The Study On Preliminary Construction Of Tissue Engineered Skin Based On The Local Micro-environment Of Dabietic Skin Ulcers

ObjectiveIt is a challenge to the treatment for chronic ulcer of diabetes, which affects the life quality seriously. In this study, according to the local acid micro- environmental of clinical diabetic skin ulcers, natural, acid sensitive macro- molecule hydrogel was designed. NGF was selected as interest protein, constructed the green fluorescent protein eucaryotic expression vector, transfected eukaryocyte and gained a cell strain which expressing interest protein steadily. Used the hydrogel material loading transgenic cell to prepare cell/scaffold three-dimensional composition. The seed cells are expected to achieve controlled release growth factors and extracellular matrix responsing to acid environment to promote the healing of diabetic skin ulcers.MethodsPart one:Selected NGF as the target gene. Genomic DNA was extracted from healthy adults, two primers with restriction enzyme site of BamH I and Nhe I were designed according that gene sequence of human NGF of Ensembl and the multiple cloning site of pSELECT-GFPzeo-MCS Vector, humanβ-NGF precursor gene was amplified by PCR; then the NGF gene was inserted into pGM-T vector to construct cloning plasmid. After sequenced and identified, inserted the NGF segment into expression plasmid to construct recombinant expression plasmid pSELECT-GFPzeo/β-NGF; transfected it into CHO-K1 and used zeocin to gain a cell strain expressing interest protein steadily; the protein expression quantity was detected by ELISA, the biological activity of the interest protein was assessed by MTT.Part two:Natural macromolecule material chitosan was choosed and quaternized by GTMAC, and a novel type of pH-sensitive hydrogel was prepared withα-β-GP. The hydrogel was characterized by FTIR spectra and scanning electron microscopy. It's degree of swelling and cytotoxicity was evaluated; transgenic seed cells were loading to construct cell/scaffold three-dimensional composition, then scaned it's ultra-microscopic structure by SEM.Results:1. The 0.8% agarose gel electrophoresis result of PCR product showed that specific proliferative band with relative molecular weight of 726 bp appeared in the prospective place;2. Connected the purified product to pGM-T cloning vector, subsequently the BamH I and Nhe I double enzyme digestion of the positive clones that obtained by blue-white selection, a band about 726 bp was got, the result of sequence analysis was in agreement with the sequence provided by Ensembl;3. Conneccted NGF gene and expression vector pSELECT-GFPzeo-MCS which were digested by BamH I and Nhe I, the positive plasmid selected by the antibiotic Zeocin, the recombinant expression plasmid pSELECT-GFPzeo/(3-NGF was confirmed correctly by enzyme digestion and sequence analysis;4. Transfected it into CH0-K1 and used the antibiotic zeocin to choose for 8 weeks, then gained a cell line which expressing interest protein steadily, with persistent expression of green fluorescent;5. The secreted NGF protein quantity is 21.78 ng/24 h/106 cells by ELISA assay; the MTT assay showed it obviously promoted NIH3T3 cell proliferation, compared with the control group, it is statistical significance (p<0.05);6. Swelling analysis showed that HTCC/GP hydrogel possessed good water swelling, could controlled release protein responsing to the changes of pH, MTT assay displayed that the hydrogel had no apparent cytotoxicity did not affect cell proliferation, there are differences between the groups (/K0.05); used it loading transgenic CHO-K1 to construct cell/scaffold three-dimensional composition, SEM showed it had network structure, well-distributed aperture, good cells adhesion.Conclusion:1. Successfully constructed the green fluorescent protein eukaryotic expression vector pSELECT-GFPzeo/β-NGF; transfected eukaryocyte and gained a cell line expressing NGF protein steadily, the secreted interest protein had good biological activity which could obviously promoted NIH3T3 cell proliferation;2. Novel pH-sensitive HTCC/GP hydrogel based on the modified material by natural macromolecule chitosan was prepared, it possessed good biocompatibility,water solubility and could controlled release protein drugs responsing to the changes of pH;3. Successfully constructed 3D hydrogel composition loading transgenic CHO-K1 cell, this scaffold favored the cell adhesion and growth, provided ideal micro-environment for communication between the cells. It is expected to form mimetic skin envrionment at the surface of uclers to repair process of diabetic ulcers, which established a certain foundation for designing tissue engineered skin on the local micro-envrionment of ulcers.