Construction of a recombinant adenovirus overexpressing human antimicrobial peptide LL37 gene and its transfection into vaginal epithelial cells

doi:10.3969/j.issn.2095-4344.2016.51.015

Abstract

Abstract:

BACKGROUND: LL37, the only antimicrobial peptide of the cathelicidin family identified from the human, not only promotes the proliferation of endothelial cells, but also plays an important role in angiogenesis and re-epithelialization.
OBJECTIVE: To construct a recombinant adenovirus overexpressing human antimicrobial peptide LL37 gene, and to detect the expression and secretion of LL37 after transfected into the canine vaginal epithelial cells.
METHODS: The cDNA encoding LL37 was amplified by PCR. Recombinant adenovirus expression plasmid encoding LL37 and green fluorescent protein (EGFP) was constructed, and identified using restriction endonuclease technology and DNA sequencing method. Adenovirus particles were generated by cotransfecting the 293-packaging cell line. The adenovirus were collected, amplified and concentrated, and viral titers were determined by end-point dilution assay by applying serial dilutions of the purified viruses to 293 cells. Primary cultured canine vaginal epithelial cells were transfected by the recombinant adenovirus. The transfection efficacy was observed by fluorescence microscope, and the cultured supernatant was collected to determine the expression of LL37 by ELISA method at 1, 2, 3, 5 and 7 days after transfection.
RESULTS AND CONCLUSION: The adenovirus vector GV314-LL37 with the titer of 3×109 pfu/mL was successfully constructed and identified by DNA sequencing methods. Canine vaginal epithelial cells were successfully isolated and cultured and grew stably. After transfection, vaginal epithelial cells could express the EGFP and LL37 efficiently in a time-dependent manner detected by fluorescence microscope and ELISA method. The transfection efficacy of EGFP reached to 89% at 72 hours. The level of LL37 in the cell culture supernatant in the transfection group was significantly higher than that in the control group, the highest expression of LL37 was found at 3 days that lasting for 7 days. In conclusion, the recombinant adenovirus overexpressing human antimicrobial peptide LL37 gene is successfully constructed, which can express and secrete LL37 after transfected into canine vaginal epithelial cells, providing a foundation for constructing the tissue-engineered vagina possessing anti-infection and neovascularization.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Vagina, Epithelial Cells, Dermcidins, Tissue Engineering

CLC Number:

R318

Shen Fu-jin, Xu Xue-xian, Zhou Li-mei. Construction of a recombinant adenovirus overexpressing human antimicrobial peptide LL37 gene and its transfection into vaginal epithelial cells[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(51): 7697-7702.

Figures/Tables 3

References

[1] Callens N, De Cuypere G, De Sutter P, et al. An update on surgical and non-surgical treatments for vaginal hypoplasia. Hum Reprod Update.2014;20(5):775-801.

[2] Londra L, Chuong FS, Kolp L. Mayer-Rokitansky- Kuster-Hauser syndrome: a review.Int J Womens Health. 2015;7:865-870.

[3] McQuillan SK, Grover SR. Dilation and surgical management in vaginal agenesis: a systematic review. Int Urogynecol J.2014; 25: 299-311.

[4] Dorin RP, Atala A, Defilippo RE. Bioengineering a vaginal replacement using a small biopsy of autologous tissue. Semin Reprod Med. 2011;29(1): 38-44.

[5] Zhu L,Zhou H,Sun Z,et al.Anatomic and sexual outcomes after vaginoplasty using tissue-engineered biomaterial graft in patients with Mayer-Rokitansky-Kuster-Hauser syndrome: a new minimally invasive and effective surgery. J Sex Med. 2013; 10: 1652-1658.

[6] Birchall MA, Seifalian AM. Tissue engineering's green shoots of disruptive innovation. Lancet. 2014; 384(9940):288-290.

[7] Ding JX, Chen XJ, Zhang XY, et al. Acellular porcine small intestinal submucosa graft for cervicovaginal reconstruction in eight patients with malformation of the uterine cervix. Hum Reprod. 2014; 29(4): 677-682.

