pcsk9siRNA对oxLDL诱导的THP-1源性巨噬细胞凋亡的影响
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摘要
人类前蛋白转化酶枯草溶菌素9 ( proprotein convertase subtilisin/kexin type 9,pcsk9)基因编码神经细胞凋亡调节转化酶-1蛋白(neural apoptosis regulated convertase 1,NARC-1),该基因属于蛋白转化酶家族。pcsk9/NARC-1通过调节细胞表面低密度脂蛋白受体,从而在胆固醇代谢中发挥重要作用;其两种不同突变类型,可导致血液中胆固醇水平完全相反的变化,出现低胆固醇血症或高胆固醇血症。最近发现pcsk9/NARC-1可能还影响神经系统分化,调节神经元凋亡。研究pcsk9与巨噬细胞凋亡之间的关系,进而探索pcsk9在动脉粥样硬化(Atherosclerosis,As)发生中的作用及机制,将有助于动脉粥样硬化发病机制的研究,也能为防治动脉粥样硬化提供新思路。本研究以THP-1源性巨噬细胞为研究对象,经过不同浓度氧化低密度脂蛋白(oxidized Low Density Lipoprotein, oxLDL)处理后,观察巨噬细胞的凋亡及pcsk9表达变化,观察pcsk9基因沉默之后对这种凋亡作用的影响,为pcsk9在动脉粥样硬化发生发展中的作用提供实验依据。
摘要
目的:观察pcsk9在不同浓度oxLDL诱导的THP-1源性巨噬细胞中表达及巨噬细胞凋亡情况,研究小干扰RNA(small interfering RNA,siRNA)对pcsk9基因抑制作用以及pcsk9 siRNA对oxLDL诱导的THP-1源性巨噬细胞凋亡的影响。
方法:THP-1源性巨噬细胞与160nmol/L的佛波酯孵育24h,诱导成贴壁的巨噬细胞后,用0μg/ml、25μg/ml、50μg/ml、75μg/ml、100μg/ml的oxLDL处理48h,Hoechst33258染色检测细胞凋亡,RT-PCR、Western blot分别检测THP-1源性巨噬细胞pcsk9 mRNA、NARC-1蛋白质的表达。设计制备三对针对pcsk9基因不同位点的siRNA,通过阳离子脂质体Lipofectamine2000转染入THP-1源性巨噬细胞。6h后荧光显微镜评价转染效率,分别在转染24h、48h后从mRNA、蛋白水平观察pcsk9沉默效率,从而筛选出最有效的siRNA转染入THP-1源性巨噬细胞24h后,加入75μg/ml oxLDL处理48h,Hoechst33258染色观察细胞形态评价细胞凋亡,流式细胞计数检测细胞凋亡率。
结果:不同浓度的oxLDL处理THP-1源性巨噬细胞48h后,Hoechst33258染色,荧光显微镜下观察细胞,在75μg/ml组出现大量的细胞核呈浓染致密的固缩形态或颗粒状荧光的凋亡细胞。同时RT-PCR、Western blotting检测发现,随着oxLDL浓度的增加,pcsk9mRNA和NARC-1蛋白质表达量均增加,75μg/ml oxLDL组增加最明显。荧光标记的siRNA (FAM-siRNA)转染细胞6h后,荧光显微镜下观察,成功转染的细胞可见绿色荧光散在分布在细胞质中,未转染的细胞未见荧光。30nmol/L、50nmol/L、80nmol/L的siRNA转染THP-1源性巨噬细胞后,RT-PCR筛选出三对siRNA的终浓度为80nmol/L均可出现明显的沉默效应。选取此浓度检测蛋白水平基因抑制情况,筛选出最有效的一对siRNA。将筛选出来的siRNA-2转染THP-1源性巨噬细胞24h后,再用oxLDL处理48h后,Hoechst33258染色及流式细胞计数结果显示转染siRNA组凋亡明显被抑制。
结论:在本实验研究的浓度范围内,随着oxLDL浓度的增加pcsk9的表达随之增加,同时THP-1源性巨噬细胞凋亡也明显增加,75μg/ml oxLDL最明显,pcsk9mRNA和蛋白的表达也在该浓度最高;pcsk9siRNA能有效抑制pcsk9基因的表达;pcsk9 siRNA能有效抑制经oxLDL诱导的THP-1源性巨噬细胞的凋亡。
AIM: To study the expression of pcsk9 and apoptosis in THP-1 derived macrophages induced by oxLDL, to investigate inhibitory effect of siRNAs(small interfering RNA,siRNA) on pcsk9 gene expression and the effect of pcsk9 siRNA on THP-1 derived macrophages apoptosis induced by oxLDL .
