Analysis of cytotoxicity of SACCHACHITIN and its effects on local immuno-modulation and inflammati

靈芝皮的細胞毒性及其在傷口癒合過程中對 傷口之發炎與免疫反應的分析

 目前有許多的生物醫學材料已被運用在醫療用途上，而松杉靈芝 (Ganoderma tsugae) 就是屬於這些生物材料中的一 種。靈芝的成份具有很多的效用，如：治療癌症，降低血壓及血醣等功能。而且目前並沒有任何報告指出食用它對 人體有何傷害，因此它是一種非常安全的中藥材料及健康食品。敷用由靈芝子實體所製成的靈芝皮 (SACCHACHITI N) 可縮短老鼠傷口的癒合時間，若將靈芝皮運用在人類的受傷皮膚上，應該也是一種很有效的促進皮膚再生之敷膜。

但在臨床應用之前，我們必需先瞭解靈芝皮對於一般細胞是否具有毒性反應、過敏性及免疫反應性，進而探討其可 能的癒合機制。

在本研究中，利用纖維母細胞來探討 SACCHACHITIN 的細胞毒性。用 H-E 染色法來觀察傷口修復的過程。利用免 疫組織染色法來觀察靈芝皮 (SACCHACHITIN) 對於第一型膠原蛋白表現的影響。以 PCNA (proliferating cell nuclear antigen) 單株抗體來觀察細胞的增生情形。組織轉麩胺  (t-TGase) 在傷口癒合及 apoptosis 的調節中扮演著某些角 色，所以利用組織轉麩胺 單株抗體來觀察組織轉麩胺 在傷口癒合過程中的表現。另外，   SACCHACHITIN 的免 疫反應性則用酵素連結免疫吸者分析法 (ELISA) 來評估。最後， SACCHACHITIN 是否會引起過敏反應則以皮下注 射 SACCHACHITIN 後的反應來決定。

SACCHACHITIN 在濃度小於 0.05% 的情況下，對纖維母細胞沒有毒性。利用皮下注射方式將 SACCHACHITIN 懸 浮液打入老鼠體內沒有過敏反應發生，也不會引發抗體反應，但是兔子的膠原蛋白則會使老鼠產生抗原引發的抗體 反應，這些結果顯示 SACCHACHITIN 對於老鼠沒有副作用。以 H-E 染色方法來觀察，皮膚切割後的第 1 天， SAC CHAHCITIN 組比控制組有更多的 neutrophils 出現在傷口的部位。包括 lymphocytes 及新生血管的 granulation tissue 則是在第 3 天較為明顯地出現在 SACCHACHITIN 組，而 scar 是在第 7 天時才形成，此時發炎反應則漸漸地消失了

，而且在 SACCHACHITIN 組所形成的 scar 區域比控制組所形成的 scar 區域厚。在第 14 天時， SACCHACHITIN 組 在傷口處幾乎由 scar tissue 來取代 granulation tissue ，而控制組則是尚未完全被 scar tissue 所取代。

在傷口癒合期間，控制組的新生血管內壁上在第 5 天時有大量組織轉麩胺 的表現，但是在 SACCHACHITIN 組則 未見有表現，它是在第 14 天時才有很強的表現。而角質層的第一型膠原蛋白在第 1 天時 SACCHACHITIN 組的表現 量就比控制組多。 SACCHACHITIN 組 scar 地區的第一型膠原蛋白在第 7 天就已有表現，而控制組則是在第 12 天才 有表現。此外，在第 1 天時， PCNA 在 SACCHACHITIN 組的角質層就有微量表現，而控制組則是在第 3 天時才有 少量的表現， SACCHACHITIN 組則是比控制組持續地大量表現，直到兩個星期才慢慢轉弱。 SACCHACHITIN 激 發的 PCNA 及第一型膠原蛋白提早表現，可加速傷口的癒合 ; 另一方面， SACCHACHITIN 促使組織轉麩胺 ，一 種 apoptosis 的指標，在較晚期表現於新生血管內壁將會增長新生血管存在期可以提供足夠的血液到傷口處，加速傷 口的癒合。綜合上述的研究結果，靈芝皮的確提供了一個有益於加速皮膚傷口癒合的機轉。

Analysis of cytotoxicity of SACCHACHITIN and its effects on local immuno-modulation and inflammati

on 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 m edical 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 tsug ae, as compared to cotton gauge (control group), significantly accelerates the process of rat skin wound healing. It is of great interest to study whether t he 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 wo und healing process. To study cell proliferation during wound healing, proliferating cell nuclear antigen (PCNA) monoclonal antibody was used to stu dy the expression of the PCNA in active proliferating cell type(s). The effect of SACCHACHITIN on the expression of type I collagen was investigate d 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 a ssay (ELISA) was used to study the immunogenicity of SACCHACHITIN. Finally, the possible allergic response of rat’s skin to SACCHACHITIN wa s also tested.

According to the obtained data, SACCHACHITIN at less than 0.05% exhibited no cytotoxicity to fibroblasts. Subcutaneous injection of the SACCHA CHITIN suspension to rats showed no allergic response. The SACCHACHITIN also did not cause antigenicity response in rats while rabbit collagen di d. 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 SACC HACHITIN group more than in the control group on day 1. Granulation tissue including angiogenesis and the lymphocytes were obviously presented i n the SACCHACHITIN group on day 3 while the neutrophils decreased. Scar formation was obvious on day 7 while the inflammatory response was d eclined and the scar area in the SACCHAHCHITIN group was thicker than in the control group. On day 14, the granulation tissue was completely repl aced 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 SAC

CHACHITIN group. On the contrary, on day 14 strong t-TGase staining was observed on the inner vessel wall of the SACCHACHITIN group. The ex

pression 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 t

ype 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 i

n 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 othe

r hand, the late expression of t-TGase, the indicator of apoptosis, on the inner wall of newly formed capillary vessels in the SACCHACHITIN group m

ay indicate a longer period of sufficient blood supply to the wound area, thus, facilitate healing. The above observed phenomena may underline the me

chanism of the beneficial effects of SACCHACHITIN on rapid wound healing.