The T-cell Response To Regenerated Bombyx Mori Silk Fibroin

Silk fibroin extracted from the natural polymer fibrin, has a good physical and chemical properties and biocompatibility. Regenerated silk fibroin is a new type of medical biological materials by physical means, epoxy agents or SD crosslinking. In recent years, researchers prepared regenerated porous silk fibroin for clinical practice needs by physical or chemical methods, which can be used for wound repair. This material has good mechanical strength and do not dissolve in water. And this porous structure of membrane has better ventilation, which can stop the invasion of wound pathogens. This material can be gradually degraded after absorbing in the body. In this study, we aim to further study T cell response to the regeneration of the different characters of Silk Fibroin after application in small animals body surface on the basis of the study the B cell response to silk fibroin after implanting in animal body, so as to provide the immunology experimental evidence for fibroin-like material application in the clinical.Objective: To investigate the immunogenicity of fibroin-like material by researching different characters silk fibroin material effect T cell activation, proliferation and differentiationMethods:1. The effect to T cells after injecting Liquid regenerated silk fibroin into vivo ICR mice were randomly divided into two groups, 6 mice each group. In experimental group every mouse was injected 1% liquid regenerated silk fibroin 0.5ml by intraperitoneal injection. In control group every mouse was injected 0.5ml sterile PBS . On the 7th and 14th day after injection, the spleen was taken sterilly. Analysis as following after isolating cells.1.1 Analysis the ratio of CD3~+CD25~+, CD4~+CD25~+and CD4~+CD25~+T cell of spleen cells The isolated spleen cells were added into flow tube, there were 3×105 cell/tube. Following the directly labeled anti-mouse antibody were added, there were there groups: the first one:CD3-FITC,CD25-APC;the second one: CD4-FITC,CD25-APC; the third one: CD4-FITC,CD25-APC and Foxp3-PE, analysis by FCM after reaction.1.2 The reaction to ConA of spleen cellThe isolated spleen cells were added into 96 holes plate, there were 2×105 cell/hole. then ConA was added, the final concentration were 0μg/ml,2.5μg/ml and 5μg/ml. Cultured for 72h, the proliferation of T cell was analysed by MTT. And the expression of CD4~+CD25~+ and CD4~+CD25~+Foxp3~+T cell was analysed by FCM.2. Study on the activation of T lymphocytes by regenerated porus silk fibrion film after local application on trauma2.1 To establish the surgical woundTo randomly select the SD rats and create a wound with 2 cm×2 cm by removing its skin of back after anesthesia. Then we covered the wound surface with a piece of regenerated porous silk fibroin film and then lay the epidermis of the excisional skin on the regenerated porous silk fibroin film. The wound was sutured .2.2 Histopathological examinationThe rats were anaesthetized at the 3rdd, 14thd, 28thd, 56thd and 90thd after operation and the skins which at the site of transplantation were stained by hematoxylin–eosin (HE). Then we analysed the infiltration of inflammatory cells and the healing of wound.2.3 Analyzing the ratio of CD3~+CD25~+T cell of PBMC, spleen and thymusFive rats belonging to either group were sacrificed by an overdose of sodium pentobarbital at the same time points as before and then obtained PBMC, spleen and thymus which were detected the ratio of CD3~+CD25~+/CD3~+T cells with two-color immunofluorescence and flow cytometry technology. At the same time immunohistochemistry was performed to analysed the positive staining of CD3 and CD25.Results:1.1 The effect to T cells after injecting Liquid regenerated silk fibroin into vivo FCM analysis results showed that: at the 7th and 14th day after injecting liquid regenerated silk fibrion in vivo the expression of CD3~+CD25~+T of spleen cells was (7.32±1.02)% and (7.58±0.98)% respectively. It is slightly higher than control ((7.262±1.31)% and (6.98±1.22)%), but there was no significantly difference. The expression of CD4~+CD25~+T of spleen cells was (7.22±0.92)% and (7.38±0.49)% respectively. It is slightly higher than control ((7.19±0.58)% and (7.03±0.69)%), but there was no significantly difference.The expression of CD4~+CD25~+Foxp3~+T of spleen cells was (3.52±1.03)% and (3.78±0.96)% respectively. It is slightly higher than control ((7.19±0.58)%及(7.03±0.69)%), but there was no significantly difference. 2. Study on the activation of T lymphocytes by regenerated Bombyx mori porus silk fibrion film after local application on trauma2.1 Histopathological examinationInflammatory cells were observed in site of implant on the 3rd and 14th day after surgery. The implanted silk fibroin film is clearly visible. The inflammatory cells reduced obviously after 28 days. There were no inflammatory cells and material after 56 and 90 days, and there were no difference from the normal skin. But there were always inflammatory cells of the skin of positive control. To 56d they decrease gradually.2.2 Analyzing the ratio of CD3~+CD25~+T cell of PBMC, spleen and thymusIn the postoperative 3rdd, 14thd, 28thd, 56thd and 90thd, the percent of CD3~+CD25~+T of PBMC, spleen cell and thymus cell which were from experiment group was showed as following Table 4. There was no significant difference between experiment group and negative group(P﹥0.05). But there was significant difference between experiment group and positive group(P<0.05). Immunohistochemical analysis also showed that: the experimental group the spleen and thymus, only small amounts of CD25 positive staining. It was consistent with the results of FCM analysis.