METHODS: THP-1 derived macrophages were induced to differentiation into macrophages by PMA treatment for 24h. The experiments were designed as follows: cells were incubated with oxLDL with a concentration of 0μg/ml, 25μg/ml, 50μg/ml, 75μg/ml,100μg/ml for 48h separately. The apoptosis of THP-1 derived macrophages was observed by staining with Hoechst33258. The expression of pcsk9 was analyzed by reverse transcription polymerase chain reaction and Western blot. siRNAs for pcsk9 gene was designed and synthesized, then be transfected into THP-1 derived macrophages by positive ion liposome Lipofectamine 2000.Transfect efficiency was assessed by fluorescence microscope assay. RT-PCR(reverse transcription polymerase chain reaction) and Western blot were conducted to detect the expression of pcsk9 after 24h,48h separately. The most efficient siRNA was selected to transfect into THP-1 derived macrophages.24h later,oxLDL was used to treat cells for 48h,and Hoechst 33258 staining and flow cytometer were conducted to determine apoptosis.
RESULTS: As shown in Hoechst33258 staining assay,the number of cells with nuclear condensation induced by 75μg/ml oxLDL increased significantly. RT-PCR and Western blot showed that, in THP-1 derived macrophages, pcsk9 was upregulated with increasing concentration of oxLDL, while 75μg/ml oxLDL increased significantly. 6h after FAM-siRNA was transfected into THP-1 derived macrophages, we collected cells and visualized green fluorescence under the fluorescence microscope whereas the normal cells non-transfected siRNA could not be seen. The result showed that siRNA was successfully transfected into cells.RT-PCR and Western blot results showed that when transfected the most effective siRNA with different concentration such as 30nmol/L,50 nmol/L,80nmol/L into cells,the effect of suppression cut down obviously at 80nmol/L. Then we selected the mostly efficient siRNA-2 transfect into THP-1 derived macrophages,after 24h add 75μg/ml oxLDL for 48h, compared with control,the apoptosis of THP-1 cell transfected siRNA was suppressed by staining with Hoechst 33258 and flow cytometer.
CONCLUSIONS: oxLDL could induce THP-1 derived macrophages apoptosis.The expression of pcsk9mRNA and protein were increased by oxLDL in a concentration-dependent manner, 75μg/ml oxLDL increased significantly.Expression of pcsk9 gene could be effectively suppressed by siRNA. THP-1 derived macrophages apoptosis induced by oxLDL could be effectively suppressed by pcsk9 siRNA.
