Transferring adenovirus vector-mediated enhanced green fluorescent protein gene into rabbit bone marrow mesenchymal stem cells: effectiveness and toxicity

doi:10.3969/j.issn.2095-4344.0515

Abstract

Abstract:

BACKGROUND: The recombinant adenovirus has certain toxic reactions to cell growth and survival, but the determination of the optimal multiplicity of infection (MOI) is not well documented.
OBJECTIVE: To study the effectiveness and toxicity of transfection of bone marrow mesenchymal stem cells (BMSCs) by adenovirus vectors with different MOIs, and to explore the effect on osteogenic capability of the cells.
METHODS: BMSCs at passage 5 were infected with enhanced green fluorescent protein (EGFP) via adenovirus vectors with different MOI (0, 50, 100, 150, 200 and 250). Cell morphology and mortality rate were observed and calculated using trypan blue staining method at 24 hours after cell transfection. The transfection efficiency and the relative mRNA expression of EGFP and Runx2 were analyzed respectively by inverted florescent microscopy and real-time quantitative PCR at 48 hours after cell transfection. The activity of BMSCs transfected with different MOIs was evaluated by MTT, and the optimal MOI was determined thereafter.
RESULTS AND CONCLUSION: With the increase of MOI, BMSCs showed decreased adherent ability, and even some cells aged and died. The mortality rate of BMSCs transfected for 24 hours at a MOI of 0 to 250 was 5.80%, 6.67%, 7.95%, 7.76%, 10.35% and 11.18%, respectively, indicating that the mortality rate of BMSCs is positively correlated with the MOI. The transfection efficiency changed insignificantly when the MOI was greater than 100. On the contrary, the cellular viability and osteogenic differentiation capability of rabbit BMSCs were receded when the MOI level was up to 200-250. The study discovered that the suitable scope of MOI to transfect BMSCs is 50- 150, and the optimal MOI is 100.

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: Bone Marrow, Mesenchymal Stem Cells, Adenoviridae, Green Fluorescent Proteins, Tissue Engineering

CLC Number:

R394.2

Wu Cheng-cong, Rong Shu, Ren Jing, Wu Zheng, Liu Tao, Liu Ke-ting, Zhu Bo, Huang He-fei, Wang Fang. Transferring adenovirus vector-mediated enhanced green fluorescent protein gene into rabbit bone marrow mesenchymal stem cells: effectiveness and toxicity[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(17): 2650-2655.

Figures/Tables 2

2.1 一般情况及死亡率骨髓间充质干细胞复苏后生长状态良好，细胞饱满，呈梭形、漩涡状生长。第5代骨髓间充质干细胞转染后24 h，实验组培养基内杂质增多，随着感染复数值增加，部分细胞体积膨胀、变薄，贴壁能力降低，培养基内漂浮细胞增多(图1)。感染复数为0组的细胞死亡率为5.80%，感染复数为50，100，150组的细胞死亡率分别为6.67%，7.95%，7.76%，与对照组相比差异无显著性意义(P > 0.05)。感染复数为200，250组的细胞死亡率为10.35%，11.18%，较对照组增高(P < 0.05，图2)。 2.2 Ad-EGFP转染效率倒置荧光显微镜下发现，转染后2 h各组即有少量荧光蛋白表达，相同感染复数下，随转染时间延长，荧光蛋白表达数量随之增加，转染24 h后替换为完全培养基，第48小时实验组细胞绿色荧光蛋白表达数量不再增加，保持稳定，荧光蛋白分布于细胞核及细胞质内。除对照组外，转染后48 h各组细胞均有绿色荧光蛋白表达，感染复数为50，100，150，200，250组的转染效率分别为62.4%，96.30%，95.72%，95.34%和96.15%。与对照组相比，腺病毒成功将EGFP基因转染进入骨髓间充质干细胞并表达增强绿色荧光蛋白，达到实验目的。而在实验组中，感染复数为50组转染效率明显低于其他各组(P < 0.01)。感染复数为100，150，200，250组转染效率并不随感染复数增加而增加，各组转染效率差异无显著性意义(P > 0.05，图3)。 2.3 Ad-EGFP转染后骨髓间充质干细胞活性腺病毒感染后各组骨髓间充质干细胞并未出现大面积死亡，细胞继续分裂、增殖，但细胞感染腺病毒后其细胞生长受到了不同程度的影响，见图4。转染后72 h内各组细胞活性并未发生明显改变(P > 0.05)。第4天开始，感染复数为250组细胞出现吸光度降低，第5天感染复数为200，250组细胞生长状态与对照组相比均出现吸光度降低，提示细胞活性降低、死亡细胞增多。而感染复数为50，100，150组骨髓间充质干细胞活性并未受到明显影响。说明随感染复数的不断增加，骨髓间充质干细胞形态和活性受到的抑制作用越明显。 2.4 转染后骨髓间充质干细胞EGFP、Runx2基因表达转染后48 h，实验组可见大量绿色荧光蛋白表达，扩增过程中未产生二聚体，转染后骨髓间充质干细胞中EGFP、Runx2基因相对表达量见表2。骨髓间充质干细胞表达大量EGFP基因，其中感染复数为50组荧光表达量明显少于其他实验组(P < 0.01)，随感染复数增加并达到感染复数为100时，EGFP基因表达量不再继续增加(P > 0.05)。相对于对照组，感染复数≤200时骨髓间充质干细胞Runx2基因表达能力无明显变化，即骨髓间充质干细胞成骨潜能无影响。而感染复数为250组Runx2基因表达量明显降低(P=0.009)，骨髓间充质干细胞成骨潜能受到明显影响。"

References

[1] 许锋,詹玉林,宋兴华. 骨形态发生蛋白2基因治疗骨缺损研究进展[J]. 医学综述,2012,18(11):1612-1614.

