Cardiac fibroblasts of the rat transfected by lenti virus-stromal cell-derived factor-1α-green fluorescent protein vector

doi:10.16098/j.issn.0529-1356.2017.01.008

Abstract

Abstract:

Objective To investigate the effect of lentivirus-stromal cell-derived factor-1α-green fluorescent protein(LV-SDF-1α-GFP) on the cardiac fibroblasts, the optimum conditions of infection, the expression and secretion of the target protein. Methods The cardiac fibroblasts of neonatal rats were primarily isolated and cultured by differential adherence methods, and were observed and identifi with immunofluorescence. LV-SDF-1α-GFP with different titers and conditions was transfected into cardiac fibroblasts. The expression of fluorescence and the optimal transfection conditions were observed. LV-SDF-1α-GFP target gene virus and negative control CON145 virus were transfected into cardiac fibroblasts. The growth curve was drawn, and the effect of transfection on the proliferation of cardiac fibroblasts was explored.The cardiac fibroblasts were transfected with the optimum transfection dose, and the expression of SDF-1α was detected by Dot-blotting. The measurement data underwent statistical analysis. Results There was no statistical difference between the cardiac fibroblasts with SDF-1α transfected lentivirus and without no-transfected SDF-1α lentivirus. The peak of the expression of SDF-1α appeared in culture day 4 and statistical analysis showed significantly difference (P<0.05). Conclusion The LV-SDF-1α-GFP vector is of higher transfection efficiency to cardiac fibroblasts with the both low cytotoxicity and ability of secreting SDF-1α protein.

Key words: Cardiac fibroblast, Lentivirus-stromal cell-derived factor-1α-green fluorescent protein vector, Transfection, Dot blotting, Neonatal rat　　　

ZHU Zhan-zhan HOU Bin SUN Wen-wen YUAN Hui-fang ZHANG Yong-chun CAI Xin-hua. Cardiac fibroblasts of the rat transfected by lenti virus-stromal cell-derived factor-1α-green fluorescent protein vector[J]. Acta Anatomica Sinica, 2017, 48(1): 43-47.

References

［1］Ma J, Ding ChH. Cardiac fibroblasts and myocardial fibrosis ［J］. Chinese Journal of Cardiology, 2014, 42 (3): 269-272.（in Chinese）

马金, 丁春华. 心脏成纤维细胞与心肌纤维化［J］. 中华心血管病杂志, 2014, 42(3):269-272.

［2］Yang Y, Gu ZhJ, Mai JT, et al. Progress and research into cardiac sources of fiber cells ［J］. Journal of Cardiology, 2015, 43 (8):750-752.（in Chinese）

杨莹, 古振杰, 麦憬霆,等. 心脏成纤维细胞的来源与研究进展［J］. 中华心血管病杂志, 2015, 43(8): 750-752.

［3］Yang WT, Shi L, Chen YB, et al. Feasibility study on cardiac fibroblast cells as gene target cells ［J］. Journal of Soochow University (Medical Science Edition), 2006, 26 (1): 24-26.（in Chinese）

杨文涛, 施立, 陈勇兵,等. 心肌成纤维细胞成为基因靶细胞的可行性研究［J］. 苏州大学学报(医学版), 2006, 26(1):24-26.

［4］Chatterjee S, Behnam Azad B, Nimmagadda S. The intricate role of CXCR4 in cancer［J］. Adv Cancer Res, 2014, 124(2):31-82.

［5］Nasri M, Karimi A, Farsani MA. Production, purification and titration of a lentivirus-based vector for gene delivery purposes ［J］. Cytotechnology, 2014, 66(6):1031-1038.

［6］Ross JS, Obuchowski N, Zepp R. The postoperative lumbar spine: evaluation of epidural scar over a 1-year period ［J］. AJNR Am J Neuroradiol, 1998, 19(1):183-186.

［7］Ibraheem D, Elaissari A, Fessi H. Gene therapy and DNA delivery systems［J］. Int J Pharm, 2014, 459(12):70-83.

［8］Tao W, Warren ST, Peng J. Toward pluripotency by reprogramming: mechanisms and application［J］. Protein Cell, 2013, 4(11):820-832.

［9］Gaffney MM, Hynes SO, Barry F, et al. Cardiovascular gene therapy: current status and therapeutic potential ［J］. Br J Pharmacol, 2007, 152(2):175-188.

［10］Conklin LD, Mcaninch RD, Kaluza GL, et al. HIV-based vectors and angiogenesis following rabbit hindlimb ischemia ［J］. J Surg Res, 2005, 123(1):55-66.

［11］Hizi A, Herschhorn A. Retroviral reverse transcriptases (other than those of HIV-1 and murine leukemia virus): a comparison of their molecular and biochemical properties［J］. Virus Res, 2008, 134(s 1-2):203-220.

［12］Mackenna AD, Summerour SR, Villarreal FJ. Role of mechanical factors in modulating cardiac fibroblast function and extracellular matrix synthesis ［J］. Early China, 1976, 2(2):257-263.

［13］Ikenishi A, Okayama H, Iwamoto N, et al. Cell cycle regulation in mouse heart during embryonic and postnatal stages［J］. DevGrowth Differ, 2012, 54(8): 731-738.

［14］Brzoska E, Kowalewska M, Markowska A, et al. The Sdf-1 (CXCL12) improves skeletal muscle regeneration via the mobilization of Cxcr4 and CD34 expressing cells ［J］. Biol Cell, 2012, 104(12): 722-737.

