利用CRISPR/Cas9系统靶向敲除猪肠上皮细胞(IPEC-J2)ace2基因及其功能分析
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摘要
关于血管紧张素转移酶2 (angiotensin converting enzyme 2, ACE2)的研究目前主要集中于啮齿类动物和人类(Homo sapiens),猪(Sus scrofa domesticus)方面的资料较少。ACE2在不同组织器官中良好的抗炎、抗损伤作用已被广泛认可,但在肠道中的作用尚不明确。本研究拟利用CRISPR/Cas9基因编辑技术建立猪肠上皮细胞(porcine intestinal epithelial cell, IPEC-J2) ace2基因敲除方法,探究ace2缺失对细胞炎性损伤的影响。首先通过Western blot和免疫荧光染色方法证实所选取的IPEC-J2细胞有ACE2蛋白表达;然后以猪ace2基因为敲除靶点,设计合成3对单导向RNA (single guide RNA, sgRNA),并插入含有Cas9骨架的pX330质粒,构建ace2基因打靶载体pX330-ace2-1、pX330-ace2-2和pX330-ace2-3。将测序正确的重组质粒pX330-ace2-2转染至IPEC-J2细胞,Western blot和免疫荧光染色结果显示ACE2蛋白表达缺失;以酶联免疫吸附测定(enzyme linked immunosorbent assay, ELISA)方法检测ace2敲除细胞的炎性因子水平,结果显示,脂多糖(lipopolysaccharide, LPS)处理组的促炎因子白介素(interleukin, IL)-1β和IL-8显著升高(P<0.01)、抗炎因子IL-10显著降低(P<0.05),细胞炎性损伤加剧,提示肠道ACE2具有一定的抗炎作用。本研究利用CRISPR/Cas9系统建立了靶向敲除猪肠上皮细胞ace2基因的方法,并初步探讨了ACE2在猪肠道的抗炎作用,为深入开展ACE2相关功能和作用机制研究提供了基础资料。
Studies about angiotensin converting enzyme 2(ACE2) have focused on rodents and humans(Homo sapiens), and there are few data on pigs(Sus scrofa domesticus). The good anti-inflammatory and antiinjury effects of ACE2 in different tissues and organs have been widely recognized, but the role of ACE2 in intestinal tract is still unclear. This study intends to use the CRISPR/Cas9 gene editing technology to establish ace2 gene knockout method in porcine intestinal epithelial cell(IPEC-J2), and to explore the effect of ace2 deletion on cellular inflammatory injury. First, the presence of ACE2 protein in selected IPEC-J2 cells was confirmed by Western blot and immunofluorescence staining. The ace2 gene was then selected as the knockout target, and 3 pairs of single guide RNA(sg RNA) were designed and synthesized, and inserted into the pX330 plasmid containing Cas9 backbone to construct the ace2 knockout targeting vector pX330-ace2-1, p X330-ace2-2 and pX330-ace2-3. The correct recombinant plasmid pX330-ace2-2 was transfected into IPEC-J2 cells.Western blot and immunofluorescence staining showed that ACE2 protein expression was absent. The inflammatory factors were detected by enzyme linked immunosorbent assay(ELISA). The results showed that lipopolysaccharide(LPS) treated ace2 knockout cells and the pro-inflammatory factors interleukin(IL)-1β and IL-8 were significantly increased(P<0.01), the anti-inflammatory factor IL-10 was significantly decreased(P<0.05), and the cellular inflammatory injury was aggravated which suggested that intestinal ACE2 might had certain anti-inflammatory effect. The present study established a method for knocking out ace2 gene in pig intestinal epithelial cells by CRISPR/Cas9 system, and preliminarily explored the anti-inflammatory effect of ACE2, which might provide basic information for further research on ACE2 related functions and mechanisms.
