過度表達紅血球生成素的 NIH/3T3 纖維母細胞株之神經元保護作用研究

Abstract

紅血球生成素(EPO) 是一種目前臨床上被廣泛應用於促進紅血球生成的細胞激素。近年來，有許多研究指出，EPO同時具有神經保護的功能，且被認為是治療神經疾病的潛力藥物。有研究指出，給予病患短時間、高劑量的EPO，雖能改善病患的神經運動能力，然而，過多的EPO可能造成血液循環系統中紅血球容積上升，進而增加血栓等心血管疾病的風險。因此，設計具有能在特定受損區域給予EPO，同時不會造成體內毒性的神經疾病治療方法，是值得嘗試的。 本研究中我們的目的主要有二：(1)建立能過度表達EPO的纖維母細胞株 (EPO-3T3-EGFP)；(2)探討並證實其神經保護效果。我們同時將小鼠EPO和綠色螢光蛋白(EGFP)的互補核酸序列(cDNA)轉殖入NIH/3T3纖維母細胞中。經過G418藥物的篩選後，我們建立了一株能過度表達EPO的細胞株(EPO-3T3-EGFP)。接著我們利用逆轉錄聚合酶鏈式反應(RT-PCR)、即時聚合酶鏈式反應(Q-PCR)、細胞免疫染色、西方墨點法以及酶聯免疫吸附試驗(ELISA)等方法對EPO-3T3-EGFP細胞進行EPO產物分析。RT-PCR和Q-PCR的分析結果顯示，EPO的mRNA表現量相較於控制組有明顯地上升。而較多的EPO產物也在細胞免疫染色及西方墨點法分析中被證實。在ELISA實驗結果中，我們更發現EPO-3T3-EGFP細胞的EPO分泌量相較於控制組是非常高且顯著的。 為了測試EPO-3T3-EGFP細胞所分泌的EPO是否有其神經保護功效，我們使用神經退化疾病模式的細胞株(PC12-INT-EGFP)來進行檢驗。之前研究發現，這株過量表達神經元中間絲蛋白α-internexin的PC12細胞經由神經生長因子(NGF)促進分化後，細胞骨架會有不正常的堆積，並且走向凋亡。收集並給予培養EPO-3T3-EGFP細胞的條件培養基後，PC12-INT-EGFP細胞的存活率有顯著的上升。相較於其他控制組， PC12-INT-EGFP細胞中部分標記有EGFP之過量表達團聚的α-internexin (α-INT-EGFP)在動態影像中呈現慢慢鬆解並且移轉至神經突出。在24-48小時間我們發現PC12-INT-EGFP細胞的神經突出生長有明顯加快的現象。最後，藉由細胞免疫染色，我們發現在給予培養EPO-3T3-EGFP細胞的條件培養基後，神經元中間絲蛋白NF-M與其磷酸化型態(p-NF-M)，以及有α-INT-EGFP大量堆積在神經突出近端靠近PC12-INT-EGFP細胞質區域情況的細胞較為減少，並且轉送分佈到神經突出。 由本研究的結果，我們可以推論EPO-3T3-EGFP細胞可以穩定且高度表達具有顯著神經保護功能性的EPO。這株EPO-3T3-EGFP細胞能在短時間內分泌大量的EPO並達到可能的生理功效。未來將可以應用在研究以細胞移植治療特定神經損傷區域的實驗動物模式中。

Erythropoietin (EPO), a well-known hematopoietic cytokine, recently has been found with neuroprotection effect and is considered as a potential therapeutic candidate for neurological diseases. Although delivery of short-term high-dose EPO seemed to improve patients’ neuromuscular functions, yet excessive EPO resulted in systematically high hematocrit and increased thrombotic risks. Therefore, new therapeutic strategy of regionally-provided EPO for neurological diseases at non-toxic level needed to be designed. In our study, we aimed to (1) establish an EPO-overexpressed NIH/3T3 fibroblast cell line, EPO-3T3-EGFP, and (2) verify its possible neuroprotection ability. Mouse EPO cDNA was subcloned into pCMS-EGFP vector and transfected into NIH/3T3 cells. After G418 stable clone selection, a stable EPO-overexpressed EPO-3T3-EGFP cell line was established. The expression of EPO was analyzed by reverse-transcriptase PCR, (RT-PCR), quantitative real-time PCR (Q-PCR), immunocytochemistry, Western blot and ELISA. We found that EPO mRNA expression level was elevated in EPO-3T3-EGFP cells analyzed by RT-PCR and Q-PCR. More EPO product from EPO-3T3-EGFP cells was demonstrated by immunocytochemical data and Western assays. We also found abundant amount of the secreted EPO from EPO-3T3-EGFP cells by ELISA analysis. In order to further confirm the neuroprotection ability of secreted EPO, a cell model of neurodegeneration, PC12-INT-EGFP cells, was used. Previous study showed that abnormal cytoskeletal aggregation and neuronal cell death could be observed after nerve growth factor (NGF) induction for PC12 cells with the overexpression of neuronal intermediate filament α-internexin. After supplementation with conditioned medium prepared from EPO-3T3-EGFP cells, cell survival rate was significantly increased in PC12-INT-EGFP cells. We found that some aggregated green fluorescent α-INT-EGFP (overexpressed-α-internexin with EGFP-tagged) of PC12-INT-EGFP cells was disaggregated and transported into neurite dynamically. Subsequently, acceleration of neurite outgrowth in PC12-INT-EGFP cells was also found during 24-48 hour treatment. Our immunostaining results also showed that the distribution of NF-M (neurofilament medium polypeptide), phosphorylated-NF-M and α-INT-EGFP in PC12-INT-EGFP cells were less aggregated in the perikaryal region and transported into neurite after supplied with conditioned medium from EPO-3T3-EGFP cells. In conclusion, our results showed effective neuroprotection of secreted EPO by the established EPO-overexpressed NIH/3T3 cell line which could provide a potential material for future in vivo studying on cell-based therapies for neurological diseases via secreting EPO on a short-term, high-dose and regional basis.