Rnd1 在 ALA-PDT 抑制 PC12 細胞神經軸突延長的角色探討

The role of Rnd1 in ALA-PDT- induced suppression of neurite elongation in PC12 cells

方佩馨(Pei-Shin Fang)

指導教授 ： 陳進庭

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.01185

摘要

中文摘要

細胞骨架重組在細胞移動、細胞分裂、吞噬作用與形態發生的過程中扮演一相當重要的角色。在許多不同的細胞型態中，Rho family GTPases 的成員是主要調控細胞骨架，而Rnd1為Rho family GTPases 新的成員之一。五-氨基酮戊酸光動力效應 (ALA-PDT) 為一新興癌症治療方式，其作用為能夠選擇性地作用於粒線體產生單態氧與活性氧分子，因而導致細胞的死亡。在本研究中我們利用五-氨基酮戊酸光動力效應做為一可特定造成粒線體傷害的工具，並採用老鼠嗜鉻細胞瘤 (Rat Pheochromocytoma cells, PC12 cells) 做為類似神經細胞之模式細胞株，探討 Rnd1 在 ALA-PDT 抑制 PC12 細胞神經軸突延長的角色。
我們發現，利用Rnd1 siRNA 降低粒腺體功能缺失的 PC12 變異株細胞 Rnd1 表現，當存在有神經生長因子（nerve growth factor）的狀況下，細胞軸突外生現象減少並且可以正常延展。在其他相關訊息調控因子中，以ALA-PDT處理後，我們發現細胞內ROS與Rnd1 表現量增加，同時觀察到MAPKs與AMPK均被活化，抑制細胞內JNK, ERK 與 AMPK後Rnd1表現量減少，而抑制p38分子活性則對Rnd1無明顯影響。

關鍵字

PC12細胞 ； Rnd1 ； 活性氧分子 ； 五-氨基酮戊酸 ； 光動力效應

並列摘要

Abstract

Cytoskeletal rearrangements play a crucial role in cell motility, cytokinesis, phagocytosis and morphogenesis. Much progress has been made in elucidating the molecular mechanisms that control the organization of the actin cytoskeleton. It has been known that members of the Rho families are key regulators of the actin cytoskeleton in various cell types. Rnd1 is a new member of Rho families. 5- aminolevulinic acid mediated photodynamic therapy (ALA-PDT) can generate single oxygen and reactive oxygen species at mitochondria. In this study, ALA-PDT was used as a tool to damage the mitochondria in Rat Pheochromocytoma cells (PC12 cells). The purpose of this study is to address the role of Rnd1 in ALA-PDT- induced suppression of neurite elongation in PC12 cell.
Here we show that, in the presence of NGF, downregulation of Rnd1 by Rnd1 siRNA suppressed neurite outgrowth and increase neurite elongation in PC12 variants. Furthermore, elevated content of ROS and Rnd1 was correlated with the activation of MAPKs and AMPK in ALA-PDT-treated PC12 cells. The upregulation of Rnd1 induced by ALA-PDT was suppressed by the inhibition of JNK, ERK and AMPK. These results indicate that the increased expression of Rnd1 might relate to the activation of ERK, JNK and AMPK, but not p38.

並列關鍵字

C12 cells ； Rnd1 ； ROS ； 5-Aminolevulinic acid ； photodynamic treatment

參考文獻

紀弘泰 (2005) 粒腺體功能異常抑制神經生長因子所調控之PC12細胞神經軸突的生長及細胞凋亡現象。碩士論文。國立台灣大學口腔生物學研究所，台北。

.Abels, C., Heil, P., Dellian, M., Kuhnle, G.E., Baumgartner, R., and Goetz, A.E. (1994). In vivo kinetics and spectra of 5-aminolaevulinic acid-induced fluorescence in an amelanotic melanoma of the hamster. Br J Cancer 70, 826-833.

Ackroyd, R., Kelty, C., Brown, N., and Reed, M. (2001). The history of photodetection and photodynamic therapy. Photochem Photobiol 74, 656-669.

Akiyama, N., Shimma, N., Takashiro, Y., Hatori, Y., Hirabayashi, T., Horie, S., Saito, T., and Murayama, T. (2005). Decrease in cytosolic phospholipase A2alpha mRNA levels by reactive oxygen species via MAP kinase pathways in PC12 cells: effects of dopaminergic neurotoxins. Cell Signal 17, 597-604.

Ames, B.N., Shigenaga, M.K., and Hagen, T.M. (1993). Oxidants, antioxidants, and the degenerative diseases of aging. Proc Natl Acad Sci U S A 90, 7915-7922.