脂质体和慢病毒介导P75神经生长因子受体及神经生长因子转染骨髓间充质干细胞的效果比较
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摘要
背景:P75神经生长因子受体(P75 neurotrophin receptor,P75~(NTR))在不同细胞中可以介导截然不同的信号通路,但是在骨髓间充质干细胞中所传导的调节作用还尚未研究;脂质体和慢病毒介导单基因转染细胞技术已趋向成熟,但是其介导双基因转染的差异比较还未见报道。目的:构建大鼠P75~(NTR)、神经生长因子(nerve growth factor,NGF)的过表达质粒及慢病毒载体,比较脂质体和慢病毒介导2种基因转染骨髓间充质干细胞的效果和实用性,以便于今后根据不同实验要求选择实验最优方案。方法:设计大鼠P75~(NTR)及NGF的基因引物,提取基因组DNA进行PCR扩增、与质粒载体重组构建表达增强绿色荧光的GV358-P75~(NTR)、表达增强红色荧光的GV492-NGF过表达质粒,转染293T细胞,加入慢病毒载体超速离心,收集目的基因过表达慢病毒,测定病毒样品滴度;选取体外培养的大鼠原代骨髓间充质干细胞,一组用脂质体Lipo3000共转染质粒GV358-P75~(NTR)、GV492-NGF,另一组用慢病毒共感染,同时设置阴性对照组和空白对照组,对转染后的骨髓间充质干细胞常规培养,第3,5,7,9,12天用荧光显微镜观察红绿荧光的表达,Westernblot检测P75~(NTR)及NGF蛋白表达,对转染后培养至7d的骨髓间充质干细胞进行消化传代培养,培养第3,5,7,9,12天用荧光显微镜观察红绿荧光的表达以及Western blot检测P75~(NTR)、NGF蛋白的表达。结果与结论:①构建的GV358-P75~(NTR)、GV492-NGF过表达质粒及慢病毒符合实验设计;②显微镜视野中红绿色荧光的丰度:脂质体共转染组先增加后降低,慢病毒共感染组持续增多;各时间点慢病毒组均多于脂质体组;脂质体转染组传代培养细胞的荧光表达相比于未传代前降低,而慢病毒转染组未见明显变化;③各时间点慢病毒感染组的P75~(NTR)及NGF蛋白表达量明显高于脂质体组,目的基因转染组的P75~(NTR)及NGF蛋白表达量均高于阴性对照组及空白对照组,差异有显著性意义(P<0.05);④P75~(NTR)、NGF双基因均能通过脂质体和慢病毒共转染方法在大鼠骨髓间充质干细胞中过表达;脂质体双基因转染操作相对简便,实验设备依赖性低,费用相对低廉,但感染效率明显低于慢病毒,而慢病毒双基因共感染后目的基因存在着持续表达,传代后基因丢失现象不明显。
BACKGROUND: As a receptor for nerve growth factor(NGF), P75 neurotrophin receptor(P75~(NTR)) can mediate distinct signaling pathways in different cells, but the regulatory effect in bone marrow mesenchymal stem cells has not been studied yet. Liposome and lentivirus-mediated single-gene transfection cell technology have matured, but the differences in mediating double gene transfection have not been reported.OBJECTIVE: To construct the overexpression plasmid and lentiviral vector of rat P75~(NTR) and NGF, and to compare the effect and practicability of transfected bone marrow mesenchymal stem cells by lipofection and lentivirus infection in order to select the best experimental solution according to different experimental requirements in the future.METHODS: The gene primers of rat P75~(NTR) and NGF were designed. Genomic DNA was extracted for PCR amplification, and GV358-P75~(NTR) and GV492-NGF overexpression plasmids were constructed by recombinant plasmid vector, transfected into 293 T cells, and ultracentrifuged by lentiviral vector to collect target genes. The lentivirus was expressed and the titer of the virus sample was determined. The rat primary bone marrow mesenchymal stem cells were cultured in vitro, and one group was co-transfected with plasmid GV358-P75~(NTR) and GV492-NGF with liposome Lipo3000, and the other group was treated with lentivirus. Negative control group and blank control group were set at the same time.The transfected bone marrow mesenchymal stem cells were routinely cultured. The expression of red and green fluorescence was observed under fluorescence microscope on days 3, 5, 7, 9, and 12, and the expression of P75~(NTR) and NGF proteins was detected by western blot.Bone marrow mesenchymal stem cells were digested and subcultured on day 7 after transfection. The expression of red and green fluorescence was observed by fluorescence microscope on days 3, 5, 7, 9, and 12, and the expression of P75~(NTR) and NGF proteins was detected by western blot.RESULTS AND CONCLUSION: The GV358-P75~(NTR) and GV492-NGF overexpression plasmids and lentivirus were constructed in accordance with the experimental design. For the abundance of red-green fluorescence in the microscope field, the liposome co-transfection group increased at first and then decreased; the lentivirus co-transfection group continued to increase; and the lentivirus co-transfection group was higher than the liposome co-transfection group at each time point. In the liposome co-transfection group, the fluorescence expression of the subcultured cells after transfection was lower than that of the non-subcultured cells, and the lentivirus co-transfection group showed no changes. The expression of P75~(NTR) and NGF proteins in the lentiviral infection group was significantly higher than that in the liposome group at each time point. The expression levels of P75~(NTR) and NGF proteins in the two target gene transfection groups were significantly higher than those in the negative control group and blank group. Both P75~(NTR) and NGF genes could be transfected into rat bone marrow mesenchymal stem cells via liposome and lentivirus co-transfection. Compared with lentivirus-mediated double gene transfection, liposome-mediated double gene transfection is a relatively simple method that has lower dependency to experimental equipment and lower cost. However, the infection efficiency is significantly lower than that of lentivirus-mediated double gene transfection. The target gene continues to express after lentivirus-mediated double gene transfection, and no gene loss occurs after subculture.
