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Purpose: To genetic engineering and tissue engineering technology continues to development, in order repairing bone defect and the promotion of spinal fusion provides a more treatment choices. Of which LIM mineralization protein (LIM mineralization protein, LMP) is the in recent years, research hotspot. This experiment by constructing eukaryotic expression plasmid pBudCE4.1-LMP-1-LMP-3, pBudCE4.1-LMP-1 and pBudCE4.1-LMP-3, and separately transfected Rabbit Bone Marrow mesenchymal stem cells, observe the experiment each group LMP-1 with the LMP-3 gene and protein expression the situation, comparative carry double gene with the carrying LMP-1 or LMP-3 single-gene of bone marrow mesenchymal stem cells in the expression of on the difference, for the further animal experiments provide theoretical support. Method: 1, will artificial synthetic-containing Hind Ⅲ / BamH Ⅰ restriction enzyme digestion-bit point person LMP-1 gene fragment and containing Xhol / kpn Ⅰ digested-bit point person LMP-3 gene fragments, respectively, with the intermediary plasmids Puc57 carried out ligation reaction, construct the plasmid Puc57 -LMP-1 and Puc57-LMP-3, parallel enzyme digestion and sequencing identification. 2, LMP-1 and LMP-3 gene, respectively, and eukaryotic pairs of expression plasmid pBudCE4.1 carried out ligation reaction, were constructed respectively plasmids pBudCE4.1-LMP-1 and pBudCE4.1-LMP-3, go through restriction digestion and sequencing confirmed, and to In the recombinant plasmid pBudCE4.1-LMP-1 based on the re carried out with the LMP-3's ligation reaction, and then by enzyme digestion and sequencing identification. 3, separation, cultured rabbit bone marrow mesenchymal stem cells, on cell carry out microscopic morphological observation. Will cells were divided into five group namely A: blank control group B: pBudCE4.1 group C: pBudCE4.1-LMP-1 group D: pBudCE4.1-LMP-3 group E: pBudCE4.1-LMP-1-LMP- 3 groups. With liposome France will four kinds plasmid was according to the grouping the in vitro, respectively, transfected into Rabbit Bone Marrow mesenchymal stem cells. Four, right the above-mentioned five group were carried out RT-PCR and Western Blot check its gene and protein expression, and compare the its differences between groups Xing. Results: 1, restriction enzyme digestion and sequencing confirmed that plasmid was Puc57-LMP-1, Puc57-LMP-3, pBudCE4.1-LMP-1, pBudCE4.1-LMP-3 and pBudCE4.1-LMP-1-LMP-3 True -core dual-expression plasmid constructed successfully. 2, The pairs expression plasmid In the vitro transfection MSCs cells after the can express LMP-1 and LMP-3 molecules. 3, on the RT-PCR and Western Blot detection results OK gray value measurement display: LMP-1 mRNA and protein levels the expression of, A, B group with the C, D, E difference between the groups there are was statistically significance (P lt; 0.05 ), C group and E group difference was not statistically significance (P gt; 0.05); LMP-3 mRNA and protein levels the expression of, A, B group with the C, D, E difference between the groups there are was statistically significance (P lt; 0.001), D group and E group difference was statistically significance (P lt; 0.05), C group and the D group In the LMP-1 and LMP-3 the expression of on the difference was not statistically significance (P gt; 0.05). Conclusions: 1, successfully constructed pBudCE4.1-LMP-1, pBudCE4.1-LMP-3 and pBudCE4.1-LMP-1-LMP-3 eukaryotic expression plasmid. 2, transfected transfected pBudCE4.1-LMP-1-LMP-3 of bone marrow mesenchymal Stem cells can in vitro simultaneous expression of LMP-1 and LMP-3 molecules. 3, double gene transfected transfection significantly improves the LMP-3 the expression level of, LMP-1 with LMP-3 exists between the interaction Expression Function.
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