The cell-matrix adhesion of hemocytes in abalone (Haliotis diversicolor) is regulated by protein kinase A signal transduction pathway

依據本實驗以下幾點發現：一、細胞附著的過程中，細胞內cAMP的量會逐漸下降；二、加入cAMP analog會抑制細胞的附著；三、Protein Kinase A受到抑制物作用後，細胞的附著率反而明顯的提高；四、細胞附著的時間越長，PKA的活性會隨著時間的增加而降低。證實PKA這條訊息傳導途徑參與並調控九孔血液細胞的附著。然而另一條由cAMP所調控的Epac訊息傳導途徑則由於對Epac具有專一性的cAMP analog對細胞的附著卻沒有任何影響，因此應與此細胞附著無關。另外，實驗亦發現，加入RGD肽胜會抑制細胞的附著，推測細胞在附著的時候是經由細胞膜上的細胞附著分子integrin與細胞間質中的蛋白質相互作用，來調節細胞附著。The adhesion of hemocytes plays an important role in the innate immunity of abalones. According to the results from this study, several conclusions were drawn about the role of PKA (protein kinase A) in the adhesion of abalone hemocytes. First, upon cell adhesion, the amount of cAMP decreased in hemocytes. Second, elevation of cAMP levels inhibited cell adhesion. Third, cell adhesion was enhanced by the PKA inhibitor KT5720. Fourth, PKA activity decreased upon adhesion was in a time-dependent manner. Therefore, these results strongly suggested that the adhesion of abalone hemocyte is regulated by PKA and the subsequent signal transduction pathway. However, another cAMP-mediated protein Epac was not involved in the regulation of the adhesion of hemocytes. Besides, since RGD peptides affected the cell adhesion suggested that the adhesion of abalone hemocytes was mediated by the interactions between integrins and fibronection.論 文 目 錄 一、中文摘要……………………………………………………………3 二、英文摘要……………………………………………………………4 三、前言……………..…………………………..……………………5 四、材料與方法…………………….…………………………………13 1. 九孔 (Haliotis Diversicolor)…………………..…..13 2. 化學試劑……………………………………………………13 3. 細胞附著…………………………………………………..15 4. 蛋白質基酶A (PKA) 活性測定…………………………..15 5. 測量細胞內cAMP的量………………………………………16 6. Dot blotting………………………………………………17 7. 呈色…………………………………………………………17 8. 統計…………………………………………………………17 五、結果…………………………………………………….…………19 9. 咖啡因 (caffeine) 調控九孔血液細胞的附著………..19 10. 九孔血液細胞內cAMP的測定………………………………19 11. 九孔血液細胞內PKA的活性測定………………………….20 12. Epac與九孔血液細胞附著的關聯性………………………20 13. RGD peptide影響細胞附著……………………………….21 14. 細胞膜上的醣分子參與在細胞附著……………………..21 六、討論……………………………………………………………….23 七、總結……………………………………………………………….30 八、參考文獻………………………………………………………….32 圖 目 錄 圖1. 九孔血液細胞的附著……………………………………………38 圖2. Caffeine調控九孔血液細胞的附著…………………………..39 圖3. 九孔血液細胞附著期間cAMP濃度變化…………………………40 圖4. dbcAMP對九孔血液細胞附著的影響……………………………41 圖5. 九孔血液細胞中存在PKA……………………………………….43 圖6. KT5720對九孔血液細胞附著的影響…………………………..44 圖7. 以水平是瓊膠 (agarose gel) 電泳分析細胞內PKA的活性…45 圖8. 九孔血液細胞內PKA活性的測定……………………………….46 圖9. 8-CPT-2Me-cAMP對九孔血液細胞附著的影響…………………47 圖10. RGD肽胜對九孔血液細胞附著的影響………………………..48 圖11. cRGD肽胜對九孔血液細胞附著的影響……………………….49 圖12. RGD肽胜與KT5720影響九孔血液細胞的附著…………………50 圖13. Hyaluronidase對九孔血液細胞附著的影響…………………51 圖14. N-acetyl-glucosaminidase對九孔血液細胞附著的影響….53 圖15. Neuraminidase對九孔血液細胞附著的影響…………………54 圖16. PKA調控九孔血液細胞附著的模式圖(Ⅰ)….……………….55 圖17. PKA調控九孔血液細胞附著的模式圖(Ⅱ)……….………….5

