慢病毒介导Notch1基因shRNA沉默大鼠骨髓间充质干细胞Notch信号通路的表达
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摘要
背景:对于骨髓间充质干细胞向心肌细胞分化中Notch信号通路的机制研究较多,但是沉默Notch信号通路在工程化心肌样组织血管网络中的机制研究还比较少。目的:构建慢病毒载体介导Notch1基因sh RNA表达体系,观察骨髓间充质干细胞中r Notch基因沉默效果。方法:构建p LV[shRNA]-EGFP:T2A:Puro-U6]{rNotch1[shRNA]_19 nt}(PLV-Notch)和p LV[sh RNA]-EGFP:T2A:Puro-U6>Scramble_shRNA(PLV-Scramble)拼装慢病毒表达载体,并将该慢病毒载体包装质粒形成混合物共转染到HEK-293T细胞中。收集慢病毒悬液,通过定量PCR检测重组病毒滴度。将构建的PLV-Notch与PLV-Scramble分别感染骨髓间充质干细胞,经荧光显微镜观察和RT-qPCR、Western blot鉴定转染后两组骨髓间充质干细胞中增强型绿色荧光蛋白基因和目的基因rNotch1表达情况。结果与结论:慢病毒载体滴度大于1×10~8 TU?mL,稳定转染筛选得到成熟的细胞株。病毒转导效果良好,荧光率达80%,rNotch1基因在干扰组中的表达量是scrambled对照组的75.2%。由此可见,慢病毒介导转染rNotch1基因的骨髓间充质干细胞稳转株构建成功,并对Notch信号通路的表达有沉默作用。
BACKGROUND: Number studies have insight into the mechanism of Notch signaling pathway in the differentiation of bone marrow mesenchymal stem cells into cardiomyocytes, but the mechanism of silencing Notch signaling pathway in the engineered myocardial-like tissue vascular network is less elucidated.OBJECTIVE: To investigate the role of Notch signaling pathway in bone marrow mesenchymal stem cells in the construction of engineered myocardial-like tissue vascular network.METHODS: The lentivirus expression vector was constructed by pLV[shRNA]-EGFP: T2A: Puro-U6] {rNotch1 [shRNA]_19 nt}(PLV-Notch)and pLV[shRNA]-EGFP: T2A: Puro-U6>Scramble_shRNA(PLV-Scramble) and packaged with the lentivirus vector. The mixture was co-transfected into HEK-293 T cells. The lentivirus suspension was collected and the titer of recombinant virus was detected by quantitative PCR. The constructed PLV-Notch and PLV-Scramble were infected with bone marrow mesenchymal stem cells respectively. The expression of enhanced green fluorescent protein gene and target gene rNotch1 in the transfected cells was identified by fluorescence microscopy,RT-qPCR and western blot.RESULTS AND CONCLUSION: The titer of lentivirus vector was more than 1×10~8 TU/mL. The mature cell lines were screened. The fluorescence rate of the two transfection groups was 80%, and the expression of rNotch1 gene in the interference group was 75.2% of that in the scrambled group. Therefore, lentiviral-mediated rNotch1 gene transfection of bone marrow mesenchymal stem cells can be successfully constructed and silence the expression of Notch signal pathway.
BACKGROUND: Number studies have insight into the mechanism of Notch signaling pathway in the differentiation of bone marrow mesenchymal stem cells into cardiomyocytes, but the mechanism of silencing Notch signaling pathway in the engineered myocardial-like tissue vascular network is less elucidated.OBJECTIVE: To investigate the role of Notch signaling pathway in bone marrow mesenchymal stem cells in the construction of engineered myocardial-like tissue vascular network.METHODS: The lentivirus expression vector was constructed by pLV[shRNA]-EGFP: T2A: Puro-U6] {rNotch1 [shRNA]_19 nt}(PLV-Notch)and pLV[shRNA]-EGFP: T2A: Puro-U6>Scramble_shRNA(PLV-Scramble) and packaged with the lentivirus vector. The mixture was co-transfected into HEK-293 T cells. The lentivirus suspension was collected and the titer of recombinant virus was detected by quantitative PCR. The constructed PLV-Notch and PLV-Scramble were infected with bone marrow mesenchymal stem cells respectively. The expression of enhanced green fluorescent protein gene and target gene rNotch1 in the transfected cells was identified by fluorescence microscopy,RT-qPCR and western blot.RESULTS AND CONCLUSION: The titer of lentivirus vector was more than 1×10~8 TU/mL. The mature cell lines were screened. The fluorescence rate of the two transfection groups was 80%, and the expression of rNotch1 gene in the interference group was 75.2% of that in the scrambled group. Therefore, lentiviral-mediated rNotch1 gene transfection of bone marrow mesenchymal stem cells can be successfully constructed and silence the expression of Notch signal pathway.
