AP、凝血酶、胰蛋白酶和类胰蛋白酶将不再能促进P815细胞内IL-13的分泌,说明PAR-1、PAR-2和PAR-4可能部分的参与了IL-13的分泌释放。
[Abstract]:Protease activated receptors (protease/proteinase-activated, receptors, PARs) belong to G protein coupled, seven transmembrane receptor family [1], 4 species of PARs found in human and mouse of the Communist Party of China at present, respectively PAR-1, PAR-2, PAR-3 and PAR-4. in PAR-1, PAR-3 and PAR-4 are [2,3,4] PAR-1 PAR-2, thrombin receptor, and PAR-4 PAR-2 is a receptor of trypsin, tryptase receptor.
RNA interference (RNA interference, RNAi) is refers to the cells to produce endogenous double stranded RNA (double stranded RNA, dsRNA) or exogenous dsRNA, the degradation of specific mRNA and dsRNA homology, resulting in gene silencing phenomenon. Due to the occurrence of this phenomenon at the post transcriptional level, so it is also called post transcriptional gene silencing (post-transcriptional gene silencing, PTGS [5].) the level of RNA gene suppression, provides a method for specific inactivation of functional genes, gene expression has become an important means of regulation and functional genomics research. The inhibition of PAR-1 by RNA interference technique, the expression of PAR-2 and PAR-4 gene, and discusses the PARs gene and IL-4, effects of IL-6 and IL-13 secretion.
Were synthesized containing 21 nucleotides PAR-1, double stranded RNA PAR-2 and the PAR-4 interference (siRNA), each gene synthesis 3 on siRNA (siRNA PAR1-1, siRNA PAR1-2 and siRNA PAR1-3; siRNA PAR2-1, siRNA PAR2-2 and siRNA PAR2-3; siRNA PAR4-1, siRNA PAR4-2 and siRNA PAR4-3). Using RNA interference technology, will more than 9 kinds of siRNA were introduced into P815 cells. The quantitative real-time PCR, Western Blot was detected by Western blot technique, flow cytometry and confocal laser techniques were used to detect PAR-1 at mRNA and protein level, the expression of PAR-4 and PAR-2; PARs activating peptide, trypsin, trypsin and thrombin respectively stimulate interference PAR-1, PAR-2 and PAR-4 expression in P815 cells 40 hours after 16 hours, the culture supernatant of IL-4 cells detected by ELISA P815, IL-6 and IL-13.
The experimental results show that: siRNA 3 PAR-1, siRNA and PAR1-1 had no significant inhibitory effect on target gene; PAR1-2 at the concentration of 3nm, the strongest inhibitory effect at 48 hours of transfection, siRNA PAR1-3 inhibition is weak; siRNA 3 PAR-2, PAR2-1, PAR2-2, PAR2-3 on the target gene had certain inhibition siRNA PAR2-3, in which the strongest inhibitory effect on PAR2, followed by siRNA PAR2-2; siRNA 3 PAR-4, the inhibitory effect of siRNA PAR4-3 transfection in different time on PAR4 were more stable, which in 48 hours after transfection, the inhibition of PAR4 with the strongest, and low concentration of siRNA inhibited the weaker inhibition.SiRNA PAR4-1 and PAR4-2 a stronger effect on gene.
PAR-2 and PAR-4 are not directly involved in the release of IL-4, IL-6 and IL-13. PAR-1 also does not participate in the release of IL-4 and IL-13. When PAR-1 is inhibited, the release of IL-6 increases, suggesting that there is a way to inhibit the release of PAR-1 by PAR-1.
PAR-1 silencing at the mRNA level, the expression of PAR-2 and PAR-4, trypsin and tryptase will no longer be able to stimulate the secretion of IL-4, indicating that the effect of tryptase on mast cell P815 possibly through activation of PARs and thrombin on mast cells. The role of P815 may be through the activation of PAR-1 and PAR-4. When the P815 cells in the expression of PAR-1 was inhibited, Il-6 release increased, suggesting that PAR-1 may be involved in certain ways through the release of IL-6. Thrombin, trypsin and tryptase induced IL-6 production at least part of the way is to play a role in the.PAR-4 induced IL-6 serine protease the release by PAR-1 and PAR-2, and this effect has amplification effect, therefore, when PAR-4 is inhibited, P815 cell IL-6 release will be significantly reduced compared with the control. PAR-1 silencing at the level of mRNA, P After AR-2 and PAR-4 expression, PAR-AP, thrombin, trypsin and tryptase will no longer promote IL-13 secretion in P815 cells, indicating that PAR-1, PAR-2 and PAR-4 may be partly involved in the secretion and release of IL-13.

【学位授予单位】：汕头大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R346

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