以3D 方式培養腫瘤細胞被認為具有腫瘤活體組織的特點，例如細胞緊密排列、延續性低氧細胞群、異質性以及功能性腫瘤增長特性等優點。本計畫為建立3D 腫瘤球體 (3D tumor spheroids)的研究平台，選擇以微生物多醣體Gellan gum 為基質與癌細胞共同培養形成類腫瘤組織。本研究採用類似 AB 膠的凝膠原理，開發Gellan-ADH/Gellan-CDI、Gellan-ADH/PEG-diNHS 兩種膠體系統。此凝膠不但具有可調控的機械性質，並且可藉由添加致癌因子或藥物做為致癌機轉或治療策略之研究平台為開發快速且具有生物安全性的凝膠系統，本研究在Gellan gum 的酸基修飾Adipic dihydrazide (以 GG-ADH 表示)。另外，也製備Gellan-CDI (1,1'-Carbonyldiimidazole) 或 PEG-diNHS。Gellan-ADH 和Gellan-CDI (或PEG-diNHS)在溫和的反應條件下，可藉由amide bond 的鍵結產生凝膠。而Gellan gum 水膠的機械模數 (Young's modulus) 則可利用Gellan-ADH/Gellan-CDI 的相對莫耳數，或是利用 PEG 的分子量進行調控。本研究開發之凝膠系統不但能應用於癌症機轉之體外研究，並可應用於癌症治療之體外模擬。本研究進一步利用Gellan gum 3D 腫瘤球體，探討機械性質與口腔鱗狀上皮細胞癌化以及對口腔癌細胞惡化之關係。為了解機械性質引起之訊息傳遞機制，本研究選擇以 RhoA/ROCK 、calcineurin/NFATc pathway 做為研究對象，探討和口腔癌之關係。另外，為改善clioquinol 在治療口腔癌之投藥方式，本計畫以Gellan gum 膠體做為clioquinol 的傳輸載體，研究clioquinol 的釋放動力學。並且製備Gellan gum-clioquinol 口腔癌治療貼片，期望此貼片可應用在口腔癌患者腫瘤組織切除後傷口的保護，以及應用在口腔癌初期口腔黏膜白化病灶處癌細胞的局部毒殺。本計畫將生醫材料研發和癌症研究結合是一種創新的構想，同時也兼具基礎研究和應用開發的價值。 3D culturing of tumor cells is thought to have all the characteristics of living tumor tissue, such as cells that are closely arranged, hypoxic cell fractions, heterogeneity and tumor proliferation. To establish the cancer research platform, we chose Gellan gum (microorganism polysaccharide) as the substrate and co-culture with cancer cell to form 3D tumor spheroids. Gellan-ADH/Gellan-CDI and Gellan-ADH/PEG-diNHS hydrogels are developed with the principle analogous to that of AB glue. This hydrogel forming system not only has mechanical properties that can be regulated, but can also merger carcinogenic factors or drugs for studying the molecular mechanisms or treatment policy of cancer. To develop a faster, nontoxic and covalent cross-linking method for constructing 3D Gellan gum hydrogels. Gellan gum will graft with adipic acid dihydrazide (Gellan-ADH) and another polymer chain as like Gellan-CDI (1,1'-Carbonyldiimidazole) or PEG-diNHS also be synthesized by a variety of methods. Gellan-CDI or PEG-diNHS can interact with primary amines of Gellan-ADH to form amide bond resulting in the formation of hydrogels. Gellan gum-based hydrogels with different Young’s modulus can be controled by adjusting the Gellan-ADH/Gellan-CDI mole ratios or the molecular weight of PEG. Further study, we evaluated the effects of hydrogel Young’s modulus on RhoA/ROCK and calcineurin/NFATc signaling during oral carcinogenesis and progression. Additionally, to chang clioquinol delivery method for improved oral cancer treatment, we use Gellan gum as drug carrier for clioquinol and study its release kinetics in vitro. We also prepare Gellan gum-clioquinol therapeutic patche for oral cancer. The patche is anticipated to protect the wounds caused by tumor excision and terminate cancer cells reproduction in oral cancer patients. The combination of biomedical material development and cancer research described in this project is a novel concept, and is valuable in both basic research and applied research.
