鼠IL-10基因腺病毒载体的构建及在骨髓间充质干细胞中的表达
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摘要
目的构建携带鼠IL-10(rIL-10)基因重组腺病毒载体,并探讨其能否在鼠骨髓间充质干细胞(MSCs)中稳定表达。方法 rIL-10引物经PCR扩增rIL-10 cDNA序列,将回收到的656 bp rIL-10 DNA片段克隆入pcDNA3.1载体中,构建pcDNA3.1-IL-10,将其与腺病毒载体共转染HEK293细胞,进行细胞内同源重组,经测序及聚合酶链反应(PCR)鉴定重组是否成功;反复冻融裂解HEK293细胞,将获得的含rIL-10基因的病毒液感染MSCs,Western blot法检测rIL-10在MSCs中的表达。结果经测序及PCR验证,rIL-10基因腺病毒载体构建成功,病毒滴度达4×109 PFU/mL。含rIL-10基因的病毒液体外感染MSCs后,可检测到IL-10的表达。结论构建重组腺病毒能够介导rIL-10基因在MSCs中的稳定表达,为下一步基因移植治疗炎症性肠病提供基础。[中国当代儿科杂志,2019,21(7):708-712]
Objective To construct the recombinant adenoviral vector carrying the rat interleukin-10(rIL-10) gene,and to investigate whether it is stably expressed in bone marrow mesenchymal stem cells. Methods The rIL-10 gene was amplified by PCR from template rIL-10 cDNA,and the recovered 656 bp rIL-10 DNA fragment was cloned into pcDNA3.1 to construct pcDNA3.1-IL-10. Then HEK293 cells were transfected with pcDNA3.1-IL-10 and adenoviral vector for homologous recombination,and sequencing and PCR were used to evaluate whether recombination was successful. HEK293 cells were lysed by repeated freeze-thaw cycles,and bone marrow mesenchymal stem cells were infected with the virus solution containing the rIL-10 gene. Western blot was used to measure the expression of rIL-10 in bone marrow mesenchymal stem cells. Results Sequencing and PCR verified that the rIL-10 adenoviral vector was successfully constructed,with a virus titer of 4×109 PFU/mL. The expression of IL-10 was detected after bone marrow mesenchymal stem cells were infected by the virus solution containing the rIL-10 gene. Conclusions The constructed rIL-10 recombinant adenovirus can mediate the stable expression of rIL-10 gene in bone marrow mesenchymal stem cells,which provides a basis for gene transplantation therapy of inflammatory bowel disease.[Chin J Contemp Pediatr,2019,21(7):708-712]
Objective To construct the recombinant adenoviral vector carrying the rat interleukin-10(rIL-10) gene,and to investigate whether it is stably expressed in bone marrow mesenchymal stem cells. Methods The rIL-10 gene was amplified by PCR from template rIL-10 cDNA,and the recovered 656 bp rIL-10 DNA fragment was cloned into pcDNA3.1 to construct pcDNA3.1-IL-10. Then HEK293 cells were transfected with pcDNA3.1-IL-10 and adenoviral vector for homologous recombination,and sequencing and PCR were used to evaluate whether recombination was successful. HEK293 cells were lysed by repeated freeze-thaw cycles,and bone marrow mesenchymal stem cells were infected with the virus solution containing the rIL-10 gene. Western blot was used to measure the expression of rIL-10 in bone marrow mesenchymal stem cells. Results Sequencing and PCR verified that the rIL-10 adenoviral vector was successfully constructed,with a virus titer of 4×109 PFU/mL. The expression of IL-10 was detected after bone marrow mesenchymal stem cells were infected by the virus solution containing the rIL-10 gene. Conclusions The constructed rIL-10 recombinant adenovirus can mediate the stable expression of rIL-10 gene in bone marrow mesenchymal stem cells,which provides a basis for gene transplantation therapy of inflammatory bowel disease.[Chin J Contemp Pediatr,2019,21(7):708-712]
引文
[1]Miheller P,Lakatos PL,Horváth G,et al.Efficacy and safety of infliximab induction therapy in Crohn's Disease in Central Europe-a Hungarian nationwide observational study[J].BMCGastroenterol,2009,9:66.
[2]Shouval DS,Biswas A,Goettel JA,et al.Interleukin-10 receptor signaling in innate immune cells regulates mucosal immune tolerance and anti-inflammatory macrophage function[J].Immunity,2014,40(5):706-719.
[3]Christodoulou K,Wiskin AE,Gibson J,et al.Next generation exome sequencing of paediatric inflammatory bowel disease patients identifies rare and novel variants in candidate genes[J].Gut,2013,62(7):977-984.