摘要
目的:观察pcsk9在不同浓度oxLDL诱导的THP-1源性巨噬细胞中表达及巨噬细胞凋亡情况,研究小干扰RNA(small interfering RNA,siRNA)对pcsk9基因抑制作用以及pcsk9 siRNA对oxLDL诱导的THP-1源性巨噬细胞凋亡的影响。
方法:THP-1源性巨噬细胞与160nmol/L的佛波酯孵育24h,诱导成贴壁的巨噬细胞后,用0μg/ml、25μg/ml、50μg/ml、75μg/ml、100μg/ml的oxLDL处理48h,Hoechst33258染色检测细胞凋亡,RT-PCR、Western blot分别检测THP-1源性巨噬细胞pcsk9 mRNA、NARC-1蛋白质的表达。设计制备三对针对pcsk9基因不同位点的siRNA,通过阳离子脂质体Lipofectamine2000转染入THP-1源性巨噬细胞。6h后荧光显微镜评价转染效率,分别在转染24h、48h后从mRNA、蛋白水平观察pcsk9沉默效率,从而筛选出最有效的siRNA转染入THP-1源性巨噬细胞24h后,加入75μg/ml oxLDL处理48h,Hoechst33258染色观察细胞形态评价细胞凋亡,流式细胞计数检测细胞凋亡率。
结果:不同浓度的oxLDL处理THP-1源性巨噬细胞48h后,Hoechst33258染色,荧光显微镜下观察细胞,在75μg/ml组出现大量的细胞核呈浓染致密的固缩形态或颗粒状荧光的凋亡细胞。同时RT-PCR、Western blotting检测发现,随着oxLDL浓度的增加,pcsk9mRNA和NARC-1蛋白质表达量均增加,75μg/ml oxLDL组增加最明显。荧光标记的siRNA (FAM-siRNA)转染细胞6h后,荧光显微镜下观察,成功转染的细胞可见绿色荧光散在分布在细胞质中,未转染的细胞未见荧光。30nmol/L、50nmol/L、80nmol/L的siRNA转染THP-1源性巨噬细胞后,RT-PCR筛选出三对siRNA的终浓度为80nmol/L均可出现明显的沉默效应。选取此浓度检测蛋白水平基因抑制情况,筛选出最有效的一对siRNA。将筛选出来的siRNA-2转染THP-1源性巨噬细胞24h后,再用oxLDL处理48h后,Hoechst33258染色及流式细胞计数结果显示转染siRNA组凋亡明显被抑制。
结论:在本实验研究的浓度范围内,随着oxLDL浓度的增加pcsk9的表达随之增加,同时THP-1源性巨噬细胞凋亡也明显增加,75μg/ml oxLDL最明显,pcsk9mRNA和蛋白的表达也在该浓度最高;pcsk9siRNA能有效抑制pcsk9基因的表达;pcsk9 siRNA能有效抑制经oxLDL诱导的THP-1源性巨噬细胞的凋亡。
AIM: To study the expression of pcsk9 and apoptosis in THP-1 derived macrophages induced by oxLDL, to investigate inhibitory effect of siRNAs(small interfering RNA,siRNA) on pcsk9 gene expression and the effect of pcsk9 siRNA on THP-1 derived macrophages apoptosis induced by oxLDL .
METHODS: THP-1 derived macrophages were induced to differentiation into macrophages by PMA treatment for 24h. The experiments were designed as follows: cells were incubated with oxLDL with a concentration of 0μg/ml, 25μg/ml, 50μg/ml, 75μg/ml,100μg/ml for 48h separately. The apoptosis of THP-1 derived macrophages was observed by staining with Hoechst33258. The expression of pcsk9 was analyzed by reverse transcription polymerase chain reaction and Western blot. siRNAs for pcsk9 gene was designed and synthesized, then be transfected into THP-1 derived macrophages by positive ion liposome Lipofectamine 2000.Transfect efficiency was assessed by fluorescence microscope assay. RT-PCR(reverse transcription polymerase chain reaction) and Western blot were conducted to detect the expression of pcsk9 after 24h,48h separately. The most efficient siRNA was selected to transfect into THP-1 derived macrophages.24h later,oxLDL was used to treat cells for 48h,and Hoechst 33258 staining and flow cytometer were conducted to determine apoptosis.
RESULTS: As shown in Hoechst33258 staining assay,the number of cells with nuclear condensation induced by 75μg/ml oxLDL increased significantly. RT-PCR and Western blot showed that, in THP-1 derived macrophages, pcsk9 was upregulated with increasing concentration of oxLDL, while 75μg/ml oxLDL increased significantly. 6h after FAM-siRNA was transfected into THP-1 derived macrophages, we collected cells and visualized green fluorescence under the fluorescence microscope whereas the normal cells non-transfected siRNA could not be seen. The result showed that siRNA was successfully transfected into cells.RT-PCR and Western blot results showed that when transfected the most effective siRNA with different concentration such as 30nmol/L,50 nmol/L,80nmol/L into cells,the effect of suppression cut down obviously at 80nmol/L. Then we selected the mostly efficient siRNA-2 transfect into THP-1 derived macrophages,after 24h add 75μg/ml oxLDL for 48h, compared with control,the apoptosis of THP-1 cell transfected siRNA was suppressed by staining with Hoechst 33258 and flow cytometer.
