臺灣博碩士論文加值系統

紅血球生成素(EPO)是一34 kDa的醣蛋白荷爾蒙，在哺乳動物體內主要是由腎臟合成，少數由肝臟合成。其主要功能是促使未成熟的血球前驅細胞存活、增生及分化。近來已有證據顯示，在一些非血球細胞中，例如人類的中樞神經系統，以及人類畸胎瘤神經母細胞株NT2中，也有紅血球生成素及其受體的mRNA之表現。然而，紅血球生成素在神經系統之作用目前仍有待釐清。在本論文中，我們探討紅血球生成素對一氧化氮(NO)、過氧化氫(H2O2)以及ceramide引起的NT2細胞死亡之保護效應。細胞存活的評估是以錐藍染色法，以及檢測細胞活性試劑WST-1來偵測。我們發現，10-11~10-3 M SNP(NO釋放劑)刺激15分鐘或24小時皆會明顯降低NT2細胞之存活率，但是若以0.31~2.5 IU/mL之紅血球生成素前處理24小時，則會顯著增加細胞存活，且此種增加存活細胞之效應，在紅血球生成素前處理0.5小時即已顯著。另外我們以西方點墨法偵測蛋白質之表現也發現，紅血球生成素的處理會明顯增加抗死亡蛋白質Bcl-2的表現。紅血球生成素也會降低與促進細胞凋亡相關的酵素caspase 3之活性。然而，在我們的系統中，紅血球生成素並無法抑制由過氧化氫以及ceramide引起的NT2細胞死亡，而由偵測與細胞增生相關之蛋白質PCNA表現，以及[3H]thymidine incorporation實驗證明，EPO並無促進NT2細胞增生之能力。因此在NT2細胞中，紅血球生成素對由一氧化氮所引起之細胞死亡具有專一性的抑制效應，且此效應可能是藉由抑制caspase 3活性，以及調升抗死亡基因Bcl-2及Bcl-xL表現來達成。

Erythropoietin (EPO), a 34-kD glycoprotein produced in the mammalian kidney and liver, was originally thought to act only on erythroid progenitor cells by stimulating their survival, proliferation, and differentiation. There is now evidence that EPO and EPO receptor mRNA are present in certain nonhematopoietic tissues such as the developing human CNS, and a human committed neuronal precursor cell line (NT2) derived from teratocarcinoma. However, the physiological role of EPO at these sites remains to be elucidated. In this study, we assessed the protective effect of EPO on nitric oxide-induced as opposed to ceramide- or H2O2-induced cytotoxicity in NT2 cells. Cell viability was monitored by vital dye (trypan blue) exclusion or by measuring mitochondrial dehydrogenase activity, using the commercial cell proliferation WST-I reagent. We found that sodium nitroprusside (SNP; a nitric oxide donor) at 10-11~10-3 M decreased NT2 cell viability after either 15 minutes or 24 hr incubation. Pretreatment of cells with recombinant human EPO at 0.31~2.5 IU/mL for 24 hr attenuated SNP- induced reduction in cell viability in a dose-related manner. The protective effect of EPO was evident at 0.5 hr after EPO exposure, the shortest time examined. EPO repressed the activity of an apoptosis-promoting enzyme, caspase 3 (CPP32), and enhanced the expression of an anti-apoptosis protein bcl-2, as assessed by Western blotting. By contrast, EPO had no protective effect on either ceramide (10-5~10-4 M)- or H2O2(100 mM)- induced reduction of cell viability. Furthermore, EPO did not alter proliferation of NT2 cells, as evidenced by proliferating cell nuclear antigen (PCNA) expression and [3H]thymidine incorporation. In conclusion, we have demonstrated that EPO appears to specifically protect NT2 cells from nitric oxide-induced cell death by down-regulating pro-apoptotic mechanisms such as the activation of CPP32, and by up-regulating anti-apoptotic signals such as bcl-2 expression.

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