[4]Shim JO,Seo JK.Very early-onset inflammatory bowel disease(IBD)in infancy is a different disease entity from adult-onset IBD;one form of interleukin-10 receptor mutations[J].J Hum Genet,2014,59(6):337-341.
[5]Lin Z,Wang Z,Hegarty JP,et al.Genetic association and epistatic interaction of the interleukin-10 signaling pathway in pediatric inflammatory bowel disease[J].World J Gastroenterol,2017,23(27):4897-4909.
[6]Kühn R,L?hler J,Rennick D,et al.Interleukin-10-deficient mice develop chronic enterocolitis[J].Cell,1993,75(2):263-274.
[7]Shim JO,Hwang S,Yang HR,et al.Interleukin-10 receptor mutations in children with neonatal-onset Crohn’s disease and intractable ulcerating enterocolitis[J].Eur J Gastroenterol Hepatol,2013,25(10):1235-1240.
[8]Doecke JD,Simms LA,Zhao ZZ,et al.Genetic susceptibility in IBD:overlap between ulcerative colitis and Crohn’s disease[J].Inflamm Bowel Dis,2013,19(2):240-245.
[9]Arthur A,Zannettino A,Gronthos S.The therapeutic applications of multipotential mesenchymal/stromal stem cells in skeletal tissue repair[J].J Cell Physiol,2009,218(2):237-245.
[10]Sémont A,Mouiseddine M,Fran?ois A,et al.Mesenchymal stem cells improve small intestinal integrity through regulation of endogenous epithelial cell homeostasis[J].Cell Death Differ,2010,17(6):952-961.
[11]Chen QQ,Yan L,Wang CZ,et al.Mesenchymal stem cells alleviate TNBS-induced colitis by modulating inflammatory and autoimmune responses[J].World J Gastroenterol,2013,19(29):4702-4717.
[12]He XW,He XS,Lian L,et al.Systemic infusion of bone marrow-derived mesenchymal stem cells for treatment of experimental colitis in mice[J].Dig Dis Sci,2012,57(12):3136-3144.
[13]Qi Y,Jiang D,Sindrilaru A,et al.TSG-6 released from intradermally injected mesenchymal stem cells accelerates wound healing and reduces tissue fibrosis in murine fullthickness skin wounds[J].J Invest Dermatol,2014,134(2):526-537.
[14]Yang J,Liu XX,Fan H,et al.Extracellular vesicles derived from bone marrow mesenchymal stem cells protect against experimental colitis via attenuating colon inflammation,oxidative stress and apoptosis[J].PLoS One,2015,10(10):e0140551.
[15]Anderson P,Souza-Moreira L,Morell M,et al.Adiposederived mesenchymal stromal cells induce immunomodulatory macrophages which protect from experimental colitis and sepsis[J].Gut,2013,62(8):1131-1141.
[16]Yanai H,Hanauer SB.Assessing response and loss of response to biological therapies in IBD[J].Am J Gastroenterol,2011,106(4):685-698.
[17]Schreiber S,Heinig T,Thiele HG,et al.Immunoregulatory role of interleukin 10 in patients with inflammatory bowel disease[J].Gastroenterology,1995,108(5):1434-1444.
[18]Moore KW,de Waal Malefyt R,Coffman RL,et al.Interleukin-10 and the interleukin-10 receptor[J].Annu Rev Immunol,2001,19:683-765.
[19]Tilg H,van Montfrans C,van den Ende A,et al.Treatment of Crohn's disease with recombinant human interleukin 10 induces the proinflammatory cytokine interferon gamma[J].Gut,2002,50(2):191-195.
[20]Steidler L,Hans W,Schotte L,et al.Treatment of murine colitis by Lactococcus lactis secreting interleukin-10[J].Science,2000,289(5483):1352-1355.
[21]Dave M,Mehta K,Luther J,et al.Mesenchymal stem cell therapy for inflammatory bowel disease:a systematic review and meta-analysis[J].Inflamm Bowel Dis,2015,21(11):2696-2707.
[22]Niu GC,Liu L,Zheng L,et al.Mesenchymal stem cell transplantation improves chronic colitis-associated complications through inhibiting the activity of toll-like receptor-4 in mice[J].BMC Gastroenterol,2018,18(1):127.
[2]Shouval DS,Biswas A,Goettel JA,et al.Interleukin-10 receptor signaling in innate immune cells regulates mucosal immune tolerance and anti-inflammatory macrophage function[J].Immunity,2014,40(5):706-719.