CONCLUSIONS: oxLDL could induce THP-1 derived macrophages apoptosis.The expression of pcsk9mRNA and protein were increased by oxLDL in a concentration-dependent manner, 75μg/ml oxLDL increased significantly.Expression of pcsk9 gene could be effectively suppressed by siRNA. THP-1 derived macrophages apoptosis induced by oxLDL could be effectively suppressed by pcsk9 siRNA.
引文
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[4]. Chiang L, Grenier J, Ettwiller L, et al.An orchestrated gene expression component of neuronal programmed cell death revealed by cDNA array analysis.PNAS 2001; 98:2814–2819
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[6]. Maxwell K N, Fisher E A, Breslow J L, et al. Over expression of pcsk9 accelerates the degradation of theLDLR in a post-endoplasmic reticulum compartment.PNAS,2005,102 (6):2069-2074
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[10]. Shioji K, Mannami T, Kokubo Y, et al. Genetic variants in pcsk9 affect the cholesterol level in Japanese. J Hum Genet, 2004, 49(2):109-114
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[12]. Berge K E, Ose L, Leren T P. Missense mutations in the pcsk9 gene are associated with hypocholesterolemia and possibly increased response to statin therapy. Arterioscler Thromb Vasc Biol, 2006,26(5):1094-1100
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[1] E1agoz A, Benjannel S, Mammarbassi A, et al. Biosynthesis and cellular trafficking of the convertase SKI-1/S1P:ectodomain shedding requires SKI-1activity [J].J Biol chem,2002,277(13):11265-11275
[2] Seidah N G, Benjannet S, Wickham L, et al. The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1):liver regeneration and neuronal differentiation[J]. Proc Natl Acad Sci USA, 2003, 100(3): 928-933
[3] Jirholt P, Adiels M, Bore'n J. How does mutant proprotein convertase neural apoptosis-regulated convertase 1 induce autosomal dominant hypercholesterolemia?[J]. Arterioscler Thromb Vasc Biol, 2004, 24(8):1334-1336
[4]叶平,陈保生.脂蛋白异常的分子遗传学[M].见:杨永宗主编,动脉粥样硬化性心血管病的基础与临床.北京:科学出版社, 2004; 436-458(YE Ping, CHENG Bao-Sheng. Molecular genetics of dyslipoproteinemia. In: YANG Yong-Zong, Basic and Clinical Researches on Atherosclerotic Cardiovascular Disease. BeiJing: Sience Press, 2004; 436-458)
[5] Dubuc G, Chamberland A, Wassef H, et al. Statins upregulate pcsk9,the gene encoding the proprotein convertase neural apoptosisregulated convertase-1 implicated in familial hypercholesterolemia[J].Arterioscler Thromb Vasc Biol, 2004, 24(8):1454-1459
[6] Maxwell K N, Soccio R E, Duncan E M, et al. Novel putative SREBP and LXR target genes identified by microarray analysis in liver of cholesterol-fed mice[J]. J Lipid Res, 2003, 44(11):2109-2119
[7] Rashid S, Curtis D E, Garuti R, et al. Decreased plasma cholesterol and hypersensitivity to statins in mice lacking Pcsk9[J].Proc Natl Acad Sci USA,2005,102(15):5374-5379
[8] Horton J D, Cohen J C, Hobbs H H. Molecular biology of pcsk9: its role in LDL metabolism[J]. Trends Biochem. Sci. 2006, 32(2):71-77
[9] Cameron J, Holla O L, Ranheim T, et al. Effect of mutations in the pcsk9 gene on the cell surface LDL receptors[J]. Hum Mol Genet. 2006; 15(9):1551-1558
[10] Timms K M, Wagner S, Samuels M E, et al. A mutation in pcsk9 causing autosomal-dominant hypercholesterolemia in a Utah pedigree[J]. Hum Genet, 2004, 114(4):349-353
[11] Leren T P. Mutations in the pcsk9 gene in Norwegian subjects with autooomal dominant hypercholesterolemia[J]. Clin Genet, 2004, 65(5):419-422