引文
[1]Zheng YH,Xiong W,Su K,et al.Multilineage differentiation of human bone marrow mesenchymal stem cells in vitro and in vivo.Exp Ther Med.2013;5(6):1576-1580.
[2]Wang K,Ding R,Ha Y,et al.Hypoxia-stressed cardiomyocytes promote early cardiac differentiation of cardiac stem cells through HIF-1α/Jagged1/Notch1 signaling.Acta Pharm Sin B.2018;8(5):795-804.
[3]Chen XQ,Chen LL,Fan L,et al.Stem cells with FGF4-bFGFfused gene enhances the expression of bFGF and improves myocardial repair in rats.Biochem Biophys Res Commun.2014;447(1):145-151.
[4]Boopathy AV,Pendergrass KD,Che PL,et al.Oxidative stress-induced Notch1 signaling promotes cardiogenic gene expression in mesenchymal stem cells.Stem Cell Res Ther.2013;4(2):43.
[5]Sun X,Altalhi W,Nunes SS.Vascularization strategies of engineered tissues and their application in cardiac regeneration.Adv Drug Deliv Rev.2016;96:183-194.
[6]Luxán G,Casanova JC,Martínez-Poveda B,et al.Mutations in the NOTCH pathway regulator MIB1 cause left ventricular noncompaction cardiomyopathy.Nat Med.2013;19(2):193-201.
[7]Polisetti N,Chaitanya VG,Babu PP,et al.Isolation,characterization and differentiation potential of rat bone marrow stromal cells.Neurol India.2010;58(2):201-208.
[8]刘艳云.PTH-FC对大鼠骨髓间充质干细胞增殖及骨向分化的影响[D].兰州:兰州大学,2018.
[9]蒋星海,赵彪,吴凯,等.血管内皮生长因子165、神经营养因子3、血管生成素1基因转染诱导骨髓间充质干细胞向神经元及血管内皮细胞分化[J].中国组织工程研究,2018,22(25):3956-3962.
[10]Li J,Chen SY,Zhao XY,et al.Rat Limbal Niche Cells Prevent Epithelial Stem/Progenitor Cells From Differentiation and Proliferation by Inhibiting Notch Signaling Pathway In Vitro.Invest Ophthalmol Vis Sci.2017;58(7):2968-2976.
[11]Shi J,Xing W,Yang J,et al.Comparison and Mechanism Research of the Differentiation of Bone Mesenchymal Stem Cells Into Cardiomyocytes Induced by 5-Azacytidine or Angiotensin II.J Biomat Tissue Eng.2015;5(5):364-371.
[12]James AW.Review of Signaling Pathways Governing MSCOsteogenic and Adipogenic Differentiation.Scientifica(Cairo).2013;2013:684736.
[13]杜红阳,李东宁,付海燕,等.Notch1(NICD)过表达真核载体构建及对大鼠BMSCs增殖分化的影响[J].天津医药,2014,42(9):883-888.
[14]Radtke F,Schweisguth F,Pear W.The Notch'gospel'.EMBORep.2005;6(12):1120-1125.
[15]张梦迪,路仲达,何易,等.Notch信号通路在骨髓间充质干细胞分化为心肌细胞过程中的作用[J].天津药学,2016,28(1):66-69.
[16]Novina CD,Sharp PA.The RNAi revolution.Nature.2004;430(6996):161-164.
[17]邓海燕,曾俊义,魏云峰,等.microRNA-1诱导大鼠骨髓间充质干细胞向心肌样细胞分化过程中Notch信号分子的表达变化[J].基础医学与临床,2014,34(9):1204-1210.
[18]靳俊杰,安静,曹涤非,等.组蛋白赖氨酸转移酶KAT6B基因shRNA慢病毒载体的构建与鉴定[J].实用肿瘤学杂志,2018,32(1):1-6.
[19]郑介柏,刘旭良,陈文雄,等.DAPT阻断Notch信号通路抑制去势大鼠骨髓间充质干细胞增殖及诱导其成骨分化的作用研究[J].河北医学,2015,21(1):9-12.
[20]何佳,邓峰美,林友胜,等.网素蛋白1和增强绿色荧光蛋白基因共表达慢病毒载体构建和鉴定及在结直肠癌细胞SW480中表达[J].中国老年学杂志,2018,38(2):302-305.
[21]廖凤玲,陈日玲,姜杉,等.Notch信号通路对VEGF促大鼠间充质干细胞增殖的作用[J].中国实验血液学杂志,2014,22(4):1068-1071.
[22]张冬,于占革.Notch信号通路与骨髓间充质干细胞分化的研究进展[J].医学综述,2013,19(22):4065-4067.
[23]任少达.Notch信号通路在骨髓来源的间充质干细胞诱导调节性DC生成中的作用及分子机制的研究[D].北京:北京协和医学院,2015.
[24]王继荣,卢艳,岳影星,等.Notch信号在脂多糖诱导的骨髓间充质干细胞中的作用[J].中国病理生理杂志,2018,34(2):308-313.