[2] Wang S, Qu X, Zhao RC. Clinical applications of mesenchymal stem cells. J Hematol Oncol. 2012;5:19.

[3] Grigsby CL, Leong KW. Balancing protection and release of DNA: tools to address a bottleneck of non-viral gene delivery. J R Soc Interface. 2010;7 Suppl 1:S67-82.

[4] 仲照东,邹萍,游泳,等. 重组腺病毒AdEGFP/hVEGF165快速构建及在骨髓移植小鼠体内的分布和表达效率[J]. 中华实验和临床病毒学杂志,2004,18(1):35-38.

[5] 武成聪,李强,陈佳滨,等. 短期冻存兔骨髓间充质干细胞对其生物特性的影响[J]. 重庆医学,2014,43(4):459-461,464.

[6] Cartier R, Reszka R. Utilization of synthetic peptides containing nuclear localization signals for nonviral gene transfer systems. Gene Ther. 2002;9(3):157-167.

[7] Laurencin CT, Attawia MA, Lu LQ, et al. Poly(lactide-co-glycolide)/hydroxyapatite delivery of BMP-2-producing cells: a regional gene therapy approach to bone regeneration. Biomaterials. 2001;22(11):1271-1277.

[8] 毛颖佳,郑源强,石艳春. 慢病毒载体及其应用的研究进展[J]. 中国生物制品学杂志,2009,22(2):196-200.

[9] Nault JC, Datta S, Imbeaud S, et al. Recurrent AAV2-related insertional mutagenesis in human hepatocellular carcinomas. Nat Genet. 2015;47(10):1187-1193.

[10] Shi S, Mercer S, Trippel SB. Effect of transfection strategy on growth factor overexpression by articular chondrocytes. J Orthop Res. 2010;28(1):103-109.

[11] 王晓旭,何敏,谭文甫,等. 腺病毒介导人TGF-β1基因转染自体腘绳肌腱重建兔前交叉韧带术后的表达[J]. 中国修复重建外科杂志,2016,30(10):1233-1237.

[12] Yu D, Jin C, Ramachandran M, et al. Adenovirus serotype 5 vectors with Tat-PTD modified hexon and serotype 35 fiber show greatly enhanced transduction capacity of primary cell cultures. PLoS One. 2013;8(1):e54952.

[13] 郝嘉,游凯,肖颖彬. 腺病毒载体,最有潜力的心血管疾病治疗载体[J]. 中国医科大学学报,2010,39(9):689-693,697.

[14] 裴树俊,徐学武,俞卫锋,等. 鞘内注射一代和三代腺病毒载体免疫反应的比较[J]. 生物技术,2009,19(3):58-61.

[15] Wiedenmann J, Oswald F, Nienhaus GU. Fluorescent proteins for live cell imaging: opportunities, limitations, and challenges. IUBMB Life. 2009;61(11):1029-1042.

[16] 张全彬,王黎明,徐燕. 重组腺病毒介导增强型绿色荧光蛋白基因转染兔骨髓间充质干细胞[J]. 中国组织工程研究与临床康复, 2009,13(23):4452-4456.

[17] Noda A, Hirai Y, Kodama Y, et al. Easy detection of GFP-positive mutants following forward mutations at specific gene locus in cultured human cells. Mutat Res. 2011;721(1): 101-107.

[18] 张琼宇,马珊珊,唐小标,等. 病毒感染复数影响腺病毒感染T淋巴细胞效率的初步研究[J]. 生命科学研究,2017,21(4):312-317.

[19] Koenig A, Norgard-Sumnicht K, Linhardt R, et al. Differential interactions of heparin and heparan sulfate glycosaminoglycans with the selectins. Implications for the use of unfractionated and low molecular weight heparins as therapeutic agents. J Clin Invest. 1998;101(4):877-889.

[20] 刘旭平,范里,朱明龙,等. MOI对HEK293细胞感染病毒后的生长代谢和腺病毒扩增效率的影响[J]. 高校化学工程学报,2010, 24(4):638-644.

[21] Tsai YC, Tsai TH, Chang CP, et al. Linear correlation between average fluorescence intensity of green fluorescent protein and the multiplicity of infection of recombinant adenovirus. J Biomed Sci. 2015;22:31.

[22] 张淑香,李东晓,朱明龙,等. 谷氨酰胺限制对杂交瘤细胞生长、代谢和单抗生产的影响[J]. 高校化学工程学报,2008,22(1):77-82.

[23] 徐练,孔清泉. 调控成骨细胞分化及骨形成关键信号通路的研究进展[J]. 中国修复重建外科杂志,2014,28(12):1484-1489.

[24] 边素艳,盖鲁粤,叶平,等. 腺病毒载体对骨髓间充质干细胞体外分化能力的影响[J]. 中国现代医学杂志,2010,20(8):1152-1156.