Experiment Studys On Ectopic Chondrogenesis Of Nude Mouse Induced By IGF-1 Gene-enhanced Tissue Engineering Technology

Objective: To explore some new methods in the repair of cartilage injury by making some innovative attempts on the use of gene-enhanced tissue engineering.We used the gene of human insulin like growth favtor-l(WGF-l) to transfect and modify human mesenchymal stem cells(hMSCs) by gene recombination technology, and observed the expression of hIGF-1.WE cultivated hMSCs transfected by hIGF-1 gene together with tissue engineering material-regenerated silk fibroin film to form the complexes of hMSCs-hIGF-1- regenerated silk fibroin film, and then implanted them into the nude mouse to determine the capacity of inducing ectopic chondrogenesis.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 chondrogenesis differentiation with phase contrast microscope, and also we have drawn the growth curve, assayed the cell phenotype with flow cytometer, determined the type- II collagen phenotype by immunohistochemical analysis.2. We designed and synthesized a pair of specific primer for the sequence encoding hIGF-1 gene reported in Genebank.We amplified hIGF-1 gene from adult human liver by Reverse Transcript Polymerase Chain Reaction (RT-PCR), and cloned it into expresion vector pSec Tag/FRT/V5-His after double enzyme digestion with NheI and BamHI to contructed a eukaryotic expression system for hIGF-1, then verified the positive clone by enzyme digest and sequenccFinally we transfected the positive plasmid to CHO cells by Lipofectamine 2000, and collected condition medium for SDS/PAGE and Western Blot analysis after transfection.3. We transferred the recombinant plasmid pSec Tag/FRT/V5-His- hIGF-1 into induced hMSCs by means of Lipofectamine, and then made a series of analysis such as RT-PCR, EIA and immunohistochemical analysis to determine the situation of hIGF-1 and type-II collagen phenotype expression.4. We seeded transfected hMSCs into regenerated silk fibroin film, and have incubated together for one week to form thr complexes of hMSCs- hIGF-1- regenerated silk fibroin film.We observed complexes under scanning electron microscope.Also we implanted complexes under the dorsal skin of nude mise about 4weeks to examine therespective specimens histologically and immunohistchemically.Results:1 . We can isolate hMSCs with discontinuous gradient centrifugation on PercoU.HMSCs showed spindle cells in shape and had potentials for proliferation and chondrogenesis differentiation while cultured in vitro.We find the phenotype of cells was similar to the characteristic of MSCs and cartilages.2. The hIGF-1 gene has been successfully cloned from adult human liver tissue and was validated by sequence analysis and DNA agarose gel electrophoresis.We constructed the eukaryotic expression system pSec Tag/FRT/V5-His- hIGF-1 and transferred it into CHO by means of Lipofectamine successfully .From molicular RNA and protein level we validated that trasfected CHO by pSec Tag/FRT/V5-His- hIGF-1 can express and secret active hIGF-1 stably.3. By means of Lipofectamine, hIGF-1 gene have been successfully exerted into induced hMSCs , which was validated by RT-PCR and EIA analysis.The cartilage type- II collagen phenotype was tesyed by immunohistochemical analysis again.4. The trasfected hMSCs can attach and grow well on the three dimensional scaffold of regenerated silk fibroin film.Histologically and immunohistochemically, the implanted complexes of hMSCs- hIGF-1- regenerated silk fibroin film in the nude mouse showed formation of new cartilage.Conclusion:1. hMSCs can be obtained by discontonuous gradient centrifugation and can be cultivated in vitro for proliferation and chondrogenesis differentiation.2. The hIGF-1 gene can be cloned from adult human liver tissue and its eukaryotic expression system pSec Tag/FRT/V5-His- hIGF-1 can be constructed successfully.3. hIGF-1 gene can be transfected into induced hMSCs by means of Lipofectamine and can be expressed to develop its biologic function.The Transfected hMSCs still express cartilage phenotype.4. Transfected hMSCs show well compatibility on the surface of regenerated silk fibroin film.The implanted complexes of hMSCs- hIGF-1- regenerated silk fibroin film under the skin of nude mise can induce ectopic chondrogenesis.