以奈米表面探討巨噬細胞及泡沫細胞的功能及活化表現

Abstract

巨噬細胞在人體的功能調控上扮演著很重要的角色，而由其分化出來的泡沫細胞更是動脈血管硬化的主要成因。在生物醫學蓬勃發展的現在，許多的人工植入物也已經被設計出來。所以考慮人工植入物表面和巨噬細胞及泡沫細胞間的交互作用情形是一個很重要的議題。本研究主要利用矽基板製備出10-nm到200-nm奈米的奈米點表面，而後取下小白鼠腹水中的巨噬細胞養於奈米表面上，並進一步以低密度脂蛋白分化為泡沫細胞。 在型態的實驗中發現各尺寸中巨噬細胞和泡沫細胞的貼覆面積跟 Flat相比,巨噬細胞在10-nm、50-nm、100-nm表面各別增加了6.17%、25.70%、9.63%，而200-nm表面則是減少了14.74%並有似凋亡的現象產生。而泡沫細胞在10nm、50nm表面則是分別增加了7.11%、12.4%，而100-nm、200-nm則是減少了6.5%、7.8%，我們推斷50-nm表面對於巨噬細胞及泡沫細胞有較佳的生物相容性，200nm表面則為較差的生物相容性。根據螢光染色的結果分析，vinculin的表現量也以50-nm為最多，和200-nm則是相對表現量較少，可以推論50-nm表面可能可以促進巨噬細胞及泡沫細胞的貼覆及骨架分布。而200-nm表面則抑制了細胞的貼覆及骨架的分布。由細胞觸角長度的統計圖可得知巨噬細胞及泡沫細胞以100-nm和200-nm的觸角延伸較為明顯，這透露出巨噬細胞及泡沫細胞可能在此兩種表面上執行他們固有的免疫功能。之後我們去做細胞密度的測試，發現巨噬細胞及泡沫細胞相對於Flat，在50-nm表面上有較高的細胞密度，而200-nm較少，則此與我們先前推論50-nm表面對於巨噬細胞及泡沫細胞有較佳之相容性一致。 在基因的實驗方面，我們利用RT-PCR來檢視巨噬細胞及泡沫細胞在各尺寸的基因表現。結果顯示出在發炎基因方面，以100-nm及200-nm對於巨噬細胞產生較嚴重的發炎現象，而泡沫細胞則在10-nm也有發炎現象的產生。 我們根據實驗的結果可以推論，不同尺寸的奈米點結構可以調控巨噬細胞及泡沫細胞的生長、貼覆、細胞密度、及免疫功能。巨噬細胞及泡沫細胞在50-nm結構表面上有較少的發炎反應及較佳的貼覆能力。未來在人工植入物的表面結構設計上可以做為參考。

Macrophage play an important role in modulating the function of human body, and foam cell differentiating from macrophage is also the major factor in Atherosclerosis. In the present day, many artificial bio-implant have been designed and applied to many category. So it is an vital issue to consider the interaction between macrophage and artificial bio-implant surface. In our research, we use silicon based substrate to build nanodot arrays ranging from 10-nm to 200-nm, and seed macrophage isolating from mouse peritoneal on nanodot arrays. Then we further differentiae macrophage into foam cells by LDL. In the morphology experiment, the outcome of cell adhesion area are that 10-nm, 50-nm,100-nm increase respectively 6.17%、25.7%、9.63% cell adhesion area compare with flat surface and 200-nm decrease visibly 14.74% ,accompanied with apoptosis-like. Foam cells increase respectively 7.11% and 12.4% cell adhesion area on 10-nm and 50-nm surface and decrease 6.5% and 7.8% on 100-nm and 200-nm surface compare with flat surface. We may imply that 50-nm surface has more bio-compatibility than 200-nm surface in terms of macrophage and foam cell . According to immunostaining, we found that 50-nm shows the more viculin and actin filament distribution in 50-nm surface than other nanodot size, which indicate that 50-nm surface promote cell adhesion and cytoskeleton organization. On the contrary, 200-nm surface hinders cells from adhesion and inhibits the organization of cytoskeleton. In terms of cell lamellipodia length, 100-nm and 200-nm shows the most extended lamellipodia of all nanodot size. It reveals that macrophage and foam cell execute their innate immune function on the surface of 100-nm and 200-nm. Then we perform a cell viability test, judging from the statistics, macrophage and foam cells seeding on 50-nm surface have the most viability and the less viability on 200-nm surface. The outcome accord with the previous inference that 50nm surface has the more bio-compatibility than 200-nm surface. In the gene experiment, we utilize RP-PCR to observe the gene expression of macrophage and foam cells. The outcome reveals that 100-nm and 200-nm surface have apparently inflammation gene expression for macrophage and 10-nm surface also have inflammation for foam cell. In our outcome, we may infer that nanostructure can modulate macrophage and foam cell in cell growth, cell density and cell spread area, immune function with size dependently. Macrophage and foam cell on 50-nm surface have less inflammation and more adhesion area. Possible application of nanostructure on the artificial implants is expected.