Basic fibroblast growth factor-transfected bone marrow mesenchymal stem cell transplantation for acute kidney injury

doi:10.3969/j.issn.2095-4344.2016.28.010

Abstract

Abstract:

BACKGROUND: It has been confirmed that basic fibroblast growth factor (bFGF) can promote the growth, proliferation, differentiation and functional expression of most cells derived from neuroderm and mesoderm.
OBJECTIVE: To investigate the effect of bFGF-transfected bone marrow mesenchymal stem cell transplantation in rats with acute kidney injury.
METHODS: bFGF genes were transfected into bone marrow mesenchymal stem cells via an adenovirus vector, and then expression of bFGF in transfected cells was identified using RT-PCR technology. Rat models of acute kidney injury were prepared by clipping bilateral renal pedicles, and then randomized into three groups (n=20): rats were given injection of bone marrow mesenchymal stem cell suspensions via tail vein as negative transfected group, those given injection of bFGF-transfected bone marrow mesenchymal stem cell suspensions via tail vein as bFGF-transfected group, and the others given injection of DMEM via tail vein as model group. Four weeks later, levels of serum creatinine and urea nitrogen were detected, expressions of connective tissue growth factor and growth factor in renal tissues were detected by Western blot assay, and morphology of renal tissues was observed using hematoxylin-eosin staining.
RESULTS AND CONCLUSION: bFGF genes were successfully transfected into bone marrow mesenchymal stem cells. Compared with the model group, the levels of serum creatinine and urea nitrogen were significantly reduced in bFGF-transfected and negative transfected groups, especially in the bFGF-transfected group (P < 0.05), while expressions of connective tissue growth factor and transforming growth factor in renal tissues in bFGF-transfected and negative transfected groups were significantly weakened in these two groups (P < 0.05), but there were no significant differences between the bFGF-transfected group and negative transfected group (P > 0.05). Besides, renal tissues necrosis and inflammatory reactions were mitigated in the negative transfected group; renal tubules with normal outlines and no overt necrotic cells could be found in the bFGF-transfected group. These findings show that bFGF-transfected bone marrow mesenchymal stem cell transplantation plays a better role in acute kidney injury repair in rats.

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 Cell Transplantation, Fibroblast Growth Factor 2, Kidney Diseases, Tissue Engineering

CLC Number:

R394.2

Cui Li-de, Li Tie-min. Basic fibroblast growth factor-transfected bone marrow mesenchymal stem cell transplantation for acute kidney injury[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(28): 4169-4175.

Figures/Tables 1

2.1 骨髓间充质干细胞形态传代后细胞生长迅速， 4 d后基本长满瓶底，形态似长梭形，随着集落数目的不断增多，第1，2，3代细胞增殖活跃，绝大多数细胞单层贴壁生长，达85%以上融合时，显微镜下可见细胞分布均匀，胞核及核仁清晰可见，细胞形态基本一致，呈漩涡状分布，第3代骨髓间充质干细胞增殖速度明显加快，细胞生长旺盛，见图1。 2.2 RT-PCR检测碱性成纤维细胞生长因子mRNA的表达腺病毒载体介导的碱性成纤维细胞生长因子基因转染48 h后，对照组和阴性转染组未出现相应大小的目的条带表达，而碱性成纤维细胞生长因子转染组出现相应大小的目的条带，提示碱性成纤维细胞生长因子基因已稳定整合入骨髓间充质干细胞，见图2。 2.3 各组大鼠血清肌酐及尿素氮水平移植后4周，与模型组比较，阴性转染组大鼠血清肌酐及尿素氮水平明显降低，碱性成纤维细胞生长因子基因转染组血清肌酐及尿素氮水平进一步降低，各组间比较差异有显著性意义(P < 0.05)，见表1，图3。 2.4 Western blot检测肾组织中结缔组织生长因子及转化生长因子β1的表达移植后4周，与模型组比较，阴性转染组及碱性成纤维细胞生长因子基因转染组肾组织结缔组织生长因子及转化生长因子β1表达明显减弱(P < 0.05)，阴性转染组及碱性成纤维细胞生长因子基因转染组间差异无显著性意义(P > 0.05)，见图4。 2.5 苏木精-伊红染色观察各组大鼠肾组织病理损伤情况移植后4周，模型组肾小管上皮细胞出现核溶解、碎裂，肾小球增生、肥大，毛细血管部分塌陷，球囊粘连，间质炎性浸润，可见萎缩、塌陷的肾小管(图5A)；阴性转染组核溶解、碎裂等细胞坏死现象得到缓解，肾小球增生、间质炎性反应明显减轻(图5B)；碱性成纤维细胞生长因子转染组可看到轮廓清晰的肾小管及较为完整的基底膜，肾组织未见明显的细胞坏死(图5C)。"