[2]Wang K,Ding R,Ha Y,et al.Hypoxia-stressed cardiomyocytes promote early cardiac differentiation of cardiac stem cells through HIF-1α/Jagged1/Notch1 signaling.Acta Pharm Sin B.2018;8(5):795-804.
[3]Chen XQ,Chen LL,Fan L,et al.Stem cells with FGF4-bFGFfused gene enhances the expression of bFGF and improves myocardial repair in rats.Biochem Biophys Res Commun.2014;447(1):145-151.
[4]Boopathy AV,Pendergrass KD,Che PL,et al.Oxidative stress-induced Notch1 signaling promotes cardiogenic gene expression in mesenchymal stem cells.Stem Cell Res Ther.2013;4(2):43.
[5]Sun X,Altalhi W,Nunes SS.Vascularization strategies of engineered tissues and their application in cardiac regeneration.Adv Drug Deliv Rev.2016;96:183-194.
[6]Luxán G,Casanova JC,Martínez-Poveda B,et al.Mutations in the NOTCH pathway regulator MIB1 cause left ventricular noncompaction cardiomyopathy.Nat Med.2013;19(2):193-201.
[7]Polisetti N,Chaitanya VG,Babu PP,et al.Isolation,characterization and differentiation potential of rat bone marrow stromal cells.Neurol India.2010;58(2):201-208.
[8]刘艳云.PTH-FC对大鼠骨髓间充质干细胞增殖及骨向分化的影响[D].兰州:兰州大学,2018.
[9]蒋星海,赵彪,吴凯,等.血管内皮生长因子165、神经营养因子3、血管生成素1基因转染诱导骨髓间充质干细胞向神经元及血管内皮细胞分化[J].中国组织工程研究,2018,22(25):3956-3962.
[10]Li J,Chen SY,Zhao XY,et al.Rat Limbal Niche Cells Prevent Epithelial Stem/Progenitor Cells From Differentiation and Proliferation by Inhibiting Notch Signaling Pathway In Vitro.Invest Ophthalmol Vis Sci.2017;58(7):2968-2976.
[11]Shi J,Xing W,Yang J,et al.Comparison and Mechanism Research of the Differentiation of Bone Mesenchymal Stem Cells Into Cardiomyocytes Induced by 5-Azacytidine or Angiotensin II.J Biomat Tissue Eng.2015;5(5):364-371.
[12]James AW.Review of Signaling Pathways Governing MSCOsteogenic and Adipogenic Differentiation.Scientifica(Cairo).2013;2013:684736.
[13]杜红阳,李东宁,付海燕,等.Notch1(NICD)过表达真核载体构建及对大鼠BMSCs增殖分化的影响[J].天津医药,2014,42(9):883-888.
[14]Radtke F,Schweisguth F,Pear W.The Notch'gospel'.EMBORep.2005;6(12):1120-1125.
[15]张梦迪,路仲达,何易,等.Notch信号通路在骨髓间充质干细胞分化为心肌细胞过程中的作用[J].天津药学,2016,28(1):66-69.
[16]Novina CD,Sharp PA.The RNAi revolution.Nature.2004;430(6996):161-164.
[17]邓海燕,曾俊义,魏云峰,等.microRNA-1诱导大鼠骨髓间充质干细胞向心肌样细胞分化过程中Notch信号分子的表达变化[J].基础医学与临床,2014,34(9):1204-1210.
[18]靳俊杰,安静,曹涤非,等.组蛋白赖氨酸转移酶KAT6B基因shRNA慢病毒载体的构建与鉴定[J].实用肿瘤学杂志,2018,32(1):1-6.
[19]郑介柏,刘旭良,陈文雄,等.DAPT阻断Notch信号通路抑制去势大鼠骨髓间充质干细胞增殖及诱导其成骨分化的作用研究[J].河北医学,2015,21(1):9-12.
[20]何佳,邓峰美,林友胜,等.网素蛋白1和增强绿色荧光蛋白基因共表达慢病毒载体构建和鉴定及在结直肠癌细胞SW480中表达[J].中国老年学杂志,2018,38(2):302-305.
[21]廖凤玲,陈日玲,姜杉,等.Notch信号通路对VEGF促大鼠间充质干细胞增殖的作用[J].中国实验血液学杂志,2014,22(4):1068-1071.
[22]张冬,于占革.Notch信号通路与骨髓间充质干细胞分化的研究进展[J].医学综述,2013,19(22):4065-4067.
[23]任少达.Notch信号通路在骨髓来源的间充质干细胞诱导调节性DC生成中的作用及分子机制的研究[D].北京:北京协和医学院,2015.
[24]王继荣,卢艳,岳影星,等.Notch信号在脂多糖诱导的骨髓间充质干细胞中的作用[J].中国病理生理杂志,2018,34(2):308-313.
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