Construction of oxalate-degrading adult bone marrow mesenchymal stem cell lines

doi:10.3969/j.issn.2095-4344.2013.27.005

Abstract

Abstract:

BACKGROUND: High oxalic acid urine is a risk factor for stone formation. Constructing cell lines with high oxalate metabolic ability using genetic engineering and stem cell technology will become the effective method to prevent and treat calcium oxalate stone.
OBJECTIVE: To construct the adult bone marrow mesenchymal stem cell lines that can decompose the oxalic acid, through co-transfecting the oxalic acid degradation genes Frc and Oxc of oxalobacter formigenes into the normal adult bone marrow mesenchymal stem cells.
METHODS: Frc and Oxc were amplified by PCR, and the eukaryotic expression vectors of pLEGFP-N1-myc-Frc and pBaBE-puro-flag-Oxc were constructed, then co-transfected into the normal adult bone marrow mesenchymal stem cells. The non-transfected bone marrow mesenchymal stem cells and the cells transfected with empty vectors were as control. Western blot was performed to detect the expression of the objective genes; the concentration of oxalate in the culture medium after transgenic was determined by ion chromatography.
RESULTS AND CONCLUSION: Restriction enzyme digestion and sequencing results showed that the Frc and Oxc genes were successfully amplified, and the vectors of pLEGFP-N1-myc-Frc and pBaBE-puro-flag-Oxc were constructed. After tranfected into the bone marrow mesenchymal stem cells, the Western blot results showed that transfected bone marrow mesenchymal stem cells could stably express the target protein myc-formyl coenzyme A transferase enzyme and the flag-oxalyl coenzyme A decarboxylase; ion chromatography test results showed with the prolonging of the culture time, the concentration of oxalic acid in the human bone marrow mesenchymal stem cell culture medium transfected with target gene was decreased gradually. While there was no target protein expression in the non-transfected human bone marrow mesenchymal stem cells as well as the cells transfected with empty vectors. The cells had the ability of oxalate-degradation. The experiment successfully constructs the adult bone marrow mesenchymal stem cell lines that can decompose the oxalic acid, and the cell lines have the ability of oxalate-degradation and can stably express the oxalate decomposition proteins Frc and Oxc.

Key words: stem cells, bone marrow-derived stem cells, bone marrow mesenchymal stem cells, oxalobacter formigenes, hyperoxal uria, retrovirus vector, oxalate-degradation, Frc, Oxc, transgenic, genetic engineering, National Natural Science Foundation of China

CLC Number:

R394.2

Yang Xu-ming, Yuan Jian, Lei Ming, Zhang Ze. Construction of oxalate-degrading adult bone marrow mesenchymal stem cell lines[J]. Chinese Journal of Tissue Engineering Research, doi: 10.3969/j.issn.2095-4344.2013.27.005.

Figures/Tables 4

References

[1] Mittal RD, Kumar R. Gut-inhabiting bacterium Oxalobacter formigenes: role in calcium oxalate urolithiasis. J Endourol. 2004;18(5):418-424.

[2] Ogawa Y, Miyazato T, Hatano T. Oxalate and urinary stones. World J Surg. 2000;24(10):1154-1159.

[3] Anantharam V, Allison MJ, Maloney PC. Oxalate: formate exchange. The basis for energy coupling in Oxalobacter. J Biol Chem. 1989;264(13):7244-7250.

[4] Baetz AL, Allison MJ. Purification and characterization of oxalyl-coenzyme A decarboxylase from Oxalobacter formigenes. J Bacteriol. 1989;171(5):2605-2608.

[5] Baetz AL, Allison MJ. Purification and characterization of formyl-coenzyme A transferase from Oxalobacter formigenes. J Bacteriol.1990;172(7):3537-3540.

[6] Ye ZQ, Kong DB, Chen ZQ, et al. Stable expression of the oxc and frc genes from Oxalobacter formigenes in human embryo kidney 293 cells: implications for gene therapy of hyperoxaluria. Int J Mol Med. 2007;20(4):521-526.