References

[1] 毛家玺,汤晓静.干细胞在急性肾损伤中的治疗作用及进展[J].中国中西医结合肾病杂志,2012,13(1):86-88.
[2] 甘露.间充质干细胞治疗急性肾损伤的临床应用可行性探讨[J].特别健康,2014,3(3):39.
[3] 李季.人类bFGF基因在大鼠骨髓间充质干细胞的表达及其分子作用机制研究[D].济南:山东大学,2004.
[4] Herrera MB, Bussolati B, Bruno S, et al. Mesenchymal stem cells contribute to the renal repair of acute tubular epithelial injury. Int J Mol Med. 2004;14(6):1035-1041.
[5] 龚圆渊.六味地黄丸联合bFGF诱导骨髓间充质干细胞向神经元样细胞分化的研究[D].成都: 成都中医药大学, 2012.
[6] 孔根现. VEGF分别与bFGF、aFGF联合诱导骨髓间充质干细胞分化为血管内皮细胞[D].广州: 南方医科大学, 2013.
[7] Lee SK, Kim Y, Kim SS, et al. Differential expression of cell surface proteins in human bone marrow mesenchymal stem cells cultured with or without basic fibroblast growth factor containing medium. Proteomics. 2009;9(18):4389-4405.
[8] Strioga M, Viswanathan S, Darinskas A, et al. Same or not the same? Comparison of adipose tissue-derived versus bone marrow-derived mesenchymal stem and stromal cells. Stem Cells Dev. 2012;21(14):2724-2752.
[9] 和小娥,任卫青,邓立新,等.生长因子对兔骨髓间充质干细胞增殖的影响[J].畜牧兽医学报,2012,43(4):553-557.
[10] 郑有华,张志光,苏凯,等.体外培养条件下人骨髓间充质干细胞碱性成纤维细胞生长因子基因的转染与鉴定[J].中国组织工程研究与临床康复,2010,14(19):3507- 3512.
[11] Xu X, Shao N, Qiao D, et al. Expression of vascular endothelial growth factor and basic fibroblast growth factor in extramammary Paget disease.Int J Clin Exp Pathol. 2015;8(3):3062-3068.
[12] 胡红林,王共先,邹丛,等.BALB/c小鼠肾缺血再灌注损伤模型的建立与评价[J].中国组织工程研究与临床康复, 2011,15(5): 870-873.
[13] 吴佳奇,冯大雄,杨天府,等.骨髓间充质干细胞经蛛网膜下腔移植治疗大鼠脊髓损伤及其对T细胞亚群影响的研究[J].中国修复重建外科杂志,2007,21(5): 492-496.
[14] 李辉映,文益民,陈克明,等.多次移植骨髓基质干细胞有利于脊髓损伤修复[J].中国矫形外科杂志,2009,17(22): 1725-1728.
[15] 徐云强,冯世庆,王沛,等.软骨素酶联合雪旺细胞移植促进脊髓损伤修复的研究[J].中华实验外科杂志,2010,27(7): 972-975.
[16] Turnbull IR, Clark AT, Stromberg PE, et al. Effects of aging on the immunopathologic response to sepsis. Crit Care Med. 2009;37(3):1018-1023.
[17] Liu WH, Huang HQ, Deng YH, et al. Effects of berberine on renal function, oxidative stress and renal aldose reductase in rats with diabetic nephropathr. Chin Pharmacol Bull. 2008;24(7):955-959.
[18] 葛成国,靳凤烁,李黔生,等.过表达Bcl-2雪旺细胞抗凋亡能力的实验研究[J].中国康复医学杂志,2004,19(2): 95-97.
[19] 牛志宏,高德轩,丁森泰,等. Cyr61,CTGF,NOV,WISP-1 基因在肾癌中的表达[J].山东大学学报:医学版, 2008, 46(7):701-703,714.
[20] 王莉. 华勒变性及少突胶质前体细胞和雪旺细胞移植[D].上海:上海交通大学,2008.
[21] 李军. 嗅鞘细胞蛛网膜下腔移植治疗脊髓损伤的实验研究[D]. 泰安:泰山医学院,2011.
[22] Yu PJ, Ferrari G, Galloway AC, et al. Basic fibroblast growth factor (FGF-2): the high molecular weight forms come of age. J Cell Biochem. 2007;100(5):1100-1108.
[23] Bhagavati S, Xu W. Isolation and enrichment of skeletal muscle progenitor cells from mouse bone marrow. Biochem Biophys Res Commun. 2004; 318(1):119-124.
[24] Fujita T, Shiba H, Van Dyke TE, et al. Differential effects of growth factors and cytokines on the synthesis of SPARC, DNA, fibronectin and alkaline phosphatase activity in human periodontal ligament cells. Cell Biol Int. 2004;28(4):281-286.
[25] Shin H, Zygourakis K, Farach-Carson MC, et al. Modulation of differentiation and mineralization of marrow stromal cells cultured on biomimetic hydrogels modified with Arg-Gly-Asp containing peptides. J Biomed Mater Res A. 2004;69(3):535-543.
