NGF基因重组慢病毒载体的构建及其在人脐带血间充质干细胞的表达
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摘要
目的:构建神经生长因子(NGF)的慢病毒表达载体,并观察其转染人脐带间充质干细胞后的表达情况。方法:采用实时定量PCR(RT-PCR)方法获取NGF基因编码片段,并将构建的慢病毒载体质粒与包装质粒和包膜质粒共转染293T细胞,包装生产慢病毒。应用相同滴度的慢病毒转导等量间充质干细胞(MSCs),观察转染后细胞的生长形态及生长曲线,再采用RT-PCR、Western Blot方法检测NGF m RNA、蛋白质的表达水平。结果:经PCR、酶切和测序结果证明成功构建NGF基因重组慢病毒载体。同时NGF基因重组慢病毒载体能够成功转染人脐带间充质干细胞,转染率达95.35%,转染后干细胞在NGF m RNA及蛋白质的表达方面较对照组明显升高,同时经倒置显微镜观察及生长曲线实验证实转染后干细胞的生长与对照组相比无明显差异。结论:重组NGF的慢病毒表达载体能够高效的转染人脐带间充质干细胞,基因转染后干细胞的增殖分化能力与未转染细胞差异无统计学意义,可作为一种高效的干细胞转染方法。
Objective: To investigate the effects of nerve growth factor gene transfection via a recombinant lentiviral virus vector on the proliferation and cell cycle of human umbilical cord mesenchymal stem cells cultured in vitro. Methods: The NGF gene was cloned into lentiviral shuttle plasmid, and identified by the enzyme cut assay, PCR and gene sequencing. It was then transfected into 293 T cells to construct the recombinant lentiviral vector plasmid. Cellular proliferation was determined by cell growth curve and Cell Counting Kit-8 assay. The infection efficiency of lentiviral virus transfection was detected by fluorescence microscope and the protein expression of NGF was tested by Western blot. Results: NGF gene recombinant lentiviral vector was successfully transfected into human umbilical cord mesenchymal stem cells, and the transfection rate reached 95.35%. The expression of NGF m RNA and protein in stem cells was significantly higher than that in the control group after transfection. It was confirmed by inverted microscope observation and growth curve experiment that the growth of stem cells after transfection was not significantly different from that of the control group. Conclusion:These findings suggest that the recombinant lentiviral virus-mediated nerve growth factor gene can transfect the umbilical cord mesenchymal stem cells. There was no significant difference in the proliferation and differentiation ability of stem cells after transfection with untransfected cells.
Objective: To investigate the effects of nerve growth factor gene transfection via a recombinant lentiviral virus vector on the proliferation and cell cycle of human umbilical cord mesenchymal stem cells cultured in vitro. Methods: The NGF gene was cloned into lentiviral shuttle plasmid, and identified by the enzyme cut assay, PCR and gene sequencing. It was then transfected into 293 T cells to construct the recombinant lentiviral vector plasmid. Cellular proliferation was determined by cell growth curve and Cell Counting Kit-8 assay. The infection efficiency of lentiviral virus transfection was detected by fluorescence microscope and the protein expression of NGF was tested by Western blot. Results: NGF gene recombinant lentiviral vector was successfully transfected into human umbilical cord mesenchymal stem cells, and the transfection rate reached 95.35%. The expression of NGF m RNA and protein in stem cells was significantly higher than that in the control group after transfection. It was confirmed by inverted microscope observation and growth curve experiment that the growth of stem cells after transfection was not significantly different from that of the control group. Conclusion:These findings suggest that the recombinant lentiviral virus-mediated nerve growth factor gene can transfect the umbilical cord mesenchymal stem cells. There was no significant difference in the proliferation and differentiation ability of stem cells after transfection with untransfected cells.
引文
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[25] Wakao S, Matsuse D, Dezawa M. Mesenchymal stem cells as a source of Schwann cells:their anticipated use in peripheral nerve regeneration[J]. Cells Tissues Organs, 2014, 200:31-41
[26] Gupta R, Tongers J, Losordo DW. Human studies of angiogenic gene therapy[J]. Circ Res, 2009, 105(8):724-736
[27] Sun K, Guo C, Deng HJ, et a1. Construction of lentivirus-based inhibitor of hsa-microRNA-338-3p with specific secondary structure[J].Acta Pharmacol Sin, 2013, 34(1):167-175
[28] Mitchell K E, Weiss M L, Mitchell B M, et al. Matrix cells from Wharton's jelly form neurons and glia[J]. Stem Cells, 2003, 21:50-60
[29]万世奇,张太平,王天笑.重组腺相关病毒载体表达抗表皮生长因子受体抗体对胰腺癌细胞株抑制作用的研究[J].中华消化外科杂志, 2011, 10(4):286-289
[30]李运军,马林,隋建丽,等.表皮生长因子受体反义重组腺病毒联合放射线对乳腺癌细胞的作用[J].中华放射肿瘤学杂志, 2005, 14(2):108-111
[2] Malavige LS, Jayaratne SD, Kathriarachchi ST, et a1. Erectile dysfunction among men with diabetes is strongly associated with premature ejaculation and reduced libido[J]. J Sex Med, 2008, 5(9):2125-2134