[7] 叶章群.泌尿系结石研究现况与展望[J].中华实验外科杂志,2005, 22(3):261-262.

[8] Stamatelou KK, Francis ME, Jones CA, et al. Time trends in reported prevalence of kidney stones in the United States:1976-1994. Kidney Int. 2003;63(5):1817-1823.

[9] Koul H, Kennington L, Honeyman T, et al. Activation of c-myc gene mediates the mitogenic effects of oxalate in LLC-PK1 cells, a line of renal epithelial cells. Kidney Int. 1996;50(5): 1525-1530.

[10] Khan SR, Shevock PN, Hackett RL. Acute hyperoxaluria, renal injury and calcium oxalate urolithiasis. J Urol. 1992; 147(1):226-230.

[11] Huang MY, Chaturvedi LS, Koul S, et al. Oxalate stimulates IL-6 production in HK-2 cells, a line of human renal proximal tubular epithelial cells. Kidney Int. 2005;68(2):497-503.

[12] Koul S, Chaturvedi LS, Sekhon A, et al. Effects of oxalate on the re-initiation of DNA synthesis in LLC-PK1 cells do not involve p42/44 MAP kinase activation. Kidney Int. 2002;61(2): 525-533.

[13] Chaturvedi LS, Koul S, Sekhon A, et al. Oxalate selectively activates p38 mitogen-activated protein kinase and c-Jun N-terminal kinase signal transduction pathways in renal epithelial cells. J Biol Chem. 2002;277(15):13321-13330.

[14] Bhandari A, Koul S, Sekhon A, et al. Effects of oxalate on HK-2 cells, a line of proximal tubular epithelial cells from normal human kidney. J Urol. 2002;168(1):253-259.

[15] Sidhu H, Schmidt ME, Cornelius JG, et al. Direct correlation between hyperoxaluria/oxalate stone disease and the absence of the gastrointestinal tract-dwelling bacterium Oxalobacter formigenes: possible prevention by gut recolonization or enzyme replacement therapy. J Am Soc Nephrol. 1999;10 Suppl 14:S334-340.

[16] Sidhu H, Allison MJ, Chow JM, et al. Rapid reversal of hyperoxaluria in a rat model after probiotic administration of Oxalobacter formigenes. J Urol. 2001;166(4):1487-1491.

[17] Hoppe B, Beck B, Gatter N, et al. Oxalobacter formigenes: a potential tool for the treatment of primary hyperoxaluria type 1. Kidney Int. 2006;70(7):1305-1311.

[18] Duncan SH, Richardson AJ, Kaul P, et al. Oxalobacter formigenes and its potential role in human health. Appl Environ Microbiol. 2002;68(8):3841-3847.

[19] Koul S, Johnson T, Pramanik S, et al. Cellular transfection to deliver alanine-glyoxylate aminotransferase to hepatocytes: a rational gene therapy for primary hyperoxaluria-1 (PH-1). Am J Nephrol. 2005;25(2):176-182.

[20] Azcarate-Peril MA, Bruno-Bárcena JM, Hassan HM, et al. Transcriptional and functional analysis of oxalyl-coenzyme A (CoA) decarboxylase and formyl-CoA transferase genes from Lactobacillus acidophilus. Appl Environ Microbiol. 2006;72(3): 1891-1899.

[21] 陈志强,刘冠琳,叶章群,等.表达产甲酸草酸杆菌草酸分解基因的人肝细胞系的构建[J].临床泌尿外科杂志,2007,22(2):143-145.

[22] 叶章群,刘冠琳,陈志强,等.可分解草酸小鼠肠干细胞群的构建[J].中华医学杂志,2009,89(16):1130-1134.

[23] 赖德辉,雷鸣,李逊,等.基因工程构建表达融合蛋白GST-FCoAT的益生大肠杆菌EcN-Frc[J].实用医学杂志,2010,26(24):4480- 4482.

[24] Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature. 2002;418(6893):41-49.

[25] 张泽,欧莉莉,曾国华,等.离子色谱法同时测定24小时尿草酸和枸橼酸的应用价值[J].中华泌尿外科杂志,2010,28(z1):75-77.