The Preparation Of TAT/LMP-3 Fusion Protein And Its Effects On Osteogenic Differentiation Of Human Marrow Mesenchymal Stem Cells

Spinal fusion is one of the commonest procedures in spinal surgery, which is widely used to treat degenerative disease, trauma and tumor of the spine. However, pseudoarthrosis formation occurs in up to 45% patients following spinal fusion. Meanwhile, the use of autologous iliac bone graft in the procedure results in additional surgical trauma and certain complications. Hence, it has become a focus of interest to further increase the success rate of spinal fusion and find ideal grafting substitute.It has been found that some biological factors play important roles in inducing osteoblast differentiation and promoting local bone formation, and these factors are called osteoinductive factors. Many osteoinductive factors have been discovered, investigated and applied to spinal fusion procedures. BMPs are the osteoinductive factors that have been studied most intensively. It was confirmed that rhBMP-2 increases the success rate of spinal fusion and attenuates surgical trauma. Nevertheless, rhBMP-2 is costly and its efficacy is dose-dependent. Normally, several milligrams of rhBMP-2 are required to achieve satisfactory clinical results.LMP is a recently discovered intracellular signaling factor that can induce osteoblast differentiation and maturation as well as bone formation. It has been shown that LMP can induce the secretion of osteoinductive factors such as BMP-2, BMP-4, BMP-6, BMP-7 and TGF-βin vitro and in vivo, and improve the responsiveness of osteogenesis-related cells to exogenous BMPs, and recruit peripheral cells to form bone. Animal experiments demonstrated that LMP-1 is more potent than BMP-2 in inducing ectopic bone formation and spinal fusion. Local gene therapy using LMP as target gene resulted in significant osteogenic effect without needing transfection of all recipient cells or long-term expression of the target gene. Hence, LMP is superior to BMPs for use in spinal fusion. These findings demonstrate that LMP-3 and LMP-1 are ideal substitutes for BMPs, and that they avoid the pitfalls in the use of large doses of BMPs alone and show more potent action to induce osteogenesis.Three LMP genotypes have been identified. Boden et al investigated the structure and function of LMP homologous proteins. According to their findings, hLMP-1 comprises 457 amino acid residues, including one PDZ domain at its N-terminal and three LIM domains at its C-terminal, hLMP-2 comprises 423 amino acid residues, including a complete PDZ domain and LIM domains, and hLMP-3 comprises 153 amino acid residues, including a complete PDZ domain, but no LIM domain. Further studies demonstrated that hLMP-2 has complete LIM domains, but has no osteogenesis-promoting effect, and that hLMP-3 has no LIM domain, but has a potential to induce osteogenesis just like hLMP-1. It was further indicated that LMP-1 and LMP-3 have a unique domain comprising 40 amino acid residues and this domain is closely related to osteogenesis.LMP gene therapy is currently mediated by plasmid and adenovirus and LMP gene over-expression is desired. The efficiency of gene transfer is relatively low in gene therapy. Moreover, gene therapy may bring some safety concerns. For instance, gene therapy might cause insertion mutations and cell malignant transformation. Through constructing recombinant LMP protein with cell transduction capability and osteogenic activity, we hope to broaden the application of LMP, particularly, to use LMP clinically.In 1988, Green and Frankle et al discovered human immunodeficiency virus-1 (HIV-1) trans-activation protein (TAT) which mediates substance transportation across cell membrane. Further researches revealed that the TAT protein contains a major domain which is a sequence of nine amino acid residues, i.e., tat49-57(RKKRRQRRR), and that this domain is the smallest fragment to conserve the functional activity of the TAT protein. An array of short peptides have been found to have similar transmembrane transporting capability, and these peptides are collectively termed protein transduction domains (PTDs). PTD transduction technology can be applied to transporting multiple biological macromolecules such as protein, without influencing the biological activity of molecules transported. Moreover, this technology is not cell type-specific. Hence, PTD transduction technology may