研究遞送具神經保護性蛋白質及胜肽至中樞神經系統

Abstract

缺血-再灌流損傷(ischemia-reperfusion injury)加劇了缺血性中風病人的腦部傷害。但至今臨床上尚未有任何治療藥品可以有效的降低再灌流所造成的腦部損傷。紅血球生成素(EPO)及擬紅血球生成素胜肽(EPO-mimetic peptides)在過去的研究中，發現其具有神經保護的作用，然而，因血腦障壁(blood-brain barrier)的阻隔，使得這些有潛力的治療藥品需高劑量及多次給藥才能發揮神經保護的效果。本研究利用具有胞吞轉送活性的載體來增加紅血球生成素及擬紅血球生成素胜肽的血腦障壁穿透性，進而增加其治療的效果。我們應用細胞穿透胜肽(cell-penetrating peptide)肝素結合凝血附著素c (heparin-binding haemagglutinin adhesin c, HBHAc)於中樞神經系統之蛋白質及胜肽遞送。 本論文之第一部份，我們順利地利用基因合成的技術，於中國倉鼠卵巢細胞(CHO-K1)培養中表現目標蛋白，再進一步利用鎳次氮基三乙酸(NiNTA)親和性管柱及粒徑篩層析管柱(size exclusion column)進行蛋白質之純化。分別利用人類血癌細胞株(TF-1 cell)及初代培養之大鼠皮質神經(primary rat cortical neuron)來評估生產之紅血球生成素及HBHAc修飾之紅血球生成素(EPO-HBHAc)的促紅血球生成(erythropoiesis activity)及神經保護活性(neuroprotective activity)，結果顯示EPO及EPO-HBHAc皆保有其生物活性。利用老鼠腦血管內皮細胞株(bEnd.3 cells)來研究EPO-HBHAc的細胞內吞作用(internalization)。在劑量依賴性細胞內吞實驗(dose-dependent cellular uptake study)中，bEnd.3與不同濃度(0.5~1.5 µg/mL)的蛋白質培養1小時。在時間依賴性細胞內吞實驗(time-dependent cellular uptake study)中，bEnd.3與1 µg/mL的蛋白質共同培養0.5至4小時。與EPO組別相比，EPO-HBHAc有較高的細胞內吞量，並呈現劑量及時間依賴性。體外血腦障壁模型可分為老鼠腦血管內皮細胞株單一培養模型(mono-culture model)及老鼠腦血管內皮細胞株及星狀細胞株共同培養模型(co-culture model)，利用跨上皮電阻值(transepithelial electrical resistance)及細胞間隙的擴散實驗(paracellular diffusion assay)來評估此兩種模型血腦障壁的完整性。與單一培養模型相比，共同培養模型具有較高的跨上皮電阻值(208±4.6 vs. 150±9.1 Ω*cm2 )及較低的細胞間隙擴散率。因此，選擇共同培養模型來評估EPO-HBHAc的胞吞轉送能力。與EPO相比，EPO-HBHAc具有16.8倍高的胞吞轉送能力，且轉送後的蛋白質仍然保有降低N-甲基-D-天冬氨酸 (N-methyl- D-aspartate, NMDA)所引起之神經毒性的活性。利用缺血-再灌流老鼠模型來評估EPO-HBHAc在活體之神經保護作用。與給予EPO的組別相比，腦部損傷的區域有明顯的下降(51.9±9.1 % vs. 31.8±8 %)，且從神經行為評估測試實驗結果中，EPO-HBHAc具有較好的治療效果。生物體內分佈(biodistribution)的實驗中，在腦脊髓液及腦組織中，EPO-HBHAc相較於EPO分別有3.8倍高及3.1倍高的濃度。而即使延遲給藥時間(再灌流後五小時給藥)，給予EPO-HBHAc的組別中，仍然可以在腦脊髓液及腦組織中偵測到較高的EPO濃度。 本論文之第二部分，利用重組基因技術成功地建立帶有HBHAc修飾之擬紅血球生成素胜肽基因的質體。近一步將其轉染至中國倉鼠卵巢細胞進行胜肽表現，但卻沒有如預期的生產並純化出目標胜肽。因此改以固相多肽合成法(solid-phase peptide synthesis)合成的HBHAc修飾之擬紅血球生成素胜肽來評估其神經保護活性及穿透血腦障壁的活性。HBHAc修飾之胜肽能有效的降低NMDA所引起的神經毒性。但將胜肽與體外血腦障壁模型培養後，無法觀測到胜肽的神經保護活性，推測可能與胜肽的安定性有關。 在本研究中，我們發現了利用HBHAc修飾，可以增強EPO穿透血腦障壁的能力，進一步提升其神經保護的效果。另一方面，HBHAc修飾後的擬紅血球生成素胜肽保有其神經保護的作用，但未來要繼續進行相關研究，必須先克服其安定性之問題。綜合本研究的結果，HBHAc是首次順利應用於遞送具生物活性之大分子藥品至中樞神經系統。

