- 學位論文
以MMP-9為藥物釋放調控因子在傷口癒合及腫瘤之診治
Wound healing and tumor therapy by regulation MMP-9 expression
摘要
基質金屬蛋白(matrix metalloproteinase; MMPs)是組織內的蛋白酵素，其中以MMP-2及MMP-9為MMPs家族中兩種分解性的蛋白脢，尤其是在受傷的組織以及腫瘤組織中扮演重要的角色。當組織受傷後，白血球聚集，釋放出IL-1，IL-6等間白質素，吸引纖維母細胞聚集，盡速修復傷口避免感染，而這過程會造成因纖維母細胞(fibroblast)增生，形成過多的細胞間質(Extra cellular matrix; ECM)增生及纖維母細胞轉化為肌母細胞(myoblast) ，而形成疤痕。同時，MMPs也在血管增生，及在腫瘤侵襲和蔓延的過程中，分解和重塑細胞間質。 本研究首先利用聚乳酸顆粒包覆prednisolone，調控 IL-1β和MMP-9在傷口癒合的表現，以達到減少傷口癒合後疤痕組織的產生。在體外試驗，利用細胞行為分析儀(ECIS)探討纖維母細胞對藥物釋放反應。當以聚乳酸微粒包覆prednisolone藥物(PPM)濃度是5 mM及50 mM時，明顯的減緩細胞的生長速率。證明以這樣的藥物釋放方式，確實會影響纖維母細胞的生長速率，進而達到減少細胞間質(ECM)的過度產生。在動物實驗方面，以大鼠作為實驗動物在大鼠背上創造乾淨的切傷，再以不同濃度的PPM塗覆後縫合。分別在術後7及14天，犧牲老鼠後，取下癒合的傷口組織，分別作組織切片和以西方點墨法分析MMP-9及IL-1的表現。在組織切片中，可以明顯的看到用0.5mM以及5 mM PPM處理的傷口組織，新生的纖維化組織面積較未處理的傷口減少6%到116％(ANOVA, p< 0.05)的細胞增生。而在MMP-9和IL-1蛋白質表現方面，當IL-1減少則MMP-9便隨之增加(ANOVA, p <0.0001)，證明確實可以藉由抑制IL-1而調控MMP-9的表現，進而達到減少ECM的產生。 本研究第二階段奈米金的實驗部份，藉由奈米金表面不同的修飾，測試細胞uptake的狀況。不同種類的細胞對不同的親疏水性奈米金有不同的攝入量，可以作為以AuNPs為model drug的參考。利用石英晶體微天平(QCM)的偵測電極做表面的修飾，使其與奈米金有良好的鍵結，以準確的量測奈米金。此一量測方式，與目前的ICP和Micro CT測量結果作一比較，QCM有較高的準確度。但是在活體或是細胞uptake奈米金之後，僅適用於Micro CT，不適用於QCM。所以在不同的細胞株以修飾過後的奈米金讓細胞uptake，再收取細胞量測金含量。 依據先前的二種實驗結果，另外設計以ECM的萃取物gelatin，包覆奈米金，探討MMPs與gelatin之間的關係，將gelatin包覆奈米金，之後以酵素作用，證明可以藉由酵素分解gelatin。再來以NIH 3T3，9c/Lac Z and Huh 7所分泌的MMP-9和MMP-2作明膠分析法，來測試腫瘤細胞中所分泌的MMP-9及MMP-2可以分解明膠，並且可以在西方點墨法中表現MMPs。
關鍵字
聚乳酸包覆Prednisolone微粒(PPM) ； 傷口癒合 ； IL-1β ； 細胞基質(ECM) ； 基質金屬蛋白9 (MMP-9) ； 奈米金 ； 耦合電漿(ICP) ； 小型電腦斷層(Micro-CT)並列摘要
MMPs (matrix metalloproteinase) are primarily thought to solely be involved in homeostasis and turnover of the extracellular matrix (ECM), but there has been increasing evidence suggesting that MMPs act on cytokines, chemokines and protein mediators to regulate various aspects of inflammation and immunity. MMPs have been speculated to play a critical role in various inflammatory disease and cancer. The precise role of these MMPs in inflammation/immunity is unknown, as it still remains unclear as to whether they are involved in the promotion or reduction of these responses. In this study, we provide a new pharmacological treatment, which may prevent scar formation on wound healing and/or plastic surgery wounds. Using the felationships of MMP-9 with cytokines such as IL-1β. We selected prednisolone as a model drug to activate the MMPs and reduce scar formation in wound excision. To prolong the drug effect, prednisolone of different amounts were encapsulated in biodegradable Poly (D,L-lactide acid; PDLL) microspheres. In the in vitro cell healing study, prednisolone was markedly effective in reducing the growth rate of fibroblast cells according to Electric Cell-Substrate Impedance Sensing (ECIS) method. At a higher concentration of prednisolone, a slower growth rate was observed. In the animal wound healing study after clear-cut wound, results show that in post-surgery days 7 and 14, all of the wound fibrosis areas of experimental rats administered with 0.5- and 5-mM of prednisolone-loaded PDLL microspheres (PPM) were decreased by 6% to 116% compared with those of the control groups (ANOVA, p< 0.05). The dosage of prednisolone used neither extends the healing time of wounds compared with the control group, nor increases the risk of infection. Adding the PPM led to reduce IL-1β but increase MMP-9 expression levels as compared with the control groups (ANOVA, p <0.0001). These results implies that using sustained releasing prednisolone microspheres can regulate ECM generated from fibroblasts, can avoid excess proliferation and reduce the formation of scar tissue during wound regeneration by inhibiting the degree of inflammation. We also choose the Au-nanoparticles (AuNPs) as a model drug for investigating the drug delivery by the gelatin nanocarriers. AuNPs were modified by 11-mercaptoundecanoic acid (MUA) and 1-decanethiol (DCT) for potential applications to drug release, protective coatings, or immunosensors. In this study, plasma deposition methods (P-D methods) were used to immobilize Au electrode on the sensor surface of a quartz crystal microbalance (QCM) to create different microenvironments. Measurements of various AuNPs obtained on the plasma-treated surface of the Au electrode compared to those obtained on an untreated Au electrode show that the linear relationship between mass change and resonant frequency shift significantly differs for AuNPs, MUA-AuNPs, and DCT-AuNPs (R2 from 0.94 to 0.965, 0.934 to 0.972, and 0.874 to 0.9514, respectively). Our results demonstrate that surface modifications measured by a QCM system for various modified AuNPs is a reliable tool for in vitro test. Finally, the gelatin-AuNPs were manufactured and fallowed by treatment with MMPs enzymes. We demonstrated that enzyme dissolved the ECM extraction of gelatin and changed the surface electric charge. Due to the reasons that the secretion MMP-9 and MMP-2 in the tumor cell are higher than that of the normal cell, NIH 3T3, 9c/Lac Z and Huh 7 cell lines were used and demonstrated that MMP-9 and MMP-2 expressed higher level in the human and mice by zymography assay. However the results are not conclusive.