[26] Ma Y, Lang L, Kiesewetter DO, et al. Purification of the precursor for the automated radiosynthesis of [18F]FCWAY by counter-current chromatography. J Chromatogr A. 2004;1034(1-2):149-153.
[27] Talbot NC, Powell AM, Caperna TJ. Comparison of colony-formation efficiency of bovine fetal fibroblast cell lines cultured with low oxygen, hydrocortisone, L-carnosine, bFGF, or different levels of FBS. Cloning Stem Cells. 2004;6(1):37-47.
[28] Haider M, Cappello J, Ghandehari H, et al. In vitro chondrogenesis of mesenchymal stem cells in recombinant silk-elastinlike hydrogels. Pharm Res. 2008;25(3):692-699.
[29] Bojin FM, Gruia AT, Cristea MI, et al. Adipocytes differentiated in vitro from rat mesenchymal stem cells lack essential free fatty acids compared to adult adipocytes. Stem Cells Dev. 2012;21(4):507-512.
[30] Jump SS, Childs TE, Zwetsloot KA, et al. Fibroblast growth factor 2-stimulated proliferation is lower in muscle precursor cells from old rats. Exp Physiol. 2009; 94(6):739-748.
[31] Tögel F, Cohen A, Zhang P, et al. Autologous and allogeneic marrow stromal cells are safe and effective for the treatment of acute kidney injury. Stem Cells Dev. 2009;18(3):475-485.
[32] Iwasaki M, Adachi Y, Minamino K, et al. Mobilization of bone marrow cells by G-CSF rescues mice from cisplatin-induced renal failure, and M-CSF enhances the effects of G-CSF. J Am Soc Nephrol. 2005;16(3): 658-666.
[33] Morigi M, Rota C, Montemurro T, et al. Life-sparing effect of human cord blood-mesenchymal stem cells in experimental acute kidney injury. Stem Cells. 2010; 28(3):513-522.
[34] Chade AR, Zhu X, Lavi R, et al. Endothelial progenitor cells restore renal function in chronic experimental renovascular disease. Circulation. 2009;119(4): 547-557.
[35] Tsuda H, Yamahara K, Ishikane S, et al. Allogenic fetal membrane-derived mesenchymal stem cells contribute to renal repair in experimental glomerulonephritis. Am J Physiol Renal Physiol. 2010;299(5):F1004-1013.
[36] Siegel N, Rosner M, Unbekandt M, et al. Contribution of human amniotic fluid stem cells to renal tissue formation depends on mTOR. Hum Mol Genet. 2010; 19(17):3320-3331.
[37] Patschan D, Krupincza K, Patschan S, et al. Dynamics of mobilization and homing of endothelial progenitor cells after acute renal ischemia: modulation by ischemic preconditioning. Am J Physiol Renal Physiol. 2006;291(1):F176-185.
[38] Rampino T, Gregorini M, Bedino G, et al. Mesenchymal stromal cells improve renal injury in anti-Thy 1 nephritis by modulating inflammatory cytokines and scatter factors. Clin Sci (Lond). 2011;120(1):25-36.
[39] Iwasaki M, Adachi Y, Minamino K, et al. Mobilization of bone marrow cells by G-CSF rescues mice from cisplatin-induced renal failure, and M-CSF enhances the effects of G-CSF. J Am Soc Nephrol. 2005;16(3): 658-666.
[40] Tögel F, Cohen A, Zhang P, et al. Autologous and allogeneic marrow stromal cells are safe and effective for the treatment of acute kidney injury. Stem Cells Dev. 2009;18(3):475-485.