BACKGROUND: As a receptor for nerve growth factor(NGF), P75 neurotrophin receptor(P75~(NTR)) can mediate distinct signaling pathways in different cells, but the regulatory effect in bone marrow mesenchymal stem cells has not been studied yet. Liposome and lentivirus-mediated single-gene transfection cell technology have matured, but the differences in mediating double gene transfection have not been reported.OBJECTIVE: To construct the overexpression plasmid and lentiviral vector of rat P75~(NTR) and NGF, and to compare the effect and practicability of transfected bone marrow mesenchymal stem cells by lipofection and lentivirus infection in order to select the best experimental solution according to different experimental requirements in the future.METHODS: The gene primers of rat P75~(NTR) and NGF were designed. Genomic DNA was extracted for PCR amplification, and GV358-P75~(NTR) and GV492-NGF overexpression plasmids were constructed by recombinant plasmid vector, transfected into 293 T cells, and ultracentrifuged by lentiviral vector to collect target genes. The lentivirus was expressed and the titer of the virus sample was determined. The rat primary bone marrow mesenchymal stem cells were cultured in vitro, and one group was co-transfected with plasmid GV358-P75~(NTR) and GV492-NGF with liposome Lipo3000, and the other group was treated with lentivirus. Negative control group and blank control group were set at the same time.The transfected bone marrow mesenchymal stem cells were routinely cultured. The expression of red and green fluorescence was observed under fluorescence microscope on days 3, 5, 7, 9, and 12, and the expression of P75~(NTR) and NGF proteins was detected by western blot.Bone marrow mesenchymal stem cells were digested and subcultured on day 7 after transfection. The expression of red and green fluorescence was observed by fluorescence microscope on days 3, 5, 7, 9, and 12, and the expression of P75~(NTR) and NGF proteins was detected by western blot.RESULTS AND CONCLUSION: The GV358-P75~(NTR) and GV492-NGF overexpression plasmids and lentivirus were constructed in accordance with the experimental design. For the abundance of red-green fluorescence in the microscope field, the liposome co-transfection group increased at first and then decreased; the lentivirus co-transfection group continued to increase; and the lentivirus co-transfection group was higher than the liposome co-transfection group at each time point. In the liposome co-transfection group, the fluorescence expression of the subcultured cells after transfection was lower than that of the non-subcultured cells, and the lentivirus co-transfection group showed no changes. The expression of P75~(NTR) and NGF proteins in the lentiviral infection group was significantly higher than that in the liposome group at each time point. The expression levels of P75~(NTR) and NGF proteins in the two target gene transfection groups were significantly higher than those in the negative control group and blank group. Both P75~(NTR) and NGF genes could be transfected into rat bone marrow mesenchymal stem cells via liposome and lentivirus co-transfection. Compared with lentivirus-mediated double gene transfection, liposome-mediated double gene transfection is a relatively simple method that has lower dependency to experimental equipment and lower cost. However, the infection efficiency is significantly lower than that of lentivirus-mediated double gene transfection. The target gene continues to express after lentivirus-mediated double gene transfection, and no gene loss occurs after subculture.