Studies about angiotensin converting enzyme 2(ACE2) have focused on rodents and humans(Homo sapiens), and there are few data on pigs(Sus scrofa domesticus). The good anti-inflammatory and antiinjury effects of ACE2 in different tissues and organs have been widely recognized, but the role of ACE2 in intestinal tract is still unclear. This study intends to use the CRISPR/Cas9 gene editing technology to establish ace2 gene knockout method in porcine intestinal epithelial cell(IPEC-J2), and to explore the effect of ace2 deletion on cellular inflammatory injury. First, the presence of ACE2 protein in selected IPEC-J2 cells was confirmed by Western blot and immunofluorescence staining. The ace2 gene was then selected as the knockout target, and 3 pairs of single guide RNA(sg RNA) were designed and synthesized, and inserted into the pX330 plasmid containing Cas9 backbone to construct the ace2 knockout targeting vector pX330-ace2-1, p X330-ace2-2 and pX330-ace2-3. The correct recombinant plasmid pX330-ace2-2 was transfected into IPEC-J2 cells.Western blot and immunofluorescence staining showed that ACE2 protein expression was absent. The inflammatory factors were detected by enzyme linked immunosorbent assay(ELISA). The results showed that lipopolysaccharide(LPS) treated ace2 knockout cells and the pro-inflammatory factors interleukin(IL)-1β and IL-8 were significantly increased(P<0.01), the anti-inflammatory factor IL-10 was significantly decreased(P<0.05), and the cellular inflammatory injury was aggravated which suggested that intestinal ACE2 might had certain anti-inflammatory effect. The present study established a method for knocking out ace2 gene in pig intestinal epithelial cells by CRISPR/Cas9 system, and preliminarily explored the anti-inflammatory effect of ACE2, which might provide basic information for further research on ACE2 related functions and mechanisms.
引文
权素玉,王睿杰,孙俊杰,等.2015.血管紧张素转化酶2和中性氨基酸转运载体B0AT1在不同日龄猪肠道中的表达差异[J].畜牧与兽医,(4):36-39.(Quan S Y,Wang R J,Sun J J,et al.2015.Differential expression of angiotensin-converting enzyme 2 and neutral amino acid transporter B0AT1 in intestinal tract of different ages[J].Journal of Animal Husbandry and Veterinary Medicine,(4):36-39.)
王鲲.2017.血管紧张素转化酶2在LPS诱导的小鼠乳腺炎症损伤中保护作用及其分子机制[D].博士学位论文,南京农业大学,导师:张源淑,pp.73-76.(Wang K.2017.Protective effect of angiotensin-converting enzyme 2 on LPS-induced mammary gland inflammatory injury in mice and its molecular mechanism[D].Thesis for Ph.D.,Nanjing Agricultural University,Supervisor:Zhang Y S,pp.73-76.)
杨维维,许媛媛,荣超,等.2015.山羊血管紧张素转换酶2基因的克隆表达与生物信息学分析[J].南京农业大学学报,38(1):120-126.(Yang W W,Xu Y Y,Rong C,et al.2015.Cloning,expression and bioinformatics analysis of goat angiotensin converting enzyme 2 gene[J].Journal of Nanjing Agricultural University,38(1):120-126.)
喻江标,吴永港,张银妆,等.2018.ACE2-Ang(1-7)-Mas受体轴在高血压射血分数保留心力衰竭中的作用[J].中南大学学报(医学版),43(7):738-746.(Yu J B,Wu YG,Zhang Y Z,et al.2018.Role of ACE2-Ang(1-7)-Mas receptor axis in heart failure with preserved ejection fraction with hypertension[J].Journal of Central South University(Medical Sciences),43(7):738-746.)
张伟,苗晋锋,王艳霞,等.2012.苏钟猪ACE2基因克隆及其遗传进化分析[J].江蘇農業學報,28(1):90-95.(Zhang W,Miao J F,Wang Y X,et al.2012.Cloning and genetic evolution analysis of ACE2 gene of Suzhong pig[J].Journal of Jiangsu Agricultural Sciences,28(1):86-91.)
王珊珊,黄瑜,张树坤,等.2013.血管紧张素转化酶2(ACE2)的基因克隆及在仔猪体内的表达分布[J].中国兽医学报,33(10):1579-1583.(Wang S S,Huang Y,Zhang S K,et al.2013.Gene cloning of angiotensin converting enzyme 2(ACE2)and its expression distribution in piglets[J].Chinese Journal of Veterinary Science.33(10):1579-1583.)
Bae E H,Konvalinka A,Fang F,et al.2015.Characterization of the intrarenal renin-angiotensin system in experimental alport syndrome[J].The American Journal of Pathology,185(5):1423-1435.
Bae E H,Fang F,Williams V R,et al.2017.Murine recombinant angiotensin-converting enzyme 2 attenuates kidney injury in experimental Alport syndrome[J].Kidney International,91(6):1347-1361.
