- 學位論文
探討雌激素與二十二碳六烯酸對於人類神經母細胞瘤細胞株SH-SY5Y神經細胞突起生長與相關蛋白質之影響
Effect of estrogen and docosahexaenoic acid on neurite outgrowth and neurite-related protein in human neuroblastoma SH-SY5Y cell line
摘要
二十二碳六烯酸（DHA, 22:6 n-3）在大腦發育時迅速累積於腦部，對於神經新生、神經分化和神經細胞突起增長扮演著重要的角色。雌激素在胎兒時期有高表現而且對於神經細胞突起增長也扮演著重要的角色。本實驗欲探討雌激素與二十二碳六烯酸在人類神經母細胞瘤細胞株SH-SY5Y中對於神經細胞突起增長和相關蛋白質表現之影響。未分化的SH-SY5Y細胞培養於含有胎牛血清或經活性炭處理之低固醇類荷爾蒙胎牛血清的培養基，再以全反式視黃酸誘導細胞分化。誘導分化時分別添加二十二碳六烯酸、雌二醇、甲型或乙型雌激素受體促效劑、拮抗劑，培養六天後觀察細胞型態以及蛋白質表現。細胞型態以顯微鏡觀察拍攝再以NIH Image J軟體分析，細胞蛋白質表現則以西方墨點法分析。 本研究發現培養於活性炭處理之胎牛血清培養基的SH-SY5Y細胞最大神經細胞突起長度比培養於胎牛血清組顯著降低。雌二醇可以顯著增加SH-SY5Y細胞的最大神經細胞突起長度，但黃體素、睪固酮、醛固酮與地塞米松皆無此效果。給予乙型雌激素受體促效劑WAY200070可顯著增加最大神經細胞突起長度，而給予甲型雌激素受體促效劑PPT則無此效果。此外給予乙型雌激素受體拮抗劑PHTPP可有效降低由雌二醇增加的最大神經細胞突起長度，給予甲型雌激素受體拮抗劑MPP則沒有此效果。單獨給予二十二碳六烯酸並不能增加最大神經細胞突起長度，但同時給予二十二碳六烯酸與雌二醇則可增加最大神經細胞突起長度。給予二十二碳六烯酸可以增加腦源性神經營養因子、乙型色胺酸受體激酶、突觸後緻密蛋白95、突觸體蛋白與細胞骨架活性調節蛋白的表現，但給予雌二醇並沒有該效果。本研究結論為雌二醇促進神經細胞突起增長主要透過乙型雌激素受體路徑而非甲型，且雌二醇並不影響神經細胞突起相關蛋白質表現；而二十二碳六烯酸可增加神經細胞突起相關蛋白質表現以及雌二醇誘導之神經細胞突起增長。
並列摘要
Docosahexaenoic acid (DHA, 22:6 n-3) accumulates rapidly during brain development and plays an important role in neurogenesis, neuronal differentiation and neurite outgrowth. Estrogen is rich in fetus and plays an important role in neurite outgrowth. The aim of this study was to examine the interaction of estradiol and DHA on neurite outgrowth and neurite-related protein expression in human neuroblastoma SH-SY5Y cell line. Undifferentiated SH-SY5Y cells were cultured in FBS or characol-dextran treated steroid hormone-free FBS medium containing 1 μM retinoic acid to induce neuronal differentiation then supplemented with or without DHA, estradiol, estrogen receptor (ER) agoinst or antagoinst for 6 days. The neuronal morphology were observed by phase-contrast photomicrographs and determined by the NIH Image J software. The expression of neuronal proteins were analyzed by Western blotting. We found that the max neurite length was significantly decreased in SH-SY5Y cells cultured in steroid hormone-free FBS medium compared to that in the FBS medium. Estradiol but not progesterone, testosterone, dexamethasone and aldosterone significantly increased the max neurite length in SH-SY5Y cells. Supplementation of ERβagonist WAY200070 but not ERαagonist PPT significantly increased max neurite length. In addition, ERβantagonist PHTPP but not ERαantagonist MPP significantly decreased estradiol-enhanced max neurite length. The max neurite length was not changed in DHA supplementation alone but was enhanced in supplementation of both DHA and estradiol. The expression of BDNF, TrkB, PSD95, synaptophysin, ARC were significantly increased by DHA but not by estradiol. It was concluded that estradiol promote neurite outgrowth is mainly through ERβbut not ERαdownstream and no effect on neurite-related protein expression. DHA increased the neurite-related protein expression and enhanced the effect of estradiol on neurite outgrowth.