Bone morphogenetic protein-2/basic fibroblast growth factor (BMP-2/bFGF) double-gene chitosan nano-microcapsules and application thereof
A technology of chitosan nanometer and BMP-2, which is applied in the field of biomedicine, can solve the problems of cumbersome acquisition of bone marrow stromal cells, limitation of source of seed cells, difficult acceptance by patients, etc., and achieve good application prospects, good effect and simple preparation method Effect
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Embodiment 1B
[0028] The preparation of embodiment 1BMP-2 / bFGF double gene chitosan nano-microcapsule
[0029] The preparation method of BMP-2 / bFGF double-gene chitosan nano-microcapsules is mainly divided into the following 4 steps:
[0030] ①Construction of BMP-2 and bFGF labeled with green and red fluorescent proteins: pEGFP-N2-Hu-BMP-2 and pTagRFP-C1-Hu-bFGF were constructed respectively, and were identified by colony PCR, double enzyme digestion and sequencing. These methods all indicate that the construction of the pTagRFP-Hu-bFGF recombinant expression vector is correct, and that Hu-bFGF and RFP (red fluorescent protein) can be expressed in high-level eukaryotic cells in the form of fusion expression with the help of Kozak sequence. The build method is as follows.
[0031] (1) Construction of pEGFP-N2-Hu-BMP-2: Total RNA of human fibroblasts was extracted with a total RNA extraction kit (Hangzhou Haoji Biotechnology Co., Ltd.), and then SuperScript TM II RTase (Invitrogen) was used ...
Embodiment 2
[0069] The identification of embodiment 2 chitosan nano microcapsules
[0070] Agarose gel electrophoresis identification of chitosan-plasmid nanoparticles: take chitosan-plasmid nanoparticles, perform 0.8% agarose gel electrophoresis, stain with ethidium bromide, and then UV imaging, P control is a pure plasmid Swimming lanes, No. 1 and No. 2 are chitosan-plasmid nanoparticle swimming lanes, because DNA and chitosan form wrapped particles and stay near the sample hole ( image 3 ).
[0071] Morphology and particle size of chitosan-plasmid nanoparticles: The prepared nanocapsules containing plasmids were directly placed under an atomic force microscope (SPI3800N) to observe that the chitosan nanocapsules were irregular spherical, compact in structure and particle size About 100-200nm, relatively uniform in size ( figure 1 ). Then chitosan-plasmid nanoparticles are diluted with 30mmol / LNa2SO4 by 1:15, get the diluted solution 5ul and fix it on the clean mica sheet, and use N...
Embodiment 3B
[0072] Example 3 Expression of BMP-2 / bFGF double-gene chitosan nanocapsules transfected into adipose stem cells
[0073] Adipose-derived stem cells were transfected with BMP-2 / bFGF dual-gene chitosan nanocapsules. Under a fluorescent microscope, green and red appeared in the same cell 24 hours after transfection, indicating that the double-gene transfer marked with red and green fluorescent proteins was transfected. expressed in the same cell ( figure 2 ).
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