引文
[1]Shen LL, Ma?ucat-Tan NB, Gao SH, et al. The ProNGF/p75NTR pathway induces tau pathology and is a therapeutic target for FTLD-tau. Mol Psychiatry. 2018;23(8):1813-1824.
[2]Zhao M, Wen X, Li G, et al. The spatiotemporal expression and mineralization regulation of p75 neurotrophin receptor in the early tooth development. Cell Prolif. 2019;52(1):e12523.
[3]吴小莹,杨斌.脑源性神经营养因子与骨/牙组织发育代谢:促成或抑制细胞的增殖与分化[J].中国组织工程研究, 2015,19(2):283-288.
[4]熊静,李志伟,韩剑虹,等. proBDNF-p75NTR通路抑制C6细胞增殖[J].医学研究杂志, 2015, 44(3):89-93.
[5]Lv GH, Jiang L, Man C, et al. The effect of nerve growth factor on the expression of BMP-2 in the healing of rabbits'mandibular fracture with partial nerve injury. Lin Chung Er Bi Yan Hou Tou Jing Wai Ke Za Zhi. 2016;30(7):535-537.
[6]Su YW, Zhou XF, Foster BK, et al. Roles of neurotrophins in skeletal tissue formation and healing. J Cell Physiol. 2018;233(3):2133-2145.
[7]崔国胜,曾剑玉,张婧,等.神经生长因子对2型糖尿病小鼠骨髓基质细胞体外成骨能力的影响[J].中华口腔医学杂志, 2018, 53(2):97-102.
[8]刘建军,黄亮,韩庆斌,等.神经生长因子对大鼠胫骨骨折愈合的影响及其机制[J].山东医药,2016,56(32):27-29.
[9]刘强和,王亮亮,王翔,等.人β-NGF重组质粒转染GFP转基因小鼠BMSC的实验研究[J].重庆医学,2014,43(10):1161-1163,1167.
[10]马乐园,赵岩,乔万庆,等.创伤性脑损伤合并骨折中加速骨折愈合过程中相关因子的表达[J].中国组织工程研究,2017, 21(32):5115-5121.
[11] Becker K, Cana A, Baumg?rtner W, et al. p75 Neurotrophin Receptor:A Double-Edged Sword in Pathology and Regeneration of the Central Nervous System. Vet Pathol. 2018;55(6):786-801.
[12] Chakraborty S, Castranova V, Perez MK, et al.Nanoparticles-induced apoptosis of human airway epithelium is mediated by proNGF/p75NTR signaling. J Toxicol Environ Health A.2017;80(1):53-68.
[13] Mitsiadis TA, Pagella P. Expression of Nerve Growth Factor(NGF),TrkA, and p75(NTR)in Developing Human Fetal Teeth. Front Physiol. 2016;7:338.
[14]李俊峰,陈莲香,吕霞,等. p75NTR蛋白体外通过TrkANGFR/p75NTR异源二聚体信号途径促进L02细胞增殖[J].中华消化杂志, 2012, 32(4):256-261.
[15] Mukai J, Hachiya T, Shoji-Hoshino S, et al. NADE, a p75NTR-associated cell death executor, is involved in signal transduction mediated by the common neurotrophin receptor p75NTR. J Biol Chem. 2000;275(23):17566-17570.
[16] Hickman FE, Stanley EM, Carter BD. Neurotrophin Responsiveness of Sympathetic Neurons Is Regulated by Rapid Mobilization of the p75 Receptor to the Cell Surface through TrkA Activation of Arf6. J Neurosci. 2018;38(24):5606-5619.
[17] Stabile A, Pistilli A, Crispoltoni L, et al. A role for NGF and its receptors TrKA and p75NTR in the progression of COPD. Biol Chem. 2016;397(2):157-163.
