Establishment of L929 Cell Line Overexpressing Nfe2l2 Gene Using Lentiviral Vectors：an Experimental Study

doi:10.12114/j.issn.1007-9572.2018.00.215

Abstract

Abstract: Objective　To establish a L929 cell line overexpressing Nfe2l2 gene using lentiviral vectors，laying a basis for investigating the role of Nfe2l2 gene in ECM remodeling.Methods　From December 2014 to May 2015，Nfe2l2 gene sequence was ligated into the GV341 vector （Ubi-MCS-3FLAG-SV40-puromycin）via restriction enzyme digestion.After amplification，transformation，verification，sequencing and plasmid extraction，the GV341 vector plasmids carrying the target gene，together with lentiviral packaging plasmids Helper 1.0 and Helper 2.0 were used to co-transfect 293T cells，and the lentiviral vector LV-Nfe2l2 was harvested after packaging and virus concentration test.After being infected by LV-Nfe2l2 for 72 h，the L929 cells were screened for stable L929/LV-Nfe2l2 cell lines.The expression of Nfe2l2 was determined by real-time qPCR.Results Sequence analysis showed that the positive clone of LV-Nfe2l2 was consistent with the target gene Nfe2l2.The optimal conditions for L929 cells to achieve 80% infectious efficiency are as follows：ENi.S medium，MOI of 8-10，infection time of 72 h.Real-time qPCR showed that the abundance of Nfe2l2 gene in L929/ LV-Nfe2l2 cells was high（ΔCt ≤12）．Conclusion In this study，we successfully established a lentiviral vector overexpressing Nfe2l2 gene；target genes with high expression levels could be obtained by LV-Nfe2l2 infected L929 cells；the screened L929/LV-Nfe2l2 cell line can be used in further experimental studies.

Key words: Transfection, Lentivirus, Nfe2l2, Fibroblasts, Extracellular matrix

摘要： 目的　利用慢病毒载体建立过表达Nfe2l2基因的L929细胞株，为探讨Nfe2l2基因在结缔组织成纤维细胞细胞外基质（ECM）重构中的作用奠定实验基础。方法　2014年12月—2015年5月，通过双酶切将Nfe2l2目的基因连接到GV341载体（Ubi-MCS-3FLAG-SV40-puromycin），经扩增、转化、验证、测序、质粒抽提，将携带目的基因的工具GV341载体质粒及病毒包装辅助质粒Helper1.0、Helper2.0转染293T细胞，完成慢病毒包装及质量检测后获取慢病毒工具载体LV-Nfe2l2，病毒感染L929细胞72 h后筛选稳定表达的细胞株L929/LV-Nfe2l2，采用Real-time qPCR测定Nfe2l2的表达。结果　经比对，构建的LV-Nfe2l2阳性克隆序列与目的基因Nfe2l2相符；L929细胞达到80%感染效率的最佳感染条件为：在ENi.S培养基中进行感染，设定感染复数（MOI）为8~10，感染时间为72 h。Real-time qPCR结果显示，经筛选的L929/LV-Nfe2l2细胞中Nfe2l2基因表达丰度为高（ΔCt值≤12）。结论　本研究成功构建了一种过表达Nfe2l2基因的慢病毒载体；LV-Nfe2l2感染L929细胞后可实现目的基因的高表达，经筛选的L929/LV-Nfe2l2细胞株可用于后续实验研究。

关键词: 转染, 慢病毒属, Nfe2l2, 成纤维细胞, 细胞外基质

LIU Cheng,TANG Jianming,LI Yang, et al. Establishment of L929 Cell Line Overexpressing Nfe2l2 Gene Using Lentiviral Vectors：an Experimental Study　[J]. Chinese General Practice, 2018, 21(30): 3723-3729. DOI: 10.12114/j.issn.1007-9572.2018.00.215.
刘成,汤剑明,李洋等. 利用慢病毒载体建立过表达Nfe2l2基因的L929细胞株实验研究[J]. 中国全科医学, 2018, 21(30): 3723-3729. DOI: 10.12114/j.issn.1007-9572.2018.00.215.