[3]Christodoulou K,Wiskin AE,Gibson J,et al.Next generation exome sequencing of paediatric inflammatory bowel disease patients identifies rare and novel variants in candidate genes[J].Gut,2013,62(7):977-984.
[4]Shim JO,Seo JK.Very early-onset inflammatory bowel disease(IBD)in infancy is a different disease entity from adult-onset IBD;one form of interleukin-10 receptor mutations[J].J Hum Genet,2014,59(6):337-341.
[5]Lin Z,Wang Z,Hegarty JP,et al.Genetic association and epistatic interaction of the interleukin-10 signaling pathway in pediatric inflammatory bowel disease[J].World J Gastroenterol,2017,23(27):4897-4909.
[6]Kühn R,L?hler J,Rennick D,et al.Interleukin-10-deficient mice develop chronic enterocolitis[J].Cell,1993,75(2):263-274.
[7]Shim JO,Hwang S,Yang HR,et al.Interleukin-10 receptor mutations in children with neonatal-onset Crohn’s disease and intractable ulcerating enterocolitis[J].Eur J Gastroenterol Hepatol,2013,25(10):1235-1240.
[8]Doecke JD,Simms LA,Zhao ZZ,et al.Genetic susceptibility in IBD:overlap between ulcerative colitis and Crohn’s disease[J].Inflamm Bowel Dis,2013,19(2):240-245.
[9]Arthur A,Zannettino A,Gronthos S.The therapeutic applications of multipotential mesenchymal/stromal stem cells in skeletal tissue repair[J].J Cell Physiol,2009,218(2):237-245.
[10]Sémont A,Mouiseddine M,Fran?ois A,et al.Mesenchymal stem cells improve small intestinal integrity through regulation of endogenous epithelial cell homeostasis[J].Cell Death Differ,2010,17(6):952-961.
[11]Chen QQ,Yan L,Wang CZ,et al.Mesenchymal stem cells alleviate TNBS-induced colitis by modulating inflammatory and autoimmune responses[J].World J Gastroenterol,2013,19(29):4702-4717.
[12]He XW,He XS,Lian L,et al.Systemic infusion of bone marrow-derived mesenchymal stem cells for treatment of experimental colitis in mice[J].Dig Dis Sci,2012,57(12):3136-3144.
[13]Qi Y,Jiang D,Sindrilaru A,et al.TSG-6 released from intradermally injected mesenchymal stem cells accelerates wound healing and reduces tissue fibrosis in murine fullthickness skin wounds[J].J Invest Dermatol,2014,134(2):526-537.
[14]Yang J,Liu XX,Fan H,et al.Extracellular vesicles derived from bone marrow mesenchymal stem cells protect against experimental colitis via attenuating colon inflammation,oxidative stress and apoptosis[J].PLoS One,2015,10(10):e0140551.
[15]Anderson P,Souza-Moreira L,Morell M,et al.Adiposederived mesenchymal stromal cells induce immunomodulatory macrophages which protect from experimental colitis and sepsis[J].Gut,2013,62(8):1131-1141.
[16]Yanai H,Hanauer SB.Assessing response and loss of response to biological therapies in IBD[J].Am J Gastroenterol,2011,106(4):685-698.
[17]Schreiber S,Heinig T,Thiele HG,et al.Immunoregulatory role of interleukin 10 in patients with inflammatory bowel disease[J].Gastroenterology,1995,108(5):1434-1444.
[18]Moore KW,de Waal Malefyt R,Coffman RL,et al.Interleukin-10 and the interleukin-10 receptor[J].Annu Rev Immunol,2001,19:683-765.
[19]Tilg H,van Montfrans C,van den Ende A,et al.Treatment of Crohn's disease with recombinant human interleukin 10 induces the proinflammatory cytokine interferon gamma[J].Gut,2002,50(2):191-195.
[20]Steidler L,Hans W,Schotte L,et al.Treatment of murine colitis by Lactococcus lactis secreting interleukin-10[J].Science,2000,289(5483):1352-1355.
[21]Dave M,Mehta K,Luther J,et al.Mesenchymal stem cell therapy for inflammatory bowel disease:a systematic review and meta-analysis[J].Inflamm Bowel Dis,2015,21(11):2696-2707.
[22]Niu GC,Liu L,Zheng L,et al.Mesenchymal stem cell transplantation improves chronic colitis-associated complications through inhibiting the activity of toll-like receptor-4 in mice[J].BMC Gastroenterol,2018,18(1):127.