［15］Wang Y, Luther K. Genetically manipulated progenitor/stem cells restore function to the infarcted heart via the SDF-1α/CXCR4 signaling pathway ［J］. Prog Mol Biol Transl Sci, 2012, 111:265-284.

［16］Masurier C, Boutin S, Veron P, et al. Enhanced lentiviral transduction of monocyte-derived dendritic cells in the presence of conditioned medium from dying monocytes ［J］. Human Gene Therapy, 2007, 18(2):161-170.

［17］Li HS, Xu Ch, Zou J. Transfection efficiency and cytotoxicity of lentivirus-GFP on rabbit adipose-derived stem cells［J］. Journal of Shanghai Jiaotong University(Medical Science Edition), 2013, 33 (4): 439-444.（in Chinese）

李宏松, 徐晨, 邹俊. 慢病毒负载GFP转染兔脂肪干细胞的效率及毒性评价［J］. 上海交通大学学报(医学版), 2013, 33(4):439-444.

[1]	LIU Yong-feng Lü Na YANG Hua CHEN Yong-hong WEI Hong-en. Method of transfection of STOP gene lentiviral vector into oligodendrocyte precursor cells of BTBR mouse model of autism [J]. Acta Anatomica Sinica, 2023, 54(1): 117-122.
[2]	LUO Hong GAO Ge ZHANG Guang-qiong LIU Huan YANG Hong-yu SHENG Xiang-chun. Effect of Smad7 deficiency on rat cardiac fibroblasts proliferation, migration, cell differentiation and collagenⅠ secretion in vitro [J]. Acta Anatomica Sinica, 2022, 53(5): 578-584.
[3]	LIU Meng-xin SUN Chang-ye Li Duo REN Ming-fen GUO ZhI-kun. Effect and mechanism of fibroblast growth factor on proliferation and transdifferentiation of cardiac fibroblasts in rats#br# [J]. Acta Anatomica Sinica, 2021, 52(5): 712-719.
[4]	MAO Zhi-rong LIU Fang DU Jie DENG Li GAO Xiao-qing. Protective effect of nuclear factor E2-related factor 2 on oxidative injury in human immortalized epidermal cells [J]. Acta Anatomica Sinica, 2021, 52(2): 225-230.
[5]	CHANG Yu-qiao JIA Yang-yang HAN Bei GUO Zhi-kun. Differences in genes expression associated with rat cardiac fibroblasts differentiation into cardiomyocyte-like cells induced by 5-azacytidine [J]. Acta Anatomica Sinica, 2020, 51(6): 861-867.
[6]	GUO Meng-yao SUN Lin LIN Xu ZHANG Wen-jing ZHANG Jing WU Jing-fang XUE Gang. Role of trefoil factor3/phosphatidylinositl 3-kinase/protein kinase B/nuclear factorκB in the proliferation and apoptosis of human papillary thyroid carcinoma cell line TPC-1 [J]. Acta Anatomica Sinica, 2020, 51(4): 528-535.
[7]	WANG Li-you ZHANG Dong-ying. Effect of alkaline phosphatase liver/bone/kidney overexpression on ventricular remodeling after acute myocardial infarction in mice#br# [J]. Acta Anatomica Sinica, 2020, 51(3): 431-436.
[8]	DING Ling GAO Jie LI Hong HU Rong SU Min. Establishment of paired box gene 6/mouse embryonic stem cell line and its stem cell biological characteristics [J]. Acta Anatomica Sinica, 2019, 50(4): 451-458.
[9]	DAI Jin LIN Xu GUO Meng-yao ZHANG Jing ZHANG Wen-jing XUE Gang WU Jing-fang. Effect and molecular mechanism of trefoil factor family 3 on the cell cycle of thyroid papillary carcinoma cell [J]. Acta Anatomica Sinica, 2019, 50(2): 211-219.
[10]	LI Jun-hua MA Yong-neng GU Rui-cai LIAO Hua. Knockdown of calcium/calmodulin-dependent protein kinaseⅣ reduce the immune characteristic of C2C12 under stimulation of interferon-γ [J]. Acta Anatomica Sinica, 2019, 50(2): 166-172.
[11]	TIAN Xiang-qin TAN Zhao-yang LI Xin-zhi MA Ke-tao LI Li SI Jun-qiang﹡. Expression of anoctamin 1 in the process of myocardial fibrosis [J]. Acta Anatomica Sinica, 2019, 50(2): 173-178.
[12]	WU Yang LIU Ying LIU Ji-tao YIN De-feng. A preliminary study on the protective effect and mechanism of paraoxonase 3 gene overexpression on hepatic injury in acutely poisoned mice [J]. Acta Anatomica Sinica, 2019, 50(2): 179-184.
[13]	HUANG Yue LAN Jiang HU Rong GAO Jie LI Hong SU Min. Effect of transfecting the vascular endothelial growth factor gene in vivo on the mice with pulmonary hypertension induced by chronic hypoxia [J]. Acta Anatomica Sinica, 2018, 49(6): 714-719.
[14]	ZHANG Jun-yue YIN Guo-tian GUO Zhi-kun . Endothelial differentiation potential of neonatal rat cardiac fibroblasts [J]. Acta Anatomica Sinica, 2018, 49(4): 443-449.
[15]	CHANG Yu-qiao LI Ci-xia JIA Yang-yang GUO Zhi-kun. Multiple phenotypes in cardiac fibroblasts [J]. Acta Anatomica Sinica, 2018, 49(3): 317-323.