[12] Abifadel M, Varret M, Rabes, J P, et al. Mutations in pcsk9 cause autosomal dominant hypercholesterolemia[J]. Nat. Genet. 2003; 34:154–156
[13] Shioji K, Mannami T, Kokubo Y, et al. Genetic variants in pcsk9 affect the cholesterol level in Japanese[J]. J Hum Genet, 2004, 49(2):109-114
[14] Bourbon M, Rato Q. Portuguese Familial Hypercholesterolemia Study: presentation of the study and preliminary results[J]. Rev Port Cardiol. 2006,25(11):999-1013
[15] Berge K E, Ose L, Leren T P. Missense mutations in the pcsk9 gene are associated with hypocholesterolemia and possibly increased response to statin therapy[J]. Arterioscler Thromb Vasc Biol, 2006,26(5):1094-1100
[16] Fasano T, Cefalu A B, Di Leo E, et al. A novel loss of function mutation of pcsk9 gene in white subjects with low-plasma low-density lipoprotein cholesterol[J]. Arterioscler Thromb Vasc Biol. 2007; 27(3):677-681
[17] Miyake Y, Kimura R, Kokubo Y, et al. Genetic variants in pcsk9 in the Japanese population: Rare genetic variants in pcsk9 might collectively contribute to plasma LDL cholesterol levels in the general population. Atherosclerosis. 2007,20
[18] Yue P, Averna M, Lin X, Schonfeld G. The c.43_44insCTG variation in pcsk9 is associated with low plasma LDL-cholesterol in a Caucasian population[J]. Hum Mutat. 2006; 27(5):460-466
[19] Cohen et al. Common inactivating mutations in pcsk9 cause low LDL cholesterol. [J] Clin Genet, 2005, 67 (6): 476-477
[20] Poirier S, Prat A, Marcinkiewicz E, et al. Implication of the proprotein convertase pcsk9/NARC-1 in the development of the nervous system[J]. J Neurochem. 2006; 98(3):838-850
[21] Chiang L, Grenier J, Ettwiller L, et al. An orchestrated gene expression component of neuronal programmed cell death revealed by cDNA array analysis[J]. PNAS 2001; 98:2814–2819
[22] Bingham B, Shen R, Kotnis S, et al. Proapoptotic effects of NARC 1 (= pcsk9), the gene encoding a novel serine proteinase.[J] Cytometry A. 2006; 69(11):1123-1131
[23] Shibata N, Ohnuma T, Higashi S, et al. No genetic association between pcsk9 polymorphisms and Alzheimer's disease and plasma cholesterol level in Japanese patients[J]. Psychiatr Genet. 2005; 15(4):239:1454-1459
[2].杨永宗主编,动脉粥样硬化性心血管病的基础与临床.北京:科学出版社, 2004; 599-600
[3]. E1agoz A, Benjannel S, Mammarbassi A, et al. Biosynthesis and cellular trafficking of the convertase SKI-1/S1P:ectodomain shedding requires SKI-1activity.J Biol chem,2002,277(13):11265-11275
[4]. Chiang L, Grenier J, Ettwiller L, et al.An orchestrated gene expression component of neuronal programmed cell death revealed by cDNA array analysis.PNAS 2001; 98:2814–2819
[5]. Maxwell K N, Breslow J L. Adenoviral-mediated expression of pcsk9 in mice results in a low-density lipoprotein receptor knockout phenotype. PNAS, 2004 ,101 (18) :7100-7105
[6]. Maxwell K N, Fisher E A, Breslow J L, et al. Over expression of pcsk9 accelerates the degradation of theLDLR in a post-endoplasmic reticulum compartment.PNAS,2005,102 (6):2069-2074
[7]. TimmsK M, Wagner S, Samuels M E, et al. A mutation in pcsk9 causing autosomal-dominant hypercholesterolemia in a Utah pedigree. Hum Genet, 2004, 114(4):349-353
[8]. Leren T P. Mutations in the pcsk9 gene in Norwegian subjects with autooomal dominant hypercholesterolemia. Clin Genet, 2004, 65(5):419-422
[9]. Abifadel M, Varret M, Rabes, J P, et al. Mutations in pcsk9 cause autosomal dominant hypercholesterolemia. Nat. Genet. 2003; 34:154–156
[10]. Shioji K, Mannami T, Kokubo Y, et al. Genetic variants in pcsk9 affect the cholesterol level in Japanese. J Hum Genet, 2004, 49(2):109-114
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