[18] Douillard T, Martinelli-Kl?y CP, Lombardi T. Nerve Growth Factor Expression and Its Receptors TrkA and p75NTR in Peri-Implantitis Lesions. Implant Dent. 2016;25(3):373-379.
[19] Amoras Eda S, Gomes ST, Freitas FB, et al. NGF and P75NTR gene expression is associated with the hepatic fibrosis stage due to viral and non-viral causes. PLoS One. 2015;10(3):e0121754.
[20]李家勇,王铭,彭称飞,等. P75NTR在兔骨折不愈合局部组织中的表达及意义[J].中国骨质疏松杂志,2017,23(4):437-440.
[21]刘宇鹏,赵德伟,王卫明,等.神经生长因子对骨折愈合中骨形态发生蛋白表达的影响[J].中华医学杂志,2014,94(23):1825-1828.
[22] Liu Y, Zhao D, Wang W, et al. Nerve growth factor modulates bone morphogenetic protein expression in rabbit fracture. Zhonghua Yi Xue Za Zhi. 2014;94(23):1825-1828.
[23] Zhuang YF, Li J. Serum EGF and NGF levels of patients with brain injury and limb fracture. Asian Pac J Trop Med. 2013;6(5):383-386.
[24] Sang XG, Wang ZY, Cheng L, et al. Analysis of the mechanism by which nerve growth factor promotes callus formation in mice with tibial fracture. Exp Ther Med. 2017;13(4):1376-1380.
[25] Bei C, Lin Z, Yang Z, et al. Study on effect of NGF on fracture healing. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2009;23(5):570-576.
[26]吴永超,郑启新,谢宗平,等.骨髓间充质干细胞表达神经营养因子及治疗脊髓损伤的研究[J].中华实验外科杂志, 2005,22(2):139-141.
[27]卢莎,谭文杰,张玲,等.脂质体转染法与电转染法在丙型肝炎病毒RNA转染人肝癌细胞中的应用效果比较[J].山东医药, 2016,56(19):15-18.
[28]张禾璇,单可人,何燕,等.脂质体法与电穿孔法转染两种细胞效率的比较[J].重庆医学,2014,43(33):4432-4433.
[29]纪玉婷,杨燕,郑荣生,等.慢病毒介导稳定高表达Cx32的Huh7细胞系建立及其对细胞增殖的影响[J].中国药理学通报, 2018, 34(2):284-289.
[30]李诗鹏,李强,石正松,等.基因重组腺病毒载体与慢病毒载体转染兔骨髓间充质干细胞的对比[J].中国组织工程研究, 2017, 21(9):1340-1345.
[2]Zhao M, Wen X, Li G, et al. The spatiotemporal expression and mineralization regulation of p75 neurotrophin receptor in the early tooth development. Cell Prolif. 2019;52(1):e12523.
[3]吴小莹,杨斌.脑源性神经营养因子与骨/牙组织发育代谢:促成或抑制细胞的增殖与分化[J].中国组织工程研究, 2015,19(2):283-288.
[4]熊静,李志伟,韩剑虹,等. proBDNF-p75NTR通路抑制C6细胞增殖[J].医学研究杂志, 2015, 44(3):89-93.
[5]Lv GH, Jiang L, Man C, et al. The effect of nerve growth factor on the expression of BMP-2 in the healing of rabbits'mandibular fracture with partial nerve injury. Lin Chung Er Bi Yan Hou Tou Jing Wai Ke Za Zhi. 2016;30(7):535-537.
[6]Su YW, Zhou XF, Foster BK, et al. Roles of neurotrophins in skeletal tissue formation and healing. J Cell Physiol. 2018;233(3):2133-2145.
[7]崔国胜,曾剑玉,张婧,等.神经生长因子对2型糖尿病小鼠骨髓基质细胞体外成骨能力的影响[J].中华口腔医学杂志, 2018, 53(2):97-102.
[8]刘建军,黄亮,韩庆斌,等.神经生长因子对大鼠胫骨骨折愈合的影响及其机制[J].山东医药,2016,56(32):27-29.
[9]刘强和,王亮亮,王翔,等.人β-NGF重组质粒转染GFP转基因小鼠BMSC的实验研究[J].重庆医学,2014,43(10):1161-1163,1167.
[10]马乐园,赵岩,乔万庆,等.创伤性脑损伤合并骨折中加速骨折愈合过程中相关因子的表达[J].中国组织工程研究,2017, 21(32):5115-5121.
