Transfection efficiency of three different lentiviruses with green fluorescent protein gene to transfect rabbit bone marrow mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2017.29.009

Abstract

Abstract:

BACKGROUND: We explore the transfection effects of different lentiviruses on bone marrow mesenchymal stem cells (BMSCs) from the same source in order to screen out appropriate cell transfection tools.
OBJECTIVE: To explore the difference in the transfection efficiency of three different lentiviruses carrying green fluorescent protein (GFP) to transfect the rabbit BMSCs.
METHODS: Bone marrow samples were extracted from the ilium of 2-week-old Japan white rabbits, and then the BMSCs were separated, purified and extended using density gradient centrifugation. Cell surface markers were identified using flow cytometry. pTomo-GFP lentivirus, Ad-GFP lentivirus and lenti-GFP lentivirus were used to transfect the BMSCs, and the transfection efficiency was tested using flow cytometry. The cell viability was detected using cell counting kit-8 before and after transfection.
RESULTS AND CONCLUSION: As the positive rates of CD44, CD34 and CD45 were 75.64%, 6.14% and 9.48%, respectively, the cultured cells were identified as BMSCs by the flow cytometry. The expression levels of GFP after transfection by pTomo-GFP, Ad-GFP and lenti-GFP were 2.64%, 16.72% and 45.24%, respectively. The cell growth curve showed that pTomo-GFP lentivirus, Ad-GFP and lenti-GFP lentivirus showed certain effects on cell growth. pTomo-GFP and Ad-GFP had an effect on cell growth at incubation period and logarithmic growth phase at 1-8 days after transfection, which may cause some interference to the viability of early cells. lenti-GFP had little effect on cell growth in the short term, but there was a change in cell growth at day 12 after lenti-GFP transfection, and a long-term effect of lenti-GFP on cell viability was considered. Therefore, lenti-GFP lentivirus could highly transfect the BMSCs and could not impact the normal growth of BMSCs, indicating that lenti-GFP lentivirus is suitable for BMSCs tracing and gene transfection.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Lentivirus, Bone Marrow, Mesenchymal Stem Cells, Green Fluorescent Proteins, Tissue Engineering

CLC Number:

R394.2

Xiao Yu, Li Yan-lin, Wang Kun, Li Long-teng, Yan Xiang-jia, Meng Xu-han. Transfection efficiency of three different lentiviruses with green fluorescent protein gene to transfect rabbit bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(29): 4642-4647.

Figures/Tables 2

2.1 骨髓间充质干细胞的分离培养结果骨髓液经Percoll离心液分离后可见浅红色上层与透明中层之间有一层薄薄的云雾状单核细胞层，骨髓间充质干细胞大部分就在这一层内。提取并接种于培养瓶内培养，3 d后在显微镜下可见有细胞贴壁，形态呈梭形，7 d可见细胞梭形生长，漩涡样分布，10 d后细胞基本铺满培养瓶底。经传代后细胞生长较快，形态均一稳定，见图1。 2.2 骨髓间充质干细胞表面标记物鉴定结果流式细胞仪检测提示第3代骨髓间充质干细胞高表达CD44，几乎不表达CD34、CD45，证实所培养细胞为骨髓间充质干细胞，见图2。 2.3 骨髓间充质干细胞分化潜能鉴定结果经成脂诱导培养，油红O染色后观察发现细胞内出现大小不等圆形红色脂滴(图3A)，证实其具有成脂分化潜能；经成骨诱导培养，茜素红染色后观察发现细胞中出现圆形红染钙结节颗粒(图3B)，证实其具有成骨分化潜能；经成软骨诱导培养，阿利新蓝染色后观察发现细胞质呈现蓝色(图3C)，证实其具有成软骨分化潜能。 2.4 三种慢病毒转染骨髓间充质干细胞效果第3代骨髓间充质干细胞分别用3种病毒转染5 d后流式细胞仪检测GFP表达情况，结果显示在MOI值为50的条件下，pTomo-GFP慢病毒转染后GFP表达率为2.64%、Ad-GFP慢病毒转染后GFP表达率为16.72%、lenti-GFP慢病毒转染后GFP的表达率为45.24%，传代后转染效果、细胞形态无明显改变，见图4。 2.5 生长曲线测定结果见图5和表1。从生长曲线可以看出，细胞接种后第1-3天处于生长潜伏期，细胞活力不高，生长缓慢；第4-8天细胞进入对数生长期，此时细胞数量增多，活力增高，铺满培养瓶底；第10-12天细胞生长进入平台期。生长曲线大致呈“S”形分布。各慢病毒在不同时期对细胞生长存在一定影响。pTomo-GFP、Ad-GFP在第1-8天对细胞生长潜伏期和对数生长期存在影响，可能对前期细胞活性造成一定干扰。短期内lenti-GFP对细胞生长影响不大，但第12天时出现了细胞生长情况变化，考虑对细胞远期活力存在影响。"

References

[1] Xu R, Xu J, Liu W. Study on the differentiation of human mesenchymal stem cells into vascular endothelial-like cells. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2014;31(2): 389-393.

[2] 王慧建,李彦林,陈建明,等, TGF-β3基因转染诱导滇南小耳猪BMSCs向软骨分化的初步研究[J].中国修复重建外科杂志, 2014, 28(2):149-154.

[3] Huang B, Tabata Y, Gao JQ. Mesenchymal stem cells as therapeutic agents and potential targeted gene delivery vehicle for brain diseases. J Control Release. 2012;162(2): 464-473.

[4] Galbraith DW, Anderson MT, Herzenberg LA. Flow cytometric analysis and FACS sorting of cells based on GFP accumulation. Methods Cell Biol. 1999;58:315-341.