Clotet-Freixas S,Soler M J,Palau V,et al.2018.Sex dimorphism in ANGⅡ-mediated crosstalk between ACE2 and ACE in diabetic nephropathy[J].Laboratory Investigation,98:1237-1249.
Cong L,Ran F A,Cox D,et al.2013.Multiplex genome engineering using CRISPR/Cas systems[J].Science,399(6121):819-823.
Donoghue M,Hsieh F,Baronas E,et al.2000.A novel angiotensin-converting enzyme-related carboxypeptidase(ACE2)converts angiotensinⅠto angiotensin 1-9[J].Circulation Research,87(5):e1-e9.
Driehuis E,Clevers H.2017.CRISPR/Cas 9 genome editing and its applications in organoids[J].American Journal of Physiology-Gastrointestinal and Liver Physiology,312(3):G257-G265.
Gootenberg J S,Abudayyeh O O,Lee J W,et al.2017.Nucleic acid detection with CRISPR-Cas13a/C2c2[J].Science,356(6336):438-442.
Harhay G P,Sonstegard T S,Keele J W,et al.2005.Characterization of 954 bovine full-CDS cDNA sequences[J].BMC Genomics,6(1):166.
Hashimoto T,Perlot T,Rehman A,et al.2012.ACE2 links amino acid malnutrition to microbial ecology and intestinal inflammation[J].Nature,487(7408):477-481.
Hsu P D,Lander E S,et al.2014.Development and applications of CRISPR-Cas9 for genome engineering[J].Cell,157(6):1262-1278.
Ip W K E,Hoshi N,Shouval D S,et al.2017.Anti-inflammatory effect of IL-10 mediated by metabolic reprogramming of macrophages[J].Science,356(6337):513-519.
Kittana N.2018.AngiotensinⅠconverting enzyme 2-angiotensin 1Ⅰ7/1Ⅰ9 system:Novel promising targets for heart failure treatment[J].Fundamental&Clinical Pharmacology,32(1):14-25.
Liu C,Xiao L,Li F,et al.2015.Generation of outbred Ace2knockout mice by RNA transfection of TALENs displaying colitis reminiscent pathophysiology and inflammation[J].Transgenic Research,24(3):433-446.
Lu X J,Xue H Y,Ke Z P,et al.2015.CRISPR-Cas9:A new and promising player in gene therapy[J].Journal of Medical Genetics,52(5):289-296.
Patel V B,Mori J,McLean,et al.2016.ACE2 deficiency worsens epicardial adipose tissue inflammation and cardiac dysfunction in response to diet-induced obesity[J].Diabetes,65(1):85-95.
Singer D,Camargo S M,Ramadan,et al.2012.Defective intestinal amino acid absorption in Ace2 null mice[J].American Journal of Physiology-Gastrointestinal and Liver Physiology,303(6):G686-G695.
Tipnis S R,Hooper N M,Hyde R,et al.2000.A human homolog of angiotensin converting enzyme-cloning and functional expression as a captopril insensitive carboxypeptidase[J].Journal of Biological Chemistry,275(43):33238-33243.
Tseng Y W,Wang,P H,et al.2010.Regulation of the expression of angiotensin-converting enzyme 2 by polyunsaturated fatty acids in porcine adipocytes[J].Journal of Animal Science,88(11):3563-3567.
Wang J,Liu R,Qi H,et al.2015.The ACE2-angiotensin-(1-7)-Mas axis protects against pancreatic cell damage in cell culture[J].Pancreas,44(2):266-272.
Wang K,Liu X,Xiao H,et al.2017.The correlation between inflammatory injury induced by LPS and RAS in EpH4-Ev cells[J].International Immunopharmacology,46:23-30.
Wong M H,Chapin O C,Johnson M D.2012.LPS-stimulated cytokine production in typeⅠcells is modulated by the Renin-Angiotensin system[J].American Journal of Respiratory Cell and Molecular Biology,46(5):641-650.
Wong T P,Ho K Y,Ng E K,et al.2012.Upregulation of ACE2-ANG-(1-7)-Mas axis in jejunal enterocytes of type 1 diabetic rats:Implications for glucose transport[J].American Journal of Physiology-Endocrinology and Metabolism,303(5):E669-E681.
W?sten-van Asperen R M,Lutter R,et al.2011.Acute respiratory distress syndrome leads to reduced ratio of ACE/ACE2 activities and is prevented by angiotensin-(1-7)or an angiotensin II receptor antagonist[J].Journal of Pathology,225(4):618-627.
