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：

Export PDF Favorites Scan Get Citation

Abstract Full text Figures/Tables Video References Cited by

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

1.	催少千, 范廣宇, 李建軍, 等. 腺病毒介導的BMP-2基因轉染兔骨髓基質干細胞對其增殖、成骨分化的影響. 東南大學學報:醫學版, 2009, 28(1):22-25.
2.	夏德林, 劉道華, 賈娟, 等. 兔骨髓基質干細胞體外分離培養及生物學特性研究. 中國美容醫學, 2011, 20(8):1251-1254.
3.	趙冰冰, 張瑋, 王琪, 等. TIZ基因全長擴增和攜帶其基因的真核表達載體的構建. 廣西醫科大學學報, 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, 劉書花, 等. 陽離子脂質體轉染效率的主要影響因素. 北方藥學, 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.	陳彥祥, 喬衛紅, 劉棟良, 等. 陽離子脂質體的轉染機制及轉染效率影響因素. 生物工程學報, 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.	張昌明, 吳永壽, 余宏. 陽離子脂質體基因載體的研究進展. 醫學綜述, 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, 張樹彪, 周集體, 等. 陽離子脂質體介導的基因轉移機制. 中國組織工程研究與臨床康復, 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, 蔣海鷹. TritonX-100在培養細胞免疫組化染色中的應用. 臨床與實驗病理學雜志, 1998, 14(2):197-199.
21.	姜篤銀, 錢春華, 周兵, 等. 不同脫細胞方法對無細胞真皮基質抗原成分的影響. 中國臨床康復, 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, 范廣宇, 李建軍, 等. 腺病毒介導的BMP-2基因轉染兔骨髓基質干細胞對其增殖、成骨分化的影響. 東南大學學報:醫學版, 2009, 28(1):22-25.
2. 夏德林, 劉道華, 賈娟, 等. 兔骨髓基質干細胞體外分離培養及生物學特性研究. 中國美容醫學, 2011, 20(8):1251-1254.
3. 趙冰冰, 張瑋, 王琪, 等. TIZ基因全長擴增和攜帶其基因的真核表達載體的構建. 廣西醫科大學學報, 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, 劉書花, 等. 陽離子脂質體轉染效率的主要影響因素. 北方藥學, 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. 陳彥祥, 喬衛紅, 劉棟良, 等. 陽離子脂質體的轉染機制及轉染效率影響因素. 生物工程學報, 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. 張昌明, 吳永壽, 余宏. 陽離子脂質體基因載體的研究進展. 醫學綜述, 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, 張樹彪, 周集體, 等. 陽離子脂質體介導的基因轉移機制. 中國組織工程研究與臨床康復, 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, 蔣海鷹. TritonX-100在培養細胞免疫組化染色中的應用. 臨床與實驗病理學雜志, 1998, 14(2):197-199.
21. 姜篤銀, 錢春華, 周兵, 等. 不同脫細胞方法對無細胞真皮基質抗原成分的影響. 中國臨床康復, 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

Abstract Full text Figures/Tables Video References Cited by

Previous Article

Next Article

Format

Content