| Objective: Observed the effects of MSCs with Rho molecular signaling pathway inhibitor fasudil hydrochloride (HA1077) on healing the full-thickness skin wounds.Methods: (1) Isolation and identification of rabbit bone mesenchymal stem cells,differentiation trend of MSCs into epidermal cells: bone marrow was extracted through iliac puncture from healthy New Zealand rabbits.MSCs were purified and cultured in vitro.The culture media was replaced 48 hours later to allow removal of non-adherent cells.Media were changed every 2 to 3 days.Cells were passaged with ratio of 1:2 or 1:3 when almost confluent.Passage 3 to 5 cells were used for further experiment. Cells were identified by immunohistochemical chemical and flow cytometry. Immunohistochemical examination was carried out to detect the positive staining of cytokeratin 19 and integrinβ1 after one week induction. (2) the effects of MSCs with HA1077 on healing the full-thickness skin wounds:①2 cm diameter circular skin defects were prepared on the back of Wistar rats and randomly grouped. Five kinds of different doses of HA1077 were injected into dermis of the experiment animal directly. To evaluate the healing quality by the wound area and histological changes.②12 New Zealand rabbits were randomly divided into six groups: the control group (group A), EGF control group (group B), MSCs group (group C), the induced cells group (Group D), MSCs + HA1077 (Group E) and HA1077 + the induced cells (group F). Three full skin loss wounds were created on each side of the back of the rabbits. Cells and drugs were injected into dermis of the experiment animal directly. Calculate WCI(wound closured index) 3,7,14 days after injury and the average healing time. Histological examination: at third, 7th, 14th, and 21st day after injury, tissues were harvested and routinely paraffin embedded and sectioned. The slides were stained with hemoxylin and eosin (HE). Statistical Analysis: In this experiment, all the data are mean±standard deviation ( x±s), using SPSS statistical analysis software for analysis of variance and t-test.Results: (1) A small amount of bone marrow mononuclear cells adhered to the flask 48 hours after plating. Colony could be seen after 5 days,and the number of colony formation, size increased significantly 10 days after plating. The cells reached homogeneous confluence in 14 days.The cells growed rapidly after passage. Cells identified by immunohistochemical chemical show displayed CD34 (-), CD44 (+), CD105 (+).The results could prove that the cultured cells are pure MSCs. Flow cytometry results further confirmed the results.The cells changed from the original formation of fibroblast-like structure to shorter and closely arranged after one week induction.The induced cells could express CK19,β1 signs. (2)①The area of rats wound reduced gradually after injury, especially in the 20μmol/L HA1077 group.②The WCI of Group C,Group D,Group E and Group F was better than the Group A and Group B(P<0.05). Group E and Group F was obviously better than the other two treatment groups (P <0.05). The average healing time in group E and group F was obviously shorter than other groups(P <0.05). The skin pathology structure of these two groups was significantly better than the other groups. However, not only WCI but also the average healing time, had the comparison of Group C and D groups, Group E and Group F no significant difference (P> 0.05).Conclusion: (1) In this study, by our culture protocol,MSCs are isolated and proliferated stably and rapidly.Cultured MSCs possess high activity of growing and amplification,and are appropriate for further research. (2) MSCs have the trend to differentiate into epidermal cells in vitro. (3) HA1077 can promote the effects of MSCs in wound healing. |
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