be used widely, for instance, in cell biology and drug development. PTD transduction exhibits obvious advantages: (1) PTD can transport substances across cell membrane quickly and efficiently; (2) most PTD complexes are not toxic within a certain concentration range and impact living cells insignificantly; (3) PTD transduction technology is easy to operate, and its transduction is efficient. Under most circumstances, the substances transported can have their biological activity not affected, and thus can exert their biological action under low concentrations.As mentioned above, TatPTD can deliver protein across cell membrane efficiently. We planned to fuse tat49-57(RKKRRQRRR) with LMP-3 and transport LMP-3 into target cells using TatPTD. Bone marrow mesenchymal stem cells (MSCs) exhibit the potential of multidirectional differentiation and are common seed cells in tissue engineering. MSCs can differentiate into osteoblasts, and they have many advantages, such as ample sources, being easy to prepare, and strong potential of proliferation and differentiation, as well as good compatibility with host bone. In the present study, we cultured human MSCs with TAT/LMP-3 fusion protein to investigate the transduction capability of the Tat sequence and the potential of TAT/LMP-3 fusion protein to promote osteogenic differentiation of human MSCs.Part 1. Prokaryotic expression and purification of TAT/LMP-3 fusion protein and preparation of polyclonal antibodies against TAT/LMP-3 fusion protein1. LMP-3 encoding sequence optimized for E. coli expression system was synthesized. TAT/LMP-3 fusion gene and LMP-3 gene were obtained by PCR amplification and prokaryotic expression vectors pET43.1a-TAT/LMP-3 and pET43.1a-LMP-3 were constructed. Sequencing results suggest successful plasmid construction, with no PCR caused mutations or frame shifts.2. The prokaryotic vectors were expressed by BL21(DE3) engineering bacteria. Under the optimized expression conditions, soluble expression of target proteins was dominated. The supernatant expression products were subjected to purification using Ni-NTA resin to obtain recombinant TAT/LMP-3 and LMP-3 proteins. The purity of the target proteins was higher than 90%.3. Rabbits were immunized with TAT/LMP-3 to obtain polyclonal antibody containing serum, and polyclonal antibodies were purified using saturated ammonium sulfate. ELISA indicated the titer of TAT/LMP-3 and LMP-3 to be higher than 1:4000, and Western blotting analysis suggested sound reactiveness of the antibody to TAT/LMP-3 and satisfactory specificity of the antibody.Part 2. Isolation, culture and identification of hMSCs 1. hMSCs were isolated and purified through Percoll density gradient centrifugation with the assistance of adherent culture, and hMSCs were subjected to flow cytometry for cell surface marker expression after two passages. The result indicates the cells were positive for CD29 and CD105, while negative for hematopoietic cell surface marker such as CD34 and CD45. The cell purity was higher than 95%.2. Under induction with the osteogenic medium containing dexamethasone (10-8 mol/L),β-sodium glycerophosphate (10mmol/L), and vitamin C (50mg/L), hMSCs showed an increase in the salkaline phosphatase activity. In addition, expression of osteocalcin in hMSCs was detected by immunocytochemical stain. After 14d of culture, alizarin red S staining suggested the formation of calcified nodules. These findings demonstrated that hMSCs can be induced to differentiate into osteoblasts under certain conditions.Part 3. TAT/LMP-3 fusion protein induces osteogenic differentiation of hMSCs1. hMSCs were cultured with TAT/LMP-3 and LMP-3, respectively. Endocytosis of TAT/LMP-3 was detected by indirect immunofluorescence method and Western blotting. The results suggested that TAT/LMP-3 can be transduced quickly and efficiently into the cell in a concentration- and time-dependent manner. The control protein LMP-3 cannot be transduced into the cell.2. hMSCs were cultured with TAT/LMP-3 and LMP-3, respectively. The efficacy of TAT/LMP-3 to induce osteogenic differentiation of hMSCs was assessed. The results suggested that 100ng/ml TAT/LMP-3 can induce hMSCs to express the marker genes of osteoblasts such as type I collagen, alkaline phosphatase, and osteocalcin. Calcified nodules were observed after 14d of culture. RT-PCR suggested that TAT/LMP-3 upregulated BMP-2, BMP-4, BMP-6, BMP-7 gene expression by hMSCs.