[8] Frasca L, Lande R. Role of defensins and cathelicidin LL37 in auto-immune and auto-inflammatory diseases. Curr Pharm Biotechnol. 2012;(10):1882-1897.

[9] Chamilos G, Gregorio J, Meller S, et al. Cytosolic sensing of extracellular self-DNA transported into monocytes by the antimicrobial peptideLL37. Blood. 2012;120(18):3699-707

[10] Thennarasu S, Tan A, Penumatchu R, et al. Antimicrobial and membrane disrupting activities of a peptide derived from the human cathelicidin antimicrobial peptide LL37. Biophys J. 2010;98(2): 248-257.

[11] Ramos R, Silva JP, Rodrigues AC, et al. Wound healing activity of the human antimicrobial peptide LL37. Peptides. 2011;32(7):1469-1476.

[12] Kittaka M, Shiba H, Kajiya M, et al. Antimicrobial peptide LL37 promotes vascular endothelial growth factor-A expression in human periodontal ligament cells.J Periodontal Res. 2013 ;48(2):228-234.

[13] Pfosser A, El-Aouni C, PfistererI, et al. NF kappaB activation in embryonic endothelial progenitor cells enhances neovascularization via PSGL-1 mediated recruitment: novel role for LL37. Stem Cells, 2010, 28(2): 376-385.

[14] 申复进,洛若愚,梁华,等.犬阴道上皮细胞和平滑肌细胞可在体外长期稳定培养[J].中国组织工程研究,2015, 19(1): 96-100.

[15] 王芳,孙蓓,李红,等.阴道上皮细胞转染抗菌肽LL-37和防御素5后对假丝酵母菌的抑制作用[J].中华妇产科杂志, 2012,47(3): 205-211.

[16] Carretero M, Escámez MJ, García M, et al. In vitro and in vivo wound healing-promoting activities of human cathelicidin LL-37.J Invest Dermatol.2008; 128: 223-236.

[17] Atala A, Kasper FK, Mikos AG.Engineering complex tissues. Sci Transl Med. 2012;4(160):160-172.

[18] Dorin RP, Atala A, Defilippo RE.Bioengineering a vaginal replacement using a small biopsy of autologous tissue. Semin Reprod Med. 2011;29(1): 38-44.

[19] De Filippo RE, Bishop CE, Filho LF, et al. Tissue engineering a complete vaginal replacement from a small biopsy of autologous tissue. Transplantation. 2008;86:208-214.

[20] Ding JX, Chen LM, Zhang XY, et al. Sexual and functional outcomes of vaginoplasty using acellular porcine small intestinal submucosa graft or laparoscopic peritoneal vaginoplasty: a comparative study. Hum Reprod. 2015 30(3):581-589.

[21] Stephan A, Batinica M, Steiger J, et al. LL37 DNA complexes provide antimicrobial activity against intracellular bacteria in human macrophages. Immunology. 2016 May 14.

[22] Ishida W, Harada Y, Fukuda K, et al. Inhibition by the Antimicrobial Peptide LL37 of Lipopolysaccharide-Induced Innate Immune Responses in Human Corneal Fibroblasts. Invest Ophthalmol Vis Sci. 2016;57(1):30-39.

[23] Chereddy KK,Her CH,Comune M,et al.PLGA nanoparticles loaded with host defense peptide LL37 promote wound healing. J Control Release. 2014;194: 138-147.

[24] Kittaka M, Shiba H, Kajiya M, et al. The antimicrobial peptide LL37 promotes bone regeneration in a rat calvarial bone defect. Peptides. 2013;46:136-142.

[25] Ghali S, Bhatt KA, Dempsey MP, et al.Treating chronic wound infections with genetically modified free flaps. Plast Reconstr Surg. 2009 ;123(4):1157-1168.

[26] Chen BS, Xie H, Zhang SL, et al. Tissue engineering of bladder using vascular endothelial growth factor gene-modified endothelial progenitor cells. Int J Artif Organs.2011;34(12): 1137-1146.

[27] Crystal RG.Adenovirus: the first effective in vivo gene delivery vector.Hum Gene Ther.2014;25(1):3-11.