| Summary: | 碩士 === 國立臺灣大學 === 化學工程學研究所 === 90 === Directed migration of cells in electric fields (EF) is termed “galvanotaxis”. Many previous studies indicated that EF affects not only the migration of cells but also other important events such as the morphology and differentiation of cells. Though the galvanotactic responses of different types of cells have been studied extensively, the detailed mechanism of galvanotaxis remains unknown. This study tried to investigate the galvanotaxis behavior of normal human dermal fibroblasts (NHDF) and to reveal the underlying mechanism. NHDF moved randomly (average directedness being —0.04) with a velocity of 5 mm/h and a speed of 25 mm/h without the influence of EF. But in the presence of EF (EF controls), they showed apparent galvanotaxis toward anode with a directedness of —0.52 in 2 V/cm EF. The speed of NHDF also increased to 45 mm/h. Comparing with the EF controls, the galvanotaxis behavior of NHDF pre-treated separately with various agents in the presence of 1 V/cm EF could be divided into two groups. The first group included NHDF pre-treated separately with EGTA (an extracellular calcium ions chelator), with PD98059 (an inhibitor of MEK1), with calphostin C (an inhibitor of both classical protein kinase C and novel protein kinase C, cPKCs and nPKCs), and with genistein (an inhibitor of receptor tyrosine kinase, RTKs). Under the influence of these agents the migration of NHDF was more unidirectional toward anode (the EGTA-treated NHDF having an average directedness of —0.66) with a greater velocity but reduced speed comparing with EF controls. Inhibition both cPKCs and nPKCs decreased the speed of NHDF to 12 mm/h, which was half of the speed of the EF controls. The other group included NHDF pre-treated separately with Go6976 (an inhibitor of cPKCs), with cytochalasin D (an inhibitor of F-actin formation), or with NAC (an antioxidant inhibiting the production of reactive oxygen species, ROS). The use of these agents caused the loss of directional migration and the decline in velocity and speed. Restraining the formation of F-actin filaments decreased the velocity of NHDF to 15 mm/h. However, inhibiting the ROS production did not affect the speed of NHDF in EF.
In summary, these results indicated that directional migration of NHDF in EF depended on F-actin filaments and that cPKCs and ROS played important roles in the signaling pathway of galvanotaxis. Inhibition of RTKs or MEK1 or both types of PKCs increased directedness and velocity but decreased the speed. This implied that RTKs, MEK1, and nPKCs appeared to be negative regulators of NHDF galvanotaxis. Moreover, the removal of extracellular calcium ions could result in significant concentration gradient of calcium ions in the cytosol, thus leading to a more pronounced galvanotactic response.
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