[5] Kim YG, Park B, Ahn JO, et al. New cell line development for antibody-producing Chinese hamster ovary cells using split green fluorescent protein. BMC Biotechnol. 2012;12:24.

[6] 中华人民共和国科学技术部.关于善待实验动物的指导性意见. 2006-09-30.

[7] Shao J, Zhang W, Yang T. Using mesenchymal stem cells as a therapy for bone regeneration and repairing. Biol Res. 2015;48: 62.

[8] Mostafa NZ, Uluda? H, Varkey M, et al. In vitro osteogenic induction of human gingival fibroblasts for bone regeneration. Open Dent J. 2011;5:139-145.

[9] Lopes JP, Fiarresga A, Silva Cunha P, et al. Mesenchymal stem cell therapy in heart disease. Rev Port Cardiol. 2013; 32(1): 43-47.

[10] Nurkovic J, Dolicanin Z, Mustafic F, et al. Mesenchymal stem cells in regenerative rehabilitation. J Phys Ther Sci. 2016; 28(6):1943-1948.

[11] Horn P, Bork S, Wagner W. Standardized isolation of human mesenchymal stromal cells with red blood cell lysis. Methods Mol Biol. 2011;698:23-35.

[12] 武海军,银和平,李树文,等.密度梯度离心和贴壁筛选法分离培养兔骨髓间充质干细胞的形态学观察[J].中华临床医师杂志:电子版, 2012,6(22):7261-7265.

[13] Horn P, Bork S, Diehlmann A, et al. Isolation of human mesenchymal stromal cells is more efficient by red blood cell lysis. Cytotherapy. 2008;10(7):676-685.

[14] Gubin AN, Reddy B, Njoroge JM, et al. Long-term, stable expression of green fluorescent protein in mammalian cells. Biochem Biophys Res Commun. 1997;236(2):347-350.

[15] Li X, Luo Q, Sun J, et al. Conditioned medium from mesenchymal stem cells enhances the migration of hepatoma cells through CXCR4 up-regulation and F-actin remodeling. Biotechnol Lett. 2015;37(3):511-521.

[16] 贾莹,陈波.两种不同方法分离、培养大鼠骨髓基质干细胞的比较研究[J].贵州医药, 2005, 29(10): 893-895.

[17] 王亦菁,张晓东,孙淏海,等.免疫磁珠筛选大鼠牙髓干细胞、外胚间充质干细胞的表型研究[J].口腔医学,2014,34(6):454-456.

[18] Gang EJ, Hong SH, Jeong JA, et al. In vitro mesengenic potential of human umbilical cord blood-derived mesenchymal stem cells. Biochem Biophys Res Commun. 2004;321(1):102-108.

[19] Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8(4):315-317.

[20] 易敏,付必莽,李志伟,等.兔骨髓间充质干细胞的培养及初步鉴定[J].昆明医科大学学报, 2015, 36(9):17-19.

[21] Rastegar F, Shenaq D, Huang J, et al. Mesenchymal stem cells: Molecular characteristics and clinical applications. World J Stem Cells. 2010;2(4):67-80.

[22] 阮征,王莲芳,胡修忠,等.新生猪骨髓间充质干细胞衍生细胞的分离、培养及生物学特性分析[J].生物技术通报, 2015(6): 170-176.

[23] 李静,张仁东.骨髓间充质干细胞研究进展[J].四川解剖学杂志, 2014, 22(1): 38-40.

[24] Platzbecker U, Ehninger G, Bornhäuser M. Allogeneic transplantation of CD34+ selected hematopoietic cells--clinical problems and current challenges. Leuk Lymphoma. 2004;45(3): 447-453.

[25] van Galen P, Kreso A, Mbong N, et al. The unfolded protein response governs integrity of the haematopoietic stem-cell pool during stress. Nature. 2014;510(7504):268-272.

[26] Kronenwett R, Martin S, Haas R. The role of cytokines and adhesion molecules for mobilization of peripheral blood stem cells. Stem Cells. 2000;18(5):320-330.

[27] Zhu H, Mitsuhashi N, Klein A,et al. The role of the hyaluronan receptor CD44 in mesenchymal stem cell migration in the extracellular matrix. Stem Cells. 2006;24(4):928-935.

[28] de Bruijn MF, Ma X, Robin C, et al. Hematopoietic stem cells localize to the endothelial cell layer in the midgestation mouse aorta. Immunity. 2002;16(5):673-683.

[29] 赵玉鑫,王福科,李彦林,等.Brdu标记VECs和ADSCs构建组织工程骨体内增殖研究[J].昆明医科大学学报, 2014, 35(3): 13-18.

[30] Zhou HX, Liu ZG, Liu XJ, et al. Umbilical cord-derived mesenchymal stem cell transplantation combined with hyperbaric oxygen treatment for repair of traumatic brain injury. Neural Regen Res. 2016;11(1):107-113.

[31] Maklakova IY, Grebnev YD, Yastrebov AP. The influence of extreme factors on homing multipotent mesenchymal stromal cells. Patol Fiziol Eksp Ter. 2015;59(4):82-86.

[32] 王鑫,李彦林,金耀峰,等. BMP-2、TGF-β3重组腺病毒载体的构建及其在滇南小耳猪BMSCs中的表达[J].中国修复重建外科杂志, 2014, 28(7): 896-902.

[33] Doi K, Nibu K, Ishida H, et al. Adenovirus-mediated gene transfer in olfactory epithelium and olfactory bulb: a long-term study. Ann Otol Rhinol Laryngol. 2005;114(8):629-633.