EFFECT OF TRITON X-100 ON LIPOSOME MEDIATED BONE MORPHOGENETIC PROTEIN 2 BY TRANSFECTION OF RAT BONE MARROW MESENCHYMAL STEM CELLS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

 XIADelin , HUANGMingke , FUGuangxing , MAZheng , WUShuangjiang , ZHOUHangyu

Department of Oral and Maxillofacial Surgery, the Affiliated Hospital of Luzhou Medical College, Luzhou Sichuan, 646000, P. R. China;

Corresponding author：

XIADelin, Email: xiadeln@163.com

Keywords：

Bone marrow mesenchymal stem cells; Triton X-100; Liposome; Gene therapy

DOI：

10.7507/1002-1892.20150016

Video：

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Objective To study the effect of Triton X-100 promoting liposome-mediated bone morphogenetic protein 2 (BMP-2) gene transfection of rat bone marrow mesenchymal stem cells (BMSCs). Methods BMSCs were separated and cultured from the femur and tibia of healthy Wistar rats (8-week-old, male). The 3rd passage BMSCs identified by detecting the surface antigen were used to transfect. The optimum concentration of Triton X-100 for liposome mediated gene transfection was determined with ELISA meter by the way of MTT. In optimum concentration of Triton X-100, liposome mediated BMP-2 gene was transfected to BMSCs. The experiment was divided into 3 groups according to types of trasfection agents:BMSCs were transfected with Triton X-100+liposome+BMP-2 (experimental group), with liposome+ BMP-2 (conventional transfection group), and untransfected BMSCs served as blank control group. After 48 hours of transfecting, the green fluorescent protein (GFP) in cells was detected through inverted fluorescence microscope. After 72 hours of transfection, real-time fluorescence quantitative PCR was applied to measure the mRNA expression of BMP-2. Results 0.01% Triton X-100 was determined to be the optimum concentration for not only making the BMSCs maintain vitality, but also achieving a certain effect on BMSCs. After trasfecting for 48 hours, GFP was observed through inverted fluorescence microscope in the experimental group and conventional transfection group, but was not observed in the blank control group. After trasfecting for 72 hours, the relative BMP-2 mRNA expression level was 5.94±0.12 in the experimental group, and was 4.99±0.08 in the conventional transfection group, showing significant difference (t=360.28, P=0.02). The transfection efficiency was increased by 19% in the experimental group. Conclusion 0.010%Triton X-100 can promote the liposome mediated BMP-2 gene transfection of rat BMSCs, and can improve the transfection efficiency.

Citation： XIADelin, HUANGMingke, FUGuangxing, MAZheng, WUShuangjiang, ZHOUHangyu. EFFECT OF TRITON X-100 ON LIPOSOME MEDIATED BONE MORPHOGENETIC PROTEIN 2 BY TRANSFECTION OF RAT BONE MARROW MESENCHYMAL STEM CELLS. Chinese Journal of Reparative and Reconstructive Surgery, 2015, 29(1): 69-73. doi: 10.7507/1002-1892.20150016 Copy

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16.	Mohammadabadi MR, El-Tamimy M, Gianello R, et al. Supramolecular assemblies of zwitterionic nanoliposomepolynucleotide complexes as gene transfer vectors:Nanolipoplex formulation and in vitro characterisation. J Liposome Res, 2009, 19(2):105-115.
17.	王冰, 张树彪, 周集体, 等. 阳离子脂质体介导的基因转移机制. 中国组织工程研究与临床康复, 2011, 15(8):1459-1462.
18.	Ko IK, Ziady A, Lu S, et al. Acid-degradable cationic methacrylamide polymerized in the presence of plasmid DNA as tunable non-viral gene carrier. Biomaterials, 2008, 29(28):3872-3881.
19.	Amidzadeh Z, Behbahani AB, Erfani N, et al. Assessment of different permeabilization methods of minimizing damage to the adherent cells for detection of intracellular RNA by flow cytometry. Avicenna J Med Biotechnol, 2014, 6(1):38-46.
20.	沈明, 蒋海鹰. TritonX-100在培养细胞免疫组化染色中的应用. 临床与实验病理学杂志, 1998, 14(2):197-199.
21.	姜笃银, 钱春华, 周兵, 等. 不同脱细胞方法对无细胞真皮基质抗原成分的影响. 中国临床康复, 2005, 9(6):35-37.
22.	Johnsson M, Bergstrand N. Phase behavior of DOPE/TritonX100(reduced) in dilute aqueous solution:aggregate structure and pHdependence. Colloids Surf B Biointerfaces, 2004, 34(2):69-76.