Wright A V,Nu?ez J K,Doudna J A,et al.2016.Biology and applications of CRISPR systems:Harnessing nature's toolbox for genome engineering[J].Cell,164(1-2):29-44.
Yang C,Yang X,Lackeyram D,et al.2016.Expression of apical Na+-l-glutamine co-transport activity,B0-system neutral amino acid co-transporter(B0AT1)and angiotensinconverting enzyme 2 along the jejunal crypt-villus axis in young pigs fed a liquid formula[J].Amino Acids,48(6):1491-1508.
Zhang J,Dong J,Martin M,et al.2018.AMPK phosphorylation of ACE2 in endothelium mitigates pulmonary hypertension[J].American Journal of Respiratory and Critical Care Medicine,198(4):509-520.
王鲲.2017.血管紧张素转化酶2在LPS诱导的小鼠乳腺炎症损伤中保护作用及其分子机制[D].博士学位论文,南京农业大学,导师:张源淑,pp.73-76.(Wang K.2017.Protective effect of angiotensin-converting enzyme 2 on LPS-induced mammary gland inflammatory injury in mice and its molecular mechanism[D].Thesis for Ph.D.,Nanjing Agricultural University,Supervisor:Zhang Y S,pp.73-76.)
杨维维,许媛媛,荣超,等.2015.山羊血管紧张素转换酶2基因的克隆表达与生物信息学分析[J].南京农业大学学报,38(1):120-126.(Yang W W,Xu Y Y,Rong C,et al.2015.Cloning,expression and bioinformatics analysis of goat angiotensin converting enzyme 2 gene[J].Journal of Nanjing Agricultural University,38(1):120-126.)
喻江标,吴永港,张银妆,等.2018.ACE2-Ang(1-7)-Mas受体轴在高血压射血分数保留心力衰竭中的作用[J].中南大学学报(医学版),43(7):738-746.(Yu J B,Wu YG,Zhang Y Z,et al.2018.Role of ACE2-Ang(1-7)-Mas receptor axis in heart failure with preserved ejection fraction with hypertension[J].Journal of Central South University(Medical Sciences),43(7):738-746.)
张伟,苗晋锋,王艳霞,等.2012.苏钟猪ACE2基因克隆及其遗传进化分析[J].江蘇農業學報,28(1):90-95.(Zhang W,Miao J F,Wang Y X,et al.2012.Cloning and genetic evolution analysis of ACE2 gene of Suzhong pig[J].Journal of Jiangsu Agricultural Sciences,28(1):86-91.)
王珊珊,黄瑜,张树坤,等.2013.血管紧张素转化酶2(ACE2)的基因克隆及在仔猪体内的表达分布[J].中国兽医学报,33(10):1579-1583.(Wang S S,Huang Y,Zhang S K,et al.2013.Gene cloning of angiotensin converting enzyme 2(ACE2)and its expression distribution in piglets[J].Chinese Journal of Veterinary Science.33(10):1579-1583.)
Bae E H,Konvalinka A,Fang F,et al.2015.Characterization of the intrarenal renin-angiotensin system in experimental alport syndrome[J].The American Journal of Pathology,185(5):1423-1435.
Bae E H,Fang F,Williams V R,et al.2017.Murine recombinant angiotensin-converting enzyme 2 attenuates kidney injury in experimental Alport syndrome[J].Kidney International,91(6):1347-1361.
Clotet-Freixas S,Soler M J,Palau V,et al.2018.Sex dimorphism in ANGⅡ-mediated crosstalk between ACE2 and ACE in diabetic nephropathy[J].Laboratory Investigation,98:1237-1249.
Cong L,Ran F A,Cox D,et al.2013.Multiplex genome engineering using CRISPR/Cas systems[J].Science,399(6121):819-823.
Donoghue M,Hsieh F,Baronas E,et al.2000.A novel angiotensin-converting enzyme-related carboxypeptidase(ACE2)converts angiotensinⅠto angiotensin 1-9[J].Circulation Research,87(5):e1-e9.
Driehuis E,Clevers H.2017.CRISPR/Cas 9 genome editing and its applications in organoids[J].American Journal of Physiology-Gastrointestinal and Liver Physiology,312(3):G257-G265.