[3] Zitzmann M, Mattern A, Hanisch J, et al. IPASS:A Study on the Tolerability and Effectiveness of Injectable Testosterone Undecanoate for the Treatment of Male Hypogonadism in a Worldwide Sample of1,438 Men[J]. J Sex Med, 2012, 10:579-588
[4] Saad MA, Eid NI, Abd El-Latif HA, et al. Potential effects of yohimbine and sildenafil on erectile dysfunction in rats[J]. Eur J Pharmacol,2013, 700:127-133
[5] Zhu H, Yu WJ, Le Y, et al. High glucose levels increase the expression of neurotrophic factors associated with p-p42/p44 MAPK in Schwann cells in vitro[J]. Mol Med Report, 2012, 6:179-184
[6] Zheng B, Li T, Chen H, et al. Correlation between ficolin-3 and vascular endothelial growth factor-to-pigment epithelium-derived factor ratio in the vitreous of eyes with proliferative diabetic retinopathy[J].Am J Ophthalmol, 2011, 152:1039-1043
[7]李锐,袁慧敏.鼠神经生长因子联合甲基强的松龙治疗AON的疗效[J].国际眼科杂志, 2011, 11(12):2230-2231
[8] Kim SJ, Park SH, Sung YC, et al. Effect of mesenchymal stem cells associated to matrixen on the erectile function in the rat model with bilateral cavernous nerve crushing injury[J]. Int Braz J Urol, 2013, 38:833-841
[9] Kovanecz I, Rivera S, Nolazco G, et al. Separate or combined treatments with daily sildenafil, molsidomine, or muscle-derived stem cells prevent erectile dysfunction in a rat model of cavernosal nerve damage[J]. J Sex Med, 2012, 9:2814-2826
[10] Hakim L, Van der Aa F, Bivalacqua TJ, et al. Emerging tools for erectile dysfunction:a role for regenerative medicine[J]. Nat Rev Urol, 2012, 9:520-536
[11] Prencipe G, Minnone G, Strippoli R, et al. Nerve growth factor downregulates inflammatory response in human monocytes through TrkA[J]. J Immunol, 2014, 192:3345-3354
[12] Nossaman BD, Gur S, Kadowitz PJ. Gene and stem cell therapy in the treatment of erectile dysfunction and pulmonary hypertension; potential treatments for the common problem of endothelial dysfunction[J]. Curr Gene Ther, 2007, 7:131-153
[13] Gur S, Kadowitz PJ, Hellstrom WJ. A review of current progress in gene and stem cell therapy for erectile dysfunction[J]. Expert Opin Biol Ther, 2008, 8:1521-1538
[14] Singh A, Singh A, Sen D. Mesenchymal stem cells in cardiac regeneration:a detailed progress report of the last 6 years(2010-2015)[J].Stem Cell Res Ther, 2016, 7:82
[15] Pandey AC, Lancaster JJ, Harris DT, et al. Cellular Therapeutics for Heart Failure:Focus on Mesenchymal Stem Cells[J]. Stem Cells Int,2017, 2017:9640108
[16] Bongso A, Fong CY. The therapeutic potential, challenges and future clinical directions of stem cells from the Wharton's jelly of the human umbilical cord[J]. Stem Cell Rev, 2013, 9:226-240
[17]毛开云,范月蕾,王跃,等.间充质干细胞治疗产品开发现状与趋势[J].中国生物工程杂志, 2017, 37(10):126-135
[18] Ramanathan R, Rupert S, Selvaraj S, et al. Role of Human Wharton's Jelly Derived Mesenchymal Stem Cells(WJ-MSCs)for Rescue of d-Galactosamine Induced Acute Liver Injury in Mice[J]. J Clin Exp Hepatol, 2017, 7:205-214
[19] Troyer DL, Weiss ML. Wharton's jelly-derived cells are a primitive stromal cell population[J]. Stem Cells, 2008, 26:591-599
[20] Cooke JP, Losordo DW. Modulating the vascular response to limb ischemia:angiogenic and cell therapies[J]. Circ Res, 2015, 116:1561-1578
[21] Ding DC, Chang YH, Shyu WC, et al. Human umbilical cord mesenchymal stem cells:a new era for stem cell therapy[J]. Cell Transplant, 2015, 24:339-347
[22] Fong CY, Subramanian A, Biswas A, et al. Freezing of Fresh Wharton's Jelly From Human Umbilical Cords Yields High Post-Thaw Mesenchymal Stem Cell Numbers for Cell-Based Therapies[J]. J Cell Biochem, 2016, 117:815-827
[23] Muheremu A, Sun JG, Wang XY, et al. Combined use of Y-tube conduits with human umbilical cord stem cells for repairing nerve bifurcation defects[J]. Neural Regen Res, 2016, 11:664-669
[24] Shivakumar SB, Bharti D, Subbarao RB, et al. DMSO-and Serum-Free Cryopreservation of Wharton's Jelly Tissue Isolated From Human Umbilical Cord[J]. J Cell Biochem, 2016, 117:2397-2412
[25] Wakao S, Matsuse D, Dezawa M. Mesenchymal stem cells as a source of Schwann cells:their anticipated use in peripheral nerve regeneration[J]. Cells Tissues Organs, 2014, 200:31-41
[26] Gupta R, Tongers J, Losordo DW. Human studies of angiogenic gene therapy[J]. Circ Res, 2009, 105(8):724-736
[27] Sun K, Guo C, Deng HJ, et a1. Construction of lentivirus-based inhibitor of hsa-microRNA-338-3p with specific secondary structure[J].Acta Pharmacol Sin, 2013, 34(1):167-175
[28] Mitchell K E, Weiss M L, Mitchell B M, et al. Matrix cells from Wharton's jelly form neurons and glia[J]. Stem Cells, 2003, 21:50-60
[29]万世奇,张太平,王天笑.重组腺相关病毒载体表达抗表皮生长因子受体抗体对胰腺癌细胞株抑制作用的研究[J].中华消化外科杂志, 2011, 10(4):286-289
[30]李运军,马林,隋建丽,等.表皮生长因子受体反义重组腺病毒联合放射线对乳腺癌细胞的作用[J].中华放射肿瘤学杂志, 2005, 14(2):108-111