| Abstract: | 糖尿病患通常都有容易發炎及免疫力功能低下的情況，當受到感染時，糖尿病的死亡人口比正常人高。先前研究中已證實，當巨噬細胞株Raw264.7受到LPS (lipopolysaccharide)的刺激，會經由與TLR-MyD88的結合，活化FAK/Src、EPS8、p38 MAPK的訊息傳遞，進而促進吞噬作用、調控細胞生長、爬行能力增加等，顯示EPS8是促進巨噬細胞的吞噬功能相互作用的關鍵。在高滲透壓的情況下，會影響RTKs的相互作用，進而調控FAK/Src、ERK1/2、p38 MAPK、JNK的磷酸化，造成細胞骨架的重組、細胞增生、凋亡、細胞質與細胞核之間的相互作用受影響。因此本研究延續上述的兩個觀點，想了解當在不同滲透壓中，Raw264.7細胞株受到LPS刺激後，是否會造成細胞在數量、型態及巨噬細胞功能的改變，也想了解當這些改變是否會影響細胞內的分子調控。
本實驗將Raw264.7細胞株培養在高濃度的葡萄糖或氯化鈉環境下，加入LPS (100 ng/ml)刺激，發現細胞型態的改變、細胞增生速度減緩使數量減少，利用細胞爬行試驗發現細胞爬行數量降低，以西方墨點法分析，實驗結果顯示，在高糖及LPS刺激下，EPS8、Src的蛋白表現量，會隨著葡萄糖濃度增加而增加，並可能因此而造成細胞爬行降低，細胞數量減少。另外，還可能藉由MEK、MSK1、SAPK及JNK的活性降低使細胞增生速率減緩；在高濃度的氯化鈉及LPS刺激下，會造成FAK、MEK、MSK1、SAPK及JNK的活性降低使細胞增生速率減緩，至於細胞數減少的現象，初步觀察並不經由細胞凋亡或自噬所造成。
In Diabetes patients, they usually have problem with easy inflammation and immune dysfunction, and the mortality rate of DM patients is higher than normal. In previous study, LPS regulated cell proliferation, phagocytosis, cell migration through singnal pathway like TLR4-MyD88, FAK/Src, EPS8 and p38 MAPK in Raw264.7 murine macrophages cell line. EPS8 is one of the critical molecular of phagocytosis regulation in macrophage. In hyperosmolarity, via FAK/Src, ERK1/2, p38 MAPK, JNK phosphorylation, these RTKs signaling can regulate the reorganization of actin cytoskeleton, cell proliferation, apoptosis. Thus , this study was to determine cell number, morphology and the functions change when cells stimulation with LPS under different osmolarity in Raw264.7, and want to know what moleculars regulated in these conditions.
We treated the macrophage cell line Raw264.7 with higher concentration of glucose or sodium chloride, than stimulated with LPS for 3day. It shows that macrophage morphology is changed, cell number decreased and cell migration ability is diminished too. Western blot analysis shows EPS8 and Src expression decrease in high level glucose and LPS treatment, which is related with macrophage migration and preloferation. It also shows the expression of well-known proliferation regulated moleculars like MEK, MSK1, SAPK and JNK are decreased. In our study, cell number decrease is seems not regulated via apoptosis or autophagy pathway. |
