| 摘要: | 血管平滑肌細胞（Vascular smooth muscle cells, VSMCs）環繞於血管外圍以維持血壓，這些細胞在健康血管中多表現為收縮型、高血壓的血管中多表現為合成型。由於 VSMCs 在體內受到脈壓的作用，週期性拉伸的應變程度對於VSMCs的表型調控具有關鍵影響。此外，有研究指出，電刺激亦可能誘導 VSMCs 由合成型轉變為收縮型表型。然而現有體外培養系統多僅能提供單一物理刺激，難以探討複合刺激對VSMCs的影響。
因此，本研究開發出一款能對 VSMCs施予多重刺激的生物反應器。在此系統中，導電性聚吡咯（polypyrrole, PPy）會以原位聚合法沉積在聚二甲基矽氧烷（polydimethylsiloxane, PDMS）薄膜上，以促進細胞黏附並實現電刺激功能。裝置上設有可移動的平台，可對 PPy/PDMS 薄膜施加不同的拉伸應變。我們將在此裝置上接種 A7r5 平滑肌細胞，施加 48 小時的循環拉伸，並以7%及15%應變分別模擬生理及病理條件。另外也分別對細胞進行每天1小時的交流(AC)或直流(DC)電場刺激，藉此進行系統性分析這兩種刺激模式其對細胞表型、蛋白表現與增殖行為之影響。
實驗結果顯示，生理性拉伸（7%）可促進 VSMCs 向收縮型表型轉換，而病理性拉伸（15%）則誘導合成型標誌蛋白上調並伴隨細胞增殖增加。另一方面，施加AC或DC電刺激均可提升收縮型標誌蛋白表現並抑制細胞增殖，其中交流電刺激之效果較直流電刺激顯著。在進行複合刺激時，電刺激不僅能在 7% 生理性拉伸條件下呈現加成效果，亦可對 15% 病理性拉伸所致的表型轉換與異常細胞增殖發揮補償作用。
本研究成功建立一套可同時調控機械與電刺激之體外培養平台，證實多重物理刺激對 VSMCs 表型具有關鍵且交互影響。此多功能生物反應器可作為未來研究血管力學生物學及相關心血管疾病機制之重要體外模型。
;Vascular smooth muscle cells (VSMCs) surround blood vessels are essential for the regulation blood pressure. In healthy vessels, VSMCs predominantly exhibit a contractile phenotype, whereas in hypertensive vessels, they tend to display a synthetic phenotype. Because VSMCs are constantly subjected to pulsatile pressure in vivo, the magnitude of cyclic stretch plays a crucial role in regulating their phenotype. In addition, previous studies indicated that electrical stimulation can induce a phenotypic transition of VSMCs from synthetic to contractile state. However, most existing in vitro systems are limited to providing a single type of physical stimulus, making it difficult to investigate the effects of combined stimuli on VSMCs.
Therefore, this study developed a bioreactor capable of applying multiple stimuli to VSMCs. In this system, conductive polypyrrole (PPy) was in situ polymerized onto a polydimethylsiloxane (PDMS) film to promote cell adhesion and enable electrical stimulation. The device is equipped a movable platform that allows different levels of stretch strain to be applied to the PPy/PDMS membrane. A7r5 smooth muscle cells were seeded onto this bioreactor and subjected cyclic stretch for 48 hours, with strain levels of 7% and 15% used to simulate physiological and pathological conditions, respectively. Additionally, cells were exposed to either alternating current (AC) or direct current (DC) stimulation for 1 hour per day. This experimental design enabled a systematic evaluation of the effects of these stimulation modalities on cell phenotype, protein expression, and proliferative behavior.
The experimental results demonstrated that physiological cyclic stretch (7%) promoted the transition of VSMCs toward a contractile phenotype, whereas pathological stretch (15%) induced the upregulation of synthetic phenotype markers accompanied by increased cell proliferation. On the other hand, both AC and DC electrical stimulation enhanced the expression of contractile markers and suppress cell proliferation, with AC stimulation exerting a more pronounced effect than DC stimulation. Regarding combined stimulations, electrical stimulation not only exhibited an additive effect under physiological stretch (7%) but also exerted a compensatory effect against phenotype switching and abnormal cell proliferation induced by pathological stretch (15%).
Overall, this study successfully established an in vitro culture platform capable of simultaneously modulating mechanical and electrical stimuli, demonstrating that multiple physical cues play critical and interactive roles in regulating VSMC phenotype. This multifunctional bioreactor may serve as a useful in vitro model for future studies on vascular mechanobiology and the mechanisms underlying cardiovascular diseases. |