References

［1］SAMPSON N，BERGER P，ZENZMAIER C.Redox signaling as a therapeutic target to inhibit myofibroblast activation in degenerative fibrotic disease［J］．Bio Med Res Int，2014，2014：131737．DOI：10.1155/2014/131737.3729
［2］SAMARAKOON R，OVERSTREET J M，HIGGINS P J.TGF-β signaling in tissue fibrosis：Redox controls，target genes and therapeutic opportunities［J］．Cell Signal，2013，25（1）：264-268．DOI：10.1016/j.cellsig.2012.10.003.3729
［3］HYBERTSON B M，GAO B，BOSE S K，et al．Oxidative stress in health and disease：the therapeutic potential of Nrf2 activation［J］．Mol Aspects Med，2011，32（4/6）：234-246．DOI：10.1016/j.mam.2011.10.006.3729
［4］CHUNG S，YOON H E，KIM S J，et al．Oleanolic acid attenuates renal fibrosis in mice with unilateral ureteral obstruction via facilitating nuclear translocation of Nrf2［J］．Nutr Metab（Lond），2014，11
（1）：2．DOI：10.1186/1743-7075-11-2.3729
［5］HUANG K，HUANG J，XIE X，et al．Sirt1 resists advanced glycation end products-induced expressions of fibronectin and TGF-beta1 by activating the Nrf2/ARE pathway in glomerular mesangial cells［J］．Free Radic Biol Med，2013，65：528-540．DOI：10.1016/j.freeradbiomed.2013.07.029.3729
［6］BOUTTEN A，GOVEN D，ARTAUD-MACARI E，et al．NRF2 targeting：a promising therapeutic strategy in chronic obstructive pulmonary disease［J］．Trends Mol Med，2011，17（7）：363-371．DOI：10.1016/j.molmed.2011.02.006.3729
［7］KOHLER U A，KURINNA S，SCHWITTER D，et al．Activated Nrf2 impairs liver regeneration in mice by activation of genes involved in cell-cycle control and apoptosis［J］．Hepatology，2014，60（2）：670-678．DOI：10.1002/hep.26964.3729
［8］KONSTANTINOPOULOS P A，SPENTZOS D，FOUNTZILAS E，
et al．Keap1 mutations and Nrf2 pathway activation in epithelial ovarian cancer［J］．Cancer Res，2011，71（15）：5081-5089．DOI：10.1158/0008-5472.CAN-10-4668.3729
［9］SPORN M B，LIBY K T.NRF2 and cancer：the good，the bad and the importance of context［J］．Nat Rev Cancer，2012，12（8）：564-571．DOI：10.1038/nrc3278.3729
［10］LIU C，WANG Y，LI B S，et al．Role of transforming growth factor beta1 in the pathogenesis of pelvic organ prolapse：a potential therapeutic target［J］．Int J Mol Med，2017，40（2）：347-356．DOI：10.3892/ijmm.2017.3042.3729
［11］CHEN B，YEH J.Alterations in connective tissue metabolism in stress incontinence and prolapse［J］．J Urol，2011，186（5）：1768-1772．DOI：10.1016/j.juro.2011.06.054.3729
［12］CHEN H，LIN Y，CHEN Y，et al．Stress urinary incontinence following vaginal trauma involves remodeling of urethral connective tissue in female mice［J］．Eur J Obstet Gynecol Reprod Biol，2012，163（2）：224-229．DOI：10.1016/j.ejogrb.2012.04.012.3729
［13］KIM E J，CHUNG N，PARK S H，et al．Involvement of oxidative stress and mitochondrial apoptosis in the pathogenesis of pelvic organ prolapse［J］．J Urol，2013，189（2）：588-594．DOI：10.1016/j.juro.2012.09.041.3729
［14］LU D，KASSAB G S.Role of shear stress and stretch in vascular mechanobiology［J］．J R Soc Interface，2011，8（63）：1379-1385．DOI：10.1098/rsif.2011.0177.3729
［15］洪莎莎，丁文娟，吴德斌，等．人子宫旁韧带成纤维细胞力作用下氧化损伤的实验研究［J］．中华临床医师杂志（电子版），2013，7（23）：10775-10779．DOI：10.3877/cma.j.issn.1674-0785.2013.23.079.3729
HONG S S，DING W J，WU D B，et al．Effects of oxidative damage in human parametrial ligament fibroblasts induced by mechanical stress［J］．Chinese Journal of Clinicians（Electronic Edition），2013，7（23）：10775-10779．DOI：10.3877/cma.j.issn.1674-0785.2013.23.079.3729
［16］BRYAN H K，OLAYANJU A，GOLDRING C E，et al．The Nrf2 cell defence pathway：Keap1-dependent and -independent mechanisms of regulation［J］．Biochem Pharmacol，2013，85（6）：705-717．DOI：10.1016/j.bcp.2012.11.016.3729
［17］KWAK M K，WAKABAYASHI N，KENSLER T W.Chemoprevention through the Keap1-Nrf2 signaling pathway by phase 2 enzyme inducers［J］．Mutat Res，2004，555（1/2）：133-148．DOI：10.1016/j.mrfmmm.2004.06.041.3729
［18］SUZUKI T，MOTOHASHI H，YAMAMOTO M.Toward clinical application of the Keap1-Nrf2 pathway［J］．Trends Pharmacol Sci，2013，34（6）：340-346．DOI：10.1016/j.tips.2013.04.005.3729
［19］HYBERTSON B M，GAO B，BOSE S K，et al．Oxidative stress in health and disease：the therapeutic potential of Nrf2 activation［J］．Mol Aspects Med，2011，32（4/6）：234-246．DOI：10.1016/j.mam.2011.10.006.3729
［20］ZHANG D D.Mechanistic studies of the Nrf2-keap1 signaling pathway［J］．Drug Metab Rev，2015，38（4）：769-789．DOI：10.1080/03602530600971974.3729
［21］HONG S，LI H，WU D，et al．Oxidative damage to human parametrial ligament fibroblasts induced by mechanical stress［J］．Mol Med Rep，2015，12（4）：5342-5348．DOI：10.3892/mmr.2015.4115.3729
［22］LI B S，GUO W J，HONG L，et al．Role of mechanical strain-activated PI3K/Akt signaling pathway in pelvic organ prolapse［J］．Mol Med Rep，2016，14（1）：243-253．DOI：10.3892/mmr.2015.4115.3729
［23］LIU C，YANG Q，FANG G，et al．Collagen metabolic disorder induced by oxidative stress in human uterosacral ligamentderived fibroblasts：a possible pathophysiological mechanism in pelvic organ prolapse［J］．Mol Med Rep，2016，13（4）：2999-3008．DOI：10.3892/mmr.2016.4919.3729
［24］索磊，杨印祥，栾佐.含人LINGO-1慢病毒干扰载体的构建与鉴定［J］．中国全科医学，2016，19（24）：2962-2966．DOI：10.3969/j.issn.1007-9572.2016.24.019.3729
SUO L，YANG Y X，LUAN Z.Construction and identification of lentiviral interference vector that including human LINGO-1［J］．Chinese General Practice，2016，19（24）：2962-2966．DOI：10.3969/j.issn.1007-9572.2016.24.019.3729

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