Analysis of cytotoxicity of SACCHACHITIN and its effects on local immuno-modulation and inflammation during skin wound healing

• Main Authors: Wei-Sheng Hung, 洪偉盛
• Other Authors: Yu-Hui Tsai
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/40113881632367129012

碩士 === 台北醫學院 === 細胞及分子生物研究所 === 88 === Many biomaterials have been used in medical application and the development of biomaterials have been one of the major focusing areas of current medical research. Components of Ganoderma tsugae, “Ling-Zhi”, have been found to have various medical effects such as anti-cancer, anti-hypertension and so on. Previous studies in this research team have shown that the SACCHACHITIN made from the residue of the fruiting body of Ganoderma tsugae, as compared to cotton gauge (control group), significantly accelerates the process of rat skin wound healing. It is of great interest to study whether the SACCHACHITIN also improves the rate of human skin wound healing. Before the application of SACCHACHITIN in clinical use, its cytotoxicity, immunogenicity, immunomodulatory effects and their healing mechanism(s) must be understood. In this study, fibroblasts were used to examine the cytotoxicity of SACCHACHITIN. H-E staining was employed to investigate the gross picture of wound healing process. To study cell proliferation during wound healing, proliferating cell nuclear antigen (PCNA) monoclonal antibody was used to study the expression of the PCNA in active proliferating cell type(s). The effect of SACCHACHITIN on the expression of type I collagen was investigated in the healing tissue by immunohistochemistry. The t-TGase has been proposed to play an important role in wound healing and also in modulation of apoptosis, t-TGase monoclonal antibody was used to access the possible involvement of the t-TGase in wound healing. Enzyme-link immunosorbent assay (ELISA) was used to study the immunogenicity of SACCHACHITIN. Finally, the possible allergic response of rat’s skin to SACCHACHITIN was also tested. According to the obtained data, SACCHACHITIN at less than 0.05% exhibited no cytotoxicity to fibroblasts. Subcutaneous injection of the SACCHACHITIN suspension to rats showed no allergic response. The SACCHACHITIN also did not cause antigenicity response in rats while rabbit collagen did. It is concluded that the SACCHACHITIN have no side effect to rats. By H-E staining, the neutrophils were found in the wound tissue of the SACCHACHITIN group more than in the control group on day 1. Granulation tissue including angiogenesis and the lymphocytes were obviously presented in the SACCHACHITIN group on day 3 while the neutrophils decreased. Scar formation was obvious on day 7 while the inflammatory response was declined and the scar area in the SACCHAHCHITIN group was thicker than in the control group. On day 14, the granulation tissue was completely replaced by scar tissue in SACCHACHITIN group but was not in control group. During this wound healing period, t-TGase was strongly expressed on inner vessel walls on day 5 of control group but not observed on that of the SACCHACHITIN group. On the contrary, on day 14 strong t-TGase staining was observed on the inner vessel wall of the SACCHACHITIN group. The expression of type I collagen was much more in the SACCHACHITIN group than the control group since day 1 at keratinocyte layer. At scar tissue, the type I collagen expression was observed in the SACCHACHITIN group on day 7 and the expression of the type I collagen was not seen till on day 12 in the control group. In addition, PCNA (proliferating cell nuclear antigen) was expressed at keratinocyte layer on day 1 in SACCHACHITIN group. In contrast, PCNA expression in the control group became obvious on day 3, and the expression of PCNA in the SACCHACHITIN group persisted. The observed stimulatory effect of SACCHACHITIN on the early expression of PCNA and type I collagen might result in accelerated healing. On the other hand, the late expression of t-TGase, the indicator of apoptosis, on the inner wall of newly formed capillary vessels in the SACCHACHITIN group may indicate a longer period of sufficient blood supply to the wound area, thus, facilitate healing. The above observed phenomena may underline the mechanism of the beneficial effects of SACCHACHITIN on rapid wound healing.