The ischemic-reperfusion injury worsens the outcome of stroke patients, however, there is no any effective approved drug for this injury. The extremely high dose and multiple administrations are required for erythropoietin (EPO) and its mimetics to elicit the neuroprotection due to the blood-brain barrier (BBB). In this study, we would like to apply heparin-binding haemagglutinin adhesin c (HBHAc), a cell-penetrating peptide (CPP), with its transcytosis ability in central nervous system (CNS) to improve the therapeutic efficacy of EPO and EPO-mimetic peptides. First, EPO and EPO-HBHAc were constructed by recombinant DNA technology and expressed in Chinese hamster ovary (CHO-K1) cells. These proteins were purified by nickel nitrilotriacetic acid (NiNTA) affinity and size exclusion columns, and the processes of purification were optimized. The erythropoiesis and neuroprotective activity of proteins were evaluated in the TF-1 cells and in the primary rat cortical neurons, respectively. Both proteins reserved their bioactivity. To evaluate the enhanced internalization of EPO-HBHAc, the cellular uptake studies were performed on bEnd.3 cells. In dose-dependent cellular uptake study, the cells were treated with various concentrations from 0.5 to 1.5 μg/mL of indicated proteins for 1 h. In time-dependent cellular uptake study, the cells were incubated with 1 μg/mL of indicated proteins for 0.5, 1, 2, and 4 h. EPO-HBHAc showed higher cellular uptake than EPO in both dose- and time-dependent study. The mono-culture and co-culture BBB models were established in vitro, and the transepithelial electrical resistance (TEER) and paracellular diffusion were used to evaluate the integrity of these models. Higher TEER values (208±4.6 vs. 150±9.1 Ω*cm2) and lower paracellular diffusion were observed in the co-culture model compared to the mono-culture model. EPO-HBHAc showed 16.8 folds higher transcytosed amount than EPO as incubated with co-culture BBB model in vitro by Western blotting analysis. In addition, the transported EPO-HBHAc in the co-culture transwell system maintained the neuroprotective activity when the transported EPO-HBHAc was administered in conditioned medium to primary rat cortical neurons undergoing NMDA-induced toxicity. The neuroprotective effect and the biodistribution of EPO-HBHAc were evaluated in ischemia-reperfusion rat model in vivo. The cerebral infarction of EPO-HBHAc (31.8±8 %) was significantly reduced compared to that of EPO (51.9±9.1 %). Moreover, the EPO amounts in cerebrospinal fluid (CSF), plasma, and organs were detected at 3 h after dosing by using EPO enzyme-linked immunosorbent assay (ELISA). EPO amounts in CSF and in brain tissue of EPO-HBHAc group were 3.8-fold and 3.1-fold higher than those of EPO group. Higher EPO amounts were detected in CSF and in brain tissue of EPO-HBHAc group than those of EPO group even when delayed dosed 5 h after reperfusion. To further explore the therapeutic applications, the plasmids of EPO-mimetic peptides and their HBHAc-modified forms were constructed. However, the expression and purification of the target peptides were unsuccessful. Therefore, peptides were generated by solid phase peptide synthesis for the study of their neuroprotective activity and BBB permeability. The HBHAc-modified peptides rescued the cortical neurons from NMDA-induced toxicity. However, the neuroprotective effect of these peptides was lost after incubated with BBB model in vitro, possible due to their instability. In summary, better efficacy of EPO-HBHAc showed not only in vitro but also in vivo. HBHAc-modified EPO-mimetic peptides reserved their neuroprotective activity, but the stability of these peptides might be a critical issue to be taken into consideration in further investigation. According to the results in this study, HBHAc is first successfully used for delivering macromolecular with biological activity into CNS.