蛋白質激酶A訊息傳導途徑調控九孔血液細胞的附著

本篇僅為摘要The adhesion of hemocytes plays an important role in the innate immunity of abalones. According to the results from this study, several conclusions about the role of PKA (protein kinase A) in the adhesion of abalone hemocytes. First, during cell adhesion, the amount of cAMP decreased in hemocytes. Second, elevation of cAMP inhibited cell adhesion. Third, the PKA inhibitor KT5720 enhanced the cell adhesion. Fourth, PKA activity decreased following the time course of incubation. Totally, these results strongly inferred that the adhesion of abalone hemocyte is regulated by PKA and the related signal transduction pathway. Horever, the another cAMP-mediated protein Epac wasn’t involved to regulate the adhesion of hemocytes. Besides, due to RGD peptide could affect the cell adhesion, it suggested that the adhesion of abalone hemocytes was mediated by the interaction between integrin & fibronection.血液細胞的附著在九孔的先天免疫機制中扮演了重要的角色。本實驗有以下幾點發現：一、細胞附著的過程中，細胞內cAMP 的量會逐漸下降;二、加入cAMP analog 會抑制細胞的附著;三、Protein Kinase A 受到抑制物作用後，細胞的附著率反而明顯的提高;四、細胞附著的時間越長，PKA 的活性會隨著時間的增加而降低。以上實驗結果皆證實PKA 這條訊息傳導途徑參與並調控九孔血液細胞的附著。而另一條由cAMP 所調控的Epac 訊息傳導途徑則由於對Epac 具有專一性的cAMP analog 對細胞的附著卻沒有任何影響，因此應與此細胞附著無關。另外，在實驗中亦發現，加入RGD 肽胜會抑制細胞的附著，推測細胞在附著的時候是經由細胞膜上的細胞附著分子integrin 與細胞間質中的fibronectin 相互作用，使得細胞可以附著

Chlorella sorokiniana-Induced Activation and Maturation of Human Monocyte-Derived Dendritic Cells through NF-κB and PI3K/MAPK Pathways

Chlorella sorokiniana (CS) is a unicellular green alga. The extracts of Chlorella have been used as treatments for relieving hypertension and modulating immune response. The detailed mechanisms are not clear yet. In this study, we sought to study the molecular mechanisms for the polysaccharide fraction of CS-induced immune response. We pulsed dendritic cells (DCs) with CS and found that CS could maturate DCs. CS-maturated DC could activate naïve T cells and stimulate T-cell proliferation and IFN-γ secretion. Furthermore, CS activated PI3K and MAPKs signaling pathways in DCs by interacting with TLR4 receptor. These CS-activated signaling pathways could further activate NF-κB and induce IL-12 production in DCs. This study provides molecular mechanisms for CS-induced DCs activation and immune response

Differential Proteomic Analysis of Cancer Stem Cell Properties in Hepatocellular Carcinomas by Isobaric Tag Labeling and Mass Spectrometry

Malignant tumors are relatively resistant to treatment due to their heterogeneous nature, drug resistance, and tendency for metastasis. Recent studies suggest that a subpopulation of cancer cells is responsible for the malignant outcomes. These cells are considered as cancer stem cells (CSC). Although a number of molecules have been identified in different cancer cells as markers for cancer stem cells, no promising markers are currently available for hepatocellular carcinoma cells. In this study, two clones of Hep3B cell lines were functionally characterized as control or CSC-like cells, based on properties including spheroid formation, drug resistance, and tumor initiation. Furthermore, their protein expression profiles were investigated by isobaric tags for relative and absolute quantitation (iTRAQ), and a total of 1,127 proteins were identified and quantified from the combined fractions; 50 proteins exhibited at least 2-fold differences between these two clones. These 50 proteins were analyzed by GeneGo and were found to be associated with liver neoplasms, hepatocellular carcinoma (HCC), and liver diseases. They were also components of metabolic pathways, immune responses, and cytoskeleton remodeling. Among these proteins, the expressions of S100P, S100A14, and vimentin were verified in several HCC cell lines, and their expressions were correlated with tumorigenicity in HCC cell lines. The functional significance of vimentin and S100A14 were also investigated and verified