- 學位論文
傷口癒合中水通道蛋白3調控細胞遷移之偵測
Detection of Cell Migration-Associated Wound Healing Regulated by Aquaporin 3
摘要
水通道蛋白(aquaporins, AQPs)是細胞膜上的通道蛋白，主要功能是傳遞水或不帶電小顆粒分子，改變細胞內的滲透壓，增加水在細胞膜通透性。在傷口癒合的過程中，AQPs會促進細胞的遷移和增生，提升傷口癒合的效果，並且藉由AQPs極化現象的形成，導致細胞內滲透壓改變，進而調控肌動蛋白(actin)的分布位置和影響重組過程中細胞特性，如細胞形態(morphology)、貼附(adhesion)和接合作用(tight junction)。本研究目的為觀察 AQP3 和 actin 對於細胞遷移和貼附之情形，並且探討傷口癒合過程中，AQP3 極化對於 actin 分布位置和重組過程之影響，建立細胞遷移的模型。 傳統傷口癒合試驗(wound heal assay)結果發現，AQP3 受到氯化鎳抑制會影響細胞遷移，進而降低傷口癒合的能力。傷口癒合過程中，抑制 AQP3 和 actin聚合造成細胞層狀偽足(lamellipodium)消失，表示 AQP3 和 actin 聚合會調控細胞層 狀 偽 足 之 形成 ， 此 與 細 胞 遷 移 與 傷 口 癒 合 有 關 。 以 免 疫 螢光 染 色(immunofluorescence)偵測 AQP3 和 actin 之分布，結果顯示在細胞遷移過程中AQP3 會均勻分布在細胞核周圍，與 actin monomers 分布在相同位置，並且 actin會持續沿著層狀偽足呈現輻射狀的聚合。當 AQP3 受到抑制而極化時，actin 呈現平行且不具方向性聚合，使得細胞延展為細長紡錘狀進而抑制細胞遷移。在電 性量測試驗(electric cell-substrate impedance sensing, ECIS)發現，AQP3 會調控細胞層狀偽足的形成，改變細胞形態，進而影響細胞遷移。另外，可藉由阻抗值變化（± 50 ohm）偵測到 AQP3 調控細胞遷移時前進和後退的現象，以及抑制 AQP3時造成阻抗值變化量明顯降低，表示 AQP3 調控細胞形態進而影響細胞遷移、延展和貼附之情形。 綜合以上結果，本研究推測並且建立傷口癒合之細胞遷移模型。當傷口形成時，首先 AQP3 誘發細胞形成層狀偽足，吸引 actin monomers 聚集在 APQ3 的位置，然後層狀偽足會持續延展，藉由 actin monomers 會形成 actin filaments 維持層狀偽足的構形，促進細胞遷移。經由電性量測的方式可即時偵測到 AQP3 調控此傷口癒合之過程。
並列摘要
Aquaporins (AQPs)are integral membrane proteins transporting water or neutral partials in cells. AQPs have been demonstrated that facilitate epidermal cells migration and proliferation during the process of wound healing.AQPs generate wound healing via polarization and osmolarity change to regulate the distribution and remodeling of actin. This study was to observe the cell migration and adhesion regulated by AQP3 and actin, and to investigate how AQP3 conferred on the phenomenon of polarization for actin location and cell remolding in wound healing in order to establish the model of cell migration through AQP3 and actin. The result of traditional wound healing assay showed that AQP3 was inhibited by nickel chloride (NiCl2)resulting in impaired cell migration and wound healing. The polymerization of AQP3 and actin were inhibited in accompanied with no lamellipodium generation. It indicated that the polymerization of AQP3 and actin mediated lamellipodium generation to facilitate cell migration during wound healing. The immunofluorescence assay explored that AQP3 generally distributed around nuclei, and actin monomers colocalized with AQP3 distribution to polymerize radically along lamellipodium. Moreover, AQP3 deficiency interfered with polarization of AQP3, and caused the location of polymerized actin parallel and undirected. The assay of electric cell-substrate impedance sensing (ECIS) detected AQP3 mediated cell lamellipodium generation to affect cell migration and distribution resulting in impaired cell migration. Otherwise,the impedance change (± 50 ohm) was detectable in the forward and backward movement during cell migration, and the impedance was significantly decreased when AQP3 was repressed. It suggested that AQP3 mediated cell morphology for cell migration, extension and adhesion. Regarding the findings of this study, it speculated and established a model of cell migration during wound healing. In the process of cell migration for wound healing, AQP3 induced cells to form lamellipodium and attract actin monomers to the location of AQP3 distribution, and lamellipodium further extended and actin filaments were formed from actin monomers to maintain the shape of lamellipodium. The ECIS assay can detect in real time that AQP3 regulated cell migration for wound healing.