[11] Becker K, Cana A, Baumg?rtner W, et al. p75 Neurotrophin Receptor:A Double-Edged Sword in Pathology and Regeneration of the Central Nervous System. Vet Pathol. 2018;55(6):786-801.
[12] Chakraborty S, Castranova V, Perez MK, et al.Nanoparticles-induced apoptosis of human airway epithelium is mediated by proNGF/p75NTR signaling. J Toxicol Environ Health A.2017;80(1):53-68.
[13] Mitsiadis TA, Pagella P. Expression of Nerve Growth Factor(NGF),TrkA, and p75(NTR)in Developing Human Fetal Teeth. Front Physiol. 2016;7:338.
[14]李俊峰,陈莲香,吕霞,等. p75NTR蛋白体外通过TrkANGFR/p75NTR异源二聚体信号途径促进L02细胞增殖[J].中华消化杂志, 2012, 32(4):256-261.
[15] Mukai J, Hachiya T, Shoji-Hoshino S, et al. NADE, a p75NTR-associated cell death executor, is involved in signal transduction mediated by the common neurotrophin receptor p75NTR. J Biol Chem. 2000;275(23):17566-17570.
[16] Hickman FE, Stanley EM, Carter BD. Neurotrophin Responsiveness of Sympathetic Neurons Is Regulated by Rapid Mobilization of the p75 Receptor to the Cell Surface through TrkA Activation of Arf6. J Neurosci. 2018;38(24):5606-5619.
[17] Stabile A, Pistilli A, Crispoltoni L, et al. A role for NGF and its receptors TrKA and p75NTR in the progression of COPD. Biol Chem. 2016;397(2):157-163.
[18] Douillard T, Martinelli-Kl?y CP, Lombardi T. Nerve Growth Factor Expression and Its Receptors TrkA and p75NTR in Peri-Implantitis Lesions. Implant Dent. 2016;25(3):373-379.
[19] Amoras Eda S, Gomes ST, Freitas FB, et al. NGF and P75NTR gene expression is associated with the hepatic fibrosis stage due to viral and non-viral causes. PLoS One. 2015;10(3):e0121754.
[20]李家勇,王铭,彭称飞,等. P75NTR在兔骨折不愈合局部组织中的表达及意义[J].中国骨质疏松杂志,2017,23(4):437-440.
[21]刘宇鹏,赵德伟,王卫明,等.神经生长因子对骨折愈合中骨形态发生蛋白表达的影响[J].中华医学杂志,2014,94(23):1825-1828.
[22] Liu Y, Zhao D, Wang W, et al. Nerve growth factor modulates bone morphogenetic protein expression in rabbit fracture. Zhonghua Yi Xue Za Zhi. 2014;94(23):1825-1828.
[23] Zhuang YF, Li J. Serum EGF and NGF levels of patients with brain injury and limb fracture. Asian Pac J Trop Med. 2013;6(5):383-386.
[24] Sang XG, Wang ZY, Cheng L, et al. Analysis of the mechanism by which nerve growth factor promotes callus formation in mice with tibial fracture. Exp Ther Med. 2017;13(4):1376-1380.
[25] Bei C, Lin Z, Yang Z, et al. Study on effect of NGF on fracture healing. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2009;23(5):570-576.
[26]吴永超,郑启新,谢宗平,等.骨髓间充质干细胞表达神经营养因子及治疗脊髓损伤的研究[J].中华实验外科杂志, 2005,22(2):139-141.
[27]卢莎,谭文杰,张玲,等.脂质体转染法与电转染法在丙型肝炎病毒RNA转染人肝癌细胞中的应用效果比较[J].山东医药, 2016,56(19):15-18.
[28]张禾璇,单可人,何燕,等.脂质体法与电穿孔法转染两种细胞效率的比较[J].重庆医学,2014,43(33):4432-4433.
[29]纪玉婷,杨燕,郑荣生,等.慢病毒介导稳定高表达Cx32的Huh7细胞系建立及其对细胞增殖的影响[J].中国药理学通报, 2018, 34(2):284-289.
[30]李诗鹏,李强,石正松,等.基因重组腺病毒载体与慢病毒载体转染兔骨髓间充质干细胞的对比[J].中国组织工程研究, 2017, 21(9):1340-1345.