Experiment Studies On Ectopic Osteogenesis Of Athymic Mouse Spine Induced By The Use Of Gene Engineering And Tissue Engineering Technology

Objective: To explore some new ideas and methods in the repair of bone injury and spine fusion by making some innovative attempts on the use of gene engineering in association with tissue engineering. We used the gene of human bone morphogenetic protein -2 (hBMP2) to transfect and modify human mesenchymal stem cells (hMSCs) by gene recombination technology, and observed the expression of hBMP2. We cultivated hMSCs transfected by hBMP2 gene together with tissue engineering material- Hydroxyapatite (HA) to form the complexes of hMSCs- hBMP2-HA, and then implanted them into the athymic mouse to determine the capacity of inducing ectopic osteogenesis.Methods: 1. We obtained hMSCs with discontinuous gradient centrifugation on Percoll. After cultivation and proliferation in vitro, we observed the morphological changes and the capacity of inducing osteogensis differentiation with phase contrast invert microscope, and also we have drawn the growth curve and assayed the cell phenotype with flow cytometer. 2. We designed and synthesized a pair of specific primers for the sequence encoding hBMP2 mature peptide gene reported in Genebank. We amplified 342bp hBMP2 mature peptide gene from adult skeletal muscle by Reverse transcript polymerase chain reaction (RT-PCR), and cloned it into PBV220 vector to form a pronucleus expression system of hBMP2. After sequence analysis, the recombinant plasmid PBV220- hBMP2 was transformed into E.coli DH5 a and incubated it at 42 癈 to express encoded protein- hBMP2, then analyzedthe expressed protein with SDS-PAGE. 3. We designed and synthesized four primers for the sequence encoding hBMP2 full-length cDNA. Using nested RT-PCR, we amplified 1188bp full-length hBMP2 gene from adult skeletal muscle and cloned into pUCM-T vector. After double enzyme digestion with Hind and Xba , we inserted full-length hBMP2 gene into pcDNA3 vector to construct a eukaryotic expression system for hBMP2. We transferred the recombinant plasmid pcDNA3- KBMP2 into hMSCs by means of Lipofectamine, and then made a series of analysis such as RT-PCR dot-ELISA immunohistochemical analysis and ALP activity to determine the situation of hBMP2 expression. 4. We seeded transfected hMSCs into HA scaffold, and have incubated together for 4 day to form the complexes of hMSCs-hBMP2-HA. We observed complexes under phase contrast invert microscope and scanning electron microscope. Also, we implanted complexes into the spinal muscle of athymic mise about 4 weeks to examine the respective specimens histologically.Results: 1. We can isolate hMSCs with discontinuous gradient centrifugation on Percoll. hMSCs showed spindle cells in shape and had potentials for proliferation and osteogensis differentiation while cultured in vitro. We find the phenotype of cells was similar to the characteristics of MSCs. 2. The hBMP2 mature peptide gene was amplified and sequenced exactly in comparison with the reported sequence in Genebank. DNA agarose gel electrophoresis showed that the product of RT-PCR was about 342bp, and double enzyme digestion of recombinant PBV220- hBMP2 plasmid corresponded with it. After the engineered E.coli DH5 a bacteria was induced for 3h 4h 5h, an anticipated 16KD protein band appeared on SDS-PAGE gel, and the expression amount achieved most at 4h induction. 3. Using nested RT-PCR and T-A Cloning, we amplified 1188bp full-lengthhBMP2 gene from adult skeletal muscle and was validated by sequence analysis and DNA agarose gel electrophoresis. We constructed the eukaryotic expression system pcDNA3- KBMP2 and transferred it into hMSCs by means of Lipofectamine successfully. From molecular RNA level and protein level, we validated that trancfected hMSCs by pcDNA3- hBMP2 can express and secrete active hBMP2 stably. 4. hMSCs can attached and growed well on the three-demension suface of HA. Histologically, the implanted complexes of hMSCs- hBMP2-HA in the athymic mouse showed formation of new bone.Conclusion: 1. hMSCs can be obtained by discontinuous gradient centrifugation and can be cultivated in vitro for proli...