The expansion of autologous adipose-derived stem cells in vitro for the functional reconstruction of nasal mucosal tissue.

BACKGROUND: I t is established that adipose-derived stem cells (ADSCs) produce and secrete cytokines/growth factors that antagonize mucosal injury. However, the exact molecular basis underlying the treatment effects exerted by ADSCs is ill understood, and whether ADSCs cooperate with adipose tissue particles to improve mucosal function in patients with empty nose syndrome (ENS) has not been explored. We investigated the impact of ADSCs on nasal mucosa, the associated mechanisms, and their use in the treatment of patients with ENS. RESULTS: The nasal endoscope and mucociliary clearance assessments were significantly improved (P < 0.05) in patients with (n = 28) and without (n = 2) a rudimentary turbinate that received ADSCs combined with fat granules transplantation. Patients experienced a significant improvement in nasal obstruction and nasal mucociliary clearance after nasal turbinate angioplasty (P < 0.05). H&E staining, Masson's staining, and AB-PAS staining confirmed that inflammation was significantly reduced, collagenous fibers became aligned, fewer deposits were observed, and the mucosal proteins generated from caliciform cells increased following treatment. After a 14-day incubation period, ADSCs developed a polygonal cobblestone shape characteristic of human epithelial cells. Furthermore, immunohistochemical analysis revealed the presence of epithelial markers such as cytokeratin-7, and cytokeratin-19. Western blot analysis showed the presence of specific epithelial cell markers including cytokeratin-7, cytokeratin-14 and cytokeratin-19 in these epithelial like cells (ELC); these markers had low expression levels of ADSCs. CONCLUSIONS: The reconstruction of mucosal function by nasal turbinate angioplasty combined with ADSCs and autologous adipose tissue particle transplantation significantly improved the symptoms of patients with ENS. This is a new procedure that will improve mucosal restoration treatment options in patients with ENS. Furthermore, we undertook preliminary explorations of the underlying mechanisms involved, and found that transplantation of ADSCs could induce epithelial cells to improve mucosa function in patients with ENS in the micro-environment of injection areas.

[Establishment of new vessels model by implantation of VEGF165 in mouse].

OBJECTIVE: To investigate the feasibility of establishing a murine new vessels model with Lentiviral vector (LVs) mediated human vascular endothelial growth factor-165 (pcDNA3.1/ VEGF165) gene. METHODS: Lentivirus plasmid carrying a human VEGF165 was constructed and was used to transfect mouse's NIH/3T3 cells. The NIH/3T3 cells with high secretion of VEGF were injected into the skeletal muscle of mouse to establish a mouse new vessels model by implantation of vascular endothelium growth factor (VEGF) gene. The external secretion of VEGF was measured with ELISA. Histological examination was carried out after injection. The expression of CD31 was assessed with immunohistochemical method. RESULTS: The lenti-VEGF165-EGFP was correctly constructed and confirmed by restriction endonuclease analysis, polymerase chain reaction and DNA sequencing analysis. Lentivirus plasmid carrying a human VEGF165 was constructed. lenti-VEGF165-EGFP was used to transfect mouse's NIH/ 3T3 cells, and human VEGF165 gene was assessed. NIH/3T3 cells mediated VEGF gene implantation can produce stable and effective mouse new vessels model. The external secretion of VEGF in peripheral blood was measured with ELISA. The legs became swollen in experimental group 14 d after injection. It found the cells expression of CD31 44 d after injection, and histological analysis showed the swollen tissue was composed of small new vessels. CONCLUSIONS: NIH/3T3 cells mediated VEGF gene implantation can produce stable and effective mouse new vessels model.

[Effect of vacuum-assisted closure on the expression of proto-oncogenes and its significance during wound healing].

OBJECTIVE: To study the effects of VAC on starting the process of wound healing and decreasing apoptosis. METHODS: To examine the variations in expression of proto-oncogenes c-myc, c-jun and Bcl-2 in pig wound model with acute full-thickness skin defect and human chronic wounds by immunohistochemistry, calculate the numbers of expressive positive cells and the labelling index (LI), and observe the process of wound healing. RESULTS: (1) In pig experiment, the wound in experimental group was very clean and without obvious exudates, many neoepiderm and granulation tissue rapidly appeared or formed after 6 days, and healed completely by the 25th day. On the contrary, in the wound of control group, more exudates and blood crust could be seen and fewer neoepiderm and granulation tissue appeared after 6 days and was healed by 30th day. Immediately after the wound was created, the expression of c-myc, c-jun and Bcl-2 was lower and mainly situated in nucleus or cytoplasma of the basilar cells. After the wound was created in control group, or after starting the VAC treatment in experimental group, their expression rapidly and obviously increased, the distribution of the positive cells also became enlarged, but the amount of expression decreased rapidly after the expressive peak have reached. In the successive 12 days following the wound was created, the expression of c-myc, c-jun and Bcl-2 in the experimental group was constantly higher than that of the control group. (2) In human chronic wounds, there wasn't obvious secretions and more healthy granulation tissue was rapidly formed after VAC treatment. The expression of c-jun was mainly located in cytoplasma of basilar cells of epithelium, dermal fibroblasts and inflammatory cells, and the positive cell and labelling index obviously decreased. The expression of c-myc and Bcl-2 was mainly in cytoplasma of basilar cells, but the amount of expression and the labelling index became obviously increased after VAC treatment. CONCLUSIONS: VAC could rapidly start the healing course of the pig' s acute skin wound and human chronic wound, decrease apoptosis of the reparative cells, so as to accelerate wound healing.

[Influence of vacuum-assisted closure technique on expression of Bcl-2 and NGF/NGFmRNA during wound healing].

OBJECTIVE: To explore the influence of vacuum-assisted closure technique (VAC) on expression of Bcl-2 and NGF during wound healing. METHODS: Eighty Sprague-Dawley rats were randomly divided into 4 groups with 20 rats of each. Group T was the experimental group; group C1, C2 and C3 were the control groups. In group T and group C1, capsaicin was injected subcutaneously to the back of the rats to destroy the sensory nerve. VAC was employed to the wound of the rats in group T and C2 three times a day at 80 mmHg negative pressure. In all the groups, tissue samples were taken from the wound edge and granulation at 1, 3, 6, 9 and 12 days after the injury. Immunohistochemistry and in situ hybridization were used to detect the expression of Bcl-2 and NGF/NGFmRNA in the samples. RESULTS: In group C2 and C3, the expression of Bcl-2 and NGF/NGFmRNA was obvious, which increased gradually and reached the peak at the 9th day. In the process of wound healing, the expression Bcl-2 and NGF/NGFmRNA was higher in the group C2 than in group C3 (P < 0.05). The expression Bcl-2 and NGF/NGFmRNA in group T and C1 was lower than group C2 and C3 (P < 0.05). CONCLUSION: The application of the vacuum-assisted closure technique during wound healing increases the expression of the apoptosic modulation related protein Bcl-2 and affects the expression of NGF/NGFmRNA, which may promote the wound healing process.