1. 催少千, 范广宇, 李建军, 等. 腺病毒介导的BMP-2基因转染兔骨髓基质干细胞对其增殖、成骨分化的影响. 东南大学学报:医学版, 2009, 28(1):22-25.
2. 夏德林, 刘道华, 贾娟, 等. 兔骨髓基质干细胞体外分离培养及生物学特性研究. 中国美容医学, 2011, 20(8):1251-1254.
3. 赵冰冰, 张玮, 王琪, 等. TIZ基因全长扩增和携带其基因的真核表达载体的构建. 广西医科大学学报, 2012, 29(1):45-47.
4. Bryksin AV, Matsumura I. Overlap extension PCR cloning:a simple and reliable way to create recombinant plasmids. Biotechniques, 2010, 48(6):463-465.
5. Minmer M, Simanek E. Nonviral Vectors for Gene Delivery. J Chem Rev, 2009, 109(2):259-302.
6. 魏云波, 高平, 刘书花, 等. 阳离子脂质体转染效率的主要影响因素. 北方药学, 2012, 9(11):86.
7. Hu YL, Huang B, Zhang TY, et al. Mesenchymal stem cells as a novel carrier for targeted delivery of gene in cancer therapy based on nonviral transfection. Mol Pharm, 2012, 9(9):2698-2709.
8. 陈彦祥, 乔卫红, 刘栋良, 等. 阳离子脂质体的转染机制及转染效率影响因素. 生物工程学报, 2007, 23(5):776-780.
9. Zhang Y, Rong Qi X, Gao Y, et al. Mechanisms of co-modified liver-targeting liposomes as gene delivery carriers based on cellular uptake and antigens inhibition effect. J Control Release, 2007, 117(2):281-290.
10. 张昌明, 吴永寿, 余宏. 阳离子脂质体基因载体的研究进展. 医学综述, 2009, 15(12):1768-1770.
11. Shin J, Quails MM, Boomer JA, et al. An eficient new route to plasmenyl-typo lipids:synthesis an d cytotoxicity of a plasmenylcholine an alogue of the an titumor ether lipid ET-18-OMe. J Am Chem Soc, 2001, 123(3):508-509.
12. Guosen H, Min F, Xin L, et al. Design, synthesis and in vitro evaluation of a novel "stealth" polymeric gene vector. Int J Pharm, 2008, 350(1):344-350.
13. Incani V, Tunis E, Clements BA, et al. Palmitic acid substitution on cationic polymers for effective delivery of plasmid DNA to bone marrow stromal cells. J Biomed Mater Res, 2007, 81(2):493-504.
14. Zabner J, Fasbender AJ, Moninger T, et al. Cellular and molecular barrier to gene transfer by a cationic lipid. J Biol Chem, 1995, 270(32):18997-19007.
15. Managit C, Kawakami S, NishikawaM, et al. Targeted and sustmned drug delivery using PEGylated galactosylated liposomes. Int J Pharm, 2003, 266(1-2):77-84.
16. Mohammadabadi MR, El-Tamimy M, Gianello R, et al. Supramolecular assemblies of zwitterionic nanoliposomepolynucleotide complexes as gene transfer vectors:Nanolipoplex formulation and in vitro characterisation. J Liposome Res, 2009, 19(2):105-115.
17. 王冰, 张树彪, 周集体, 等. 阳离子脂质体介导的基因转移机制. 中国组织工程研究与临床康复, 2011, 15(8):1459-1462.
18. Ko IK, Ziady A, Lu S, et al. Acid-degradable cationic methacrylamide polymerized in the presence of plasmid DNA as tunable non-viral gene carrier. Biomaterials, 2008, 29(28):3872-3881.
19. Amidzadeh Z, Behbahani AB, Erfani N, et al. Assessment of different permeabilization methods of minimizing damage to the adherent cells for detection of intracellular RNA by flow cytometry. Avicenna J Med Biotechnol, 2014, 6(1):38-46.
20. 沈明, 蒋海鹰. TritonX-100在培养细胞免疫组化染色中的应用. 临床与实验病理学杂志, 1998, 14(2):197-199.
21. 姜笃银, 钱春华, 周兵, 等. 不同脱细胞方法对无细胞真皮基质抗原成分的影响. 中国临床康复, 2005, 9(6):35-37.
22. Johnsson M, Bergstrand N. Phase behavior of DOPE/TritonX100(reduced) in dilute aqueous solution:aggregate structure and pHdependence. Colloids Surf B Biointerfaces, 2004, 34(2):69-76.

Chinese Journal of Reparative and Reconstructive Surgery

EFFECT OF TRITON X-100 ON LIPOSOME MEDIATED BONE MORPHOGENETIC PROTEIN 2 BY TRANSFECTION OF RAT BONE MARROW MESENCHYMAL STEM CELLS

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