灵芝皮的细胞毒性及其在伤口愈合过程中对伤口之发炎与免疫反应的

灵芝皮的细胞毒性及其在伤口愈合过程中对伤口之发炎与免疫反应的 灵芝皮的细胞毒性及其在伤口愈合过程中对 伤口之发炎与免疫反应的分析 目前有 许多的生物医学材料已被运用在医疗用途上，而松杉灵芝Ganoderma tsugae就是属於 这些生物材料中的一种。 灵芝的成份具有很多的效用，如:治疗癌症，降低血压及 血醣等功能。而且目前并没有任何指出食用它对人体 有何伤害，因此它是一种 非常安全的中药材料及健康食品。敷用由灵芝子实体所制成的灵芝皮 SACCHACHITIN可 缩短老鼠伤口的愈合时间，若将灵芝皮运用在人类的受伤皮肤 上，应该也是一种很有效的促进皮肤再生之敷膜。但 在临床应用之前，我们必需先 了解灵芝皮对於一般细胞是否具有毒性反应、过敏性及免疫反应性，进而探讨其可 能 的愈合机制。 在本研究中，利用纤维母细胞来探讨SACCHACHITIN的细胞毒性。 用H-E染色法来观察伤口修复的过程。利用免疫 组织染色法来观察灵芝皮 SACCHACHITIN对於第一型胶原蛋白现的影响。以PCNA proliferating cell nuclear antigen 单株抗体来观察细胞的增生情形。组织转麸胺 t-TGase 在伤口愈合及 apoptosis的调节中扮演著某些角色， 所以利用组织转麸胺单株抗体来观察组织转麸 胺在伤口愈合过程中的表现。另外， SACCHACHITIN的免疫反 应性则用酵素连结 免疫吸者分析法ELISA来评估。最后，SACCHACHITIN是否会引起过敏反应则以皮 下注射 SACCHACHITIN后的反应来决定。 SACCHACHITIN在浓度小於0.05的情 况下，对纤维母细胞没有毒性。利用皮下注射方式将SACCHACHITIN悬浮液 打入 老鼠体内没有过敏反应发生，也不会引发抗体反应，但是兔子的胶原蛋白则会使老 鼠产生抗原引发的抗体反应， 这些结果显示SACCHACHITIN对於老鼠没有副作用。 以H-E染色方法来观察，皮肤切割后的第1天， SACCHAHCITIN组比控制组有更多 的neutrophils出现在伤口的部位。包括lymphocytes及新生血管的granulation tissue 则 是在第3天较为明显地出现在SACCHACHITIN组，而scar是在第7天时才形成，此时 发炎反应则渐渐地消失了，而 且在SACCHACHITIN组所形成的scar区域比控制组 所形成的scar区域厚。在第14天时，SACCHACHITIN组在伤口处 几乎由scar tissue来取代granulation tissue，而控制组则是尚未完全被scar tissue所取代。 在伤口愈合 期间，控制组的新生血管内壁上在第5天时有大量组织转麸胺的表现，但是在 SACCHACHITIN组则未 见有表现，它是在第14天时才有很强的表现。而角质层的 第一型胶原蛋白在第1天时SACCHACHITIN组的表现量就 比控制组多。 SACCHACHITIN组scar地区的第一型胶原蛋白在第7天就已有表现，而控制组则是 在第12天才有表现。 此外，在第1天时，PCNA在SACCHACHITIN组的角质层就有 微量表现，而控制组则是在第3天时才有少量的表现， SACCHACHITIN组则是比控 制组持续地大量表现，直到两个星期才慢慢转弱。SACCHACHITIN激发的PCNA及 第一 型胶原蛋白提早表现，可加速伤口的愈合另一方面，SACCHACHITIN促使组 织转麸胺，一种apoptosis的指标，在 较晚期表现於新生血管内壁将会增长新生血管 存在期可以提供足够的血液到伤口处，加速伤口的愈合。综合上述的 研究结果，灵 芝皮的确提供了一个有益於加速皮肤伤口愈合的机转。 Analysis of cytotoxicity of SACCHACHITIN and its effects on local immuno-modulation and inflammation during skin wound healing 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 mechanisms 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 types. 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.