本文選題：蛋白酶激活受體(PARs)　切入點(diǎn)：RNA干擾　出處：《汕頭大學(xué)》2008年博士論文　論文類型：學(xué)位論文

【摘要】： 蛋白酶激活受體(protease/proteinase-activated receptors, PARs)屬于與G蛋白相偶聯(lián)、有七個(gè)跨膜單位的受體家族[1],目前在人和小鼠中共發(fā)現(xiàn)4種PARs,分別為PAR-1、PAR-2、PAR-3和PAR-4。其中PAR-1, PAR-3和PAR-4是凝血酶受體[2,3,4],PAR-1, PAR-2和PAR-4是胰蛋白酶受體,PAR-2是類胰蛋白酶受體。 RNA干擾(RNA interference,RNAi )是是指細(xì)胞產(chǎn)生內(nèi)源性雙鏈RNA(double stranded RNA,dsRNA )或?qū)胪庠葱詃sRNA后,與dsRNA同源的內(nèi)源性mRNA發(fā)生特異性的降解,從而導(dǎo)致基因表達(dá)沉默的現(xiàn)象。因這種現(xiàn)象發(fā)生在轉(zhuǎn)錄后水平,故又稱為轉(zhuǎn)錄后基因沉默(post-transcriptional gene silencing, PTGS )[5]。這種RNA水平上的基因抑制,提供了一種特異性失活功能基因的方法,成為基因表達(dá)調(diào)控和功能基因組學(xué)研究的一個(gè)重要手段。本研究通過(guò)RNA干擾技術(shù),抑制PAR-1、PAR-2和PAR-4基因的表達(dá),并探討了PARs基因與IL-4、IL-6和IL-13分泌的影響。 分別合成了含有21個(gè)核苷酸PAR-1, PAR-2和PAR-4的的小雙鏈干擾RNA(siRNA),每種基因合成3對(duì)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干擾技術(shù),將以上9種siRNA分別導(dǎo)入P815細(xì)胞中。通過(guò)實(shí)時(shí)定量PCR、Western Blot免疫印記檢測(cè)技術(shù)、流式細(xì)胞術(shù)和激光共聚焦技術(shù)在mRNA水平和蛋白水平分別檢測(cè)PAR-1,PAR-2和PAR-4表達(dá)情況;用PARs的激動(dòng)肽、胰蛋白酶、類胰蛋白酶和凝血酶分別激發(fā)干擾了PAR-1、PAR-2和PAR-4表達(dá)40小時(shí)后的P815細(xì)胞16小時(shí),用ELISA檢測(cè)P815細(xì)胞細(xì)胞培養(yǎng)上清液中的IL-4、IL-6和IL-13。 實(shí)驗(yàn)結(jié)果顯示:3種PAR-1的siRNA中,siRNA PAR1-1對(duì)目的基因無(wú)明顯抑制作用;PAR1-2在濃度為3nm,轉(zhuǎn)染48小時(shí)時(shí)的抑制效果最強(qiáng),siRNA PAR1-3有較弱的抑制作用;3種PAR-2的siRNA中,PAR2-1、PAR2-2、PAR2-3對(duì)目的基因均有一定的抑制作,其中siRNA PAR2-3對(duì)PAR2的抑制作用最強(qiáng),其次為siRNA PAR2-2;3種PAR-4的siRNA中,siRNA PAR4-3在轉(zhuǎn)染不同的時(shí)間對(duì)PAR4均有較穩(wěn)定的抑制作用,其中在轉(zhuǎn)染48小時(shí)時(shí),對(duì)PAR4的抑制作用最強(qiáng),且較低濃度的siRNA抑制作用更強(qiáng)。siRNA PAR4-1和PAR4-2對(duì)目的基因的抑制作用較弱。 PAR-2和PAR-4不直接參與IL-4、IL-6和IL-13的釋放;PAR-1也不參與IL-4和IL-13的釋放;PAR-1被抑制時(shí),IL-6的釋放量增加,暗示存在某種途徑可以通過(guò)PAR-1來(lái)抑制IL-6的釋放。 在mRNA水平上沉默PAR-1、PAR-2和PAR-4的表達(dá)后,胰蛋白酶和類胰蛋白酶將不再能促進(jìn)IL-4的分泌,說(shuō)明胰蛋白酶和類胰蛋白酶對(duì)肥大細(xì)胞P815的作用極可能通過(guò)激活PARs實(shí)現(xiàn)。而凝血酶對(duì)肥大細(xì)胞P815的作用有可能是通過(guò)激活PAR-1和PAR-4實(shí)現(xiàn)的。當(dāng)P815細(xì)胞中的PAR-1的表達(dá)被抑制時(shí),Il-6的釋放增加,提示PAR-1可能通過(guò)某種途徑參與了IL-6的釋放。凝血酶、胰蛋白酶和類胰蛋白酶引起的IL-6的產(chǎn)生至少有部分途徑是通過(guò)PAR-1和PAR-2而起作用。PAR-4參與了這些絲氨酸蛋白酶引起的IL-6的釋放,并且這種作用具有放大效應(yīng),故當(dāng)PAR-4被抑制時(shí),P815細(xì)胞的IL-6的釋放與對(duì)照相比會(huì)明顯減少。在mRNA水平上沉默PAR-1、PAR-2和PAR-4的表達(dá)后,PAR-AP、凝血酶、胰蛋白酶和類胰蛋白酶將不再能促進(jìn)P815細(xì)胞內(nèi)IL-13的分泌,說(shuō)明PAR-1、PAR-2和PAR-4可能部分的參與了IL-13的分泌釋放。
[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.

【學(xué)位授予單位】：汕頭大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R346

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