Gootenberg J S,Abudayyeh O O,Lee J W,et al.2017.Nucleic acid detection with CRISPR-Cas13a/C2c2[J].Science,356(6336):438-442.
Harhay G P,Sonstegard T S,Keele J W,et al.2005.Characterization of 954 bovine full-CDS cDNA sequences[J].BMC Genomics,6(1):166.
Hashimoto T,Perlot T,Rehman A,et al.2012.ACE2 links amino acid malnutrition to microbial ecology and intestinal inflammation[J].Nature,487(7408):477-481.
Hsu P D,Lander E S,et al.2014.Development and applications of CRISPR-Cas9 for genome engineering[J].Cell,157(6):1262-1278.
Ip W K E,Hoshi N,Shouval D S,et al.2017.Anti-inflammatory effect of IL-10 mediated by metabolic reprogramming of macrophages[J].Science,356(6337):513-519.
Kittana N.2018.AngiotensinⅠconverting enzyme 2-angiotensin 1Ⅰ7/1Ⅰ9 system:Novel promising targets for heart failure treatment[J].Fundamental&Clinical Pharmacology,32(1):14-25.
Liu C,Xiao L,Li F,et al.2015.Generation of outbred Ace2knockout mice by RNA transfection of TALENs displaying colitis reminiscent pathophysiology and inflammation[J].Transgenic Research,24(3):433-446.
Lu X J,Xue H Y,Ke Z P,et al.2015.CRISPR-Cas9:A new and promising player in gene therapy[J].Journal of Medical Genetics,52(5):289-296.
Patel V B,Mori J,McLean,et al.2016.ACE2 deficiency worsens epicardial adipose tissue inflammation and cardiac dysfunction in response to diet-induced obesity[J].Diabetes,65(1):85-95.
Singer D,Camargo S M,Ramadan,et al.2012.Defective intestinal amino acid absorption in Ace2 null mice[J].American Journal of Physiology-Gastrointestinal and Liver Physiology,303(6):G686-G695.
Tipnis S R,Hooper N M,Hyde R,et al.2000.A human homolog of angiotensin converting enzyme-cloning and functional expression as a captopril insensitive carboxypeptidase[J].Journal of Biological Chemistry,275(43):33238-33243.
Tseng Y W,Wang,P H,et al.2010.Regulation of the expression of angiotensin-converting enzyme 2 by polyunsaturated fatty acids in porcine adipocytes[J].Journal of Animal Science,88(11):3563-3567.
Wang J,Liu R,Qi H,et al.2015.The ACE2-angiotensin-(1-7)-Mas axis protects against pancreatic cell damage in cell culture[J].Pancreas,44(2):266-272.
Wang K,Liu X,Xiao H,et al.2017.The correlation between inflammatory injury induced by LPS and RAS in EpH4-Ev cells[J].International Immunopharmacology,46:23-30.
Wong M H,Chapin O C,Johnson M D.2012.LPS-stimulated cytokine production in typeⅠcells is modulated by the Renin-Angiotensin system[J].American Journal of Respiratory Cell and Molecular Biology,46(5):641-650.
Wong T P,Ho K Y,Ng E K,et al.2012.Upregulation of ACE2-ANG-(1-7)-Mas axis in jejunal enterocytes of type 1 diabetic rats:Implications for glucose transport[J].American Journal of Physiology-Endocrinology and Metabolism,303(5):E669-E681.
W?sten-van Asperen R M,Lutter R,et al.2011.Acute respiratory distress syndrome leads to reduced ratio of ACE/ACE2 activities and is prevented by angiotensin-(1-7)or an angiotensin II receptor antagonist[J].Journal of Pathology,225(4):618-627.
Wright A V,Nu?ez J K,Doudna J A,et al.2016.Biology and applications of CRISPR systems:Harnessing nature's toolbox for genome engineering[J].Cell,164(1-2):29-44.
Yang C,Yang X,Lackeyram D,et al.2016.Expression of apical Na+-l-glutamine co-transport activity,B0-system neutral amino acid co-transporter(B0AT1)and angiotensinconverting enzyme 2 along the jejunal crypt-villus axis in young pigs fed a liquid formula[J].Amino Acids,48(6):1491-1508.
Zhang J,Dong J,Martin M,et al.2018.AMPK phosphorylation of ACE2 in endothelium mitigates pulmonary hypertension[J].American Journal of Respiratory and Critical Care Medicine,198(4):509-520.