| 摘要: | 近年來，富含生長因子(GF)的血小板裂解液(PL)被認為是一有力的臨床級添加物，將其添加至細胞培養基中可使間葉幹細胞(MSC)進行擴增，且有促進癒合的作用，故也可視為一治療產品。然而在再生醫學中，此類血液製品有必要被確保其安全性以及最佳品質，因此需考慮如血小板衍生微粒(PMP)以及病原性病毒的傳染等的影響。PMP表面雙層脂質膜上的磷脂絲胺酸，被認為會引起血栓和發炎等副作用，需利用奈米過濾去除血液中存在的微粒；而病原性病毒的傳染，包括C型肝炎病毒，會影響血液製品的安全性，需透過化學劑(S/D)、熱處理或奈米過濾等專門消毒方法去除病毒的活性。
論文首先探討是否能透過消毒技術來提高再生醫療中血液製品的安全性：(a) 利用75奈米過濾去除PMP; (b) 透過S/D或56°C加熱處理滅活HCV或透過奈米過濾去除HCV。其次探討是否能開發和改進臨床級血小板沉澱裂解液(PPL)，使之能應用於神經再生。我們假設客製化的PPL富含多種神經滋養生長因子，且透過熱處理使其不含血漿蛋白（特別是纖維蛋白原），如此一來，此客製化的PPL能提升多巴胺神經元的神經保護作用，可作為神經退化性疾病的新治療策略。研究結果如下：
第一，透過消毒技術來提高再生醫療中血液製品的安全性：(a)透過特殊生物檢測方法可發現，經由75奈米過濾後， PMP 被確切移除，且避免了體外PMP相關的凝血酶的產生，但依舊保留血漿蛋白、脂蛋白、凝血因子含量和球蛋白凝血活性。(b) 根據訊號強度分析指出，將具有冷光標記感染性細胞衍生的HCV (HCVcc)顆粒加入血漿中，使血漿受到感染後，再將該HCVcc 在31°C的作用環境下透過S/D處理，30分鐘內即可發現其病毒感染力完全喪失，且偵測不到病毒蛋白NS5A。綜合上述結論可知，透過S/D處理和奈米過濾技術可有效降低血栓形成機率和HCV的傳染風險，進而提高輸血之安全性。
第二，開發和改進臨床級PPL，使之能應用於神經再生醫學：(a)根據ELISA分析和蛋白質組學研究顯示，經由56°C熱處理，30分鐘後的蛋白質組成會被修飾且更具有神經保護活性。故熱處理能改善神經保護活性，若進一步使用S/D和奈米過濾等方法將有助於HCV失活。(b)在帕金森氏症模型的離體實驗（以MPP+神經毒素刺激LUHMES細胞），和動物實驗（給予小鼠MPTP神經毒素）中，PPL皆被證實有顯著的神經保護作用。(c)在黑質緻密物中酪胺酸羥化酶(TH)蛋白表現的研究顯示，透過鼻腔給予 (i.n.) PPL的方式可作為未來神經退行性疾病的另一種治療策略。(d)根據LUHMES研究顯示，訊息轉導途徑的特異性抑制會調控PPL的神經保護作用。(e)先前研究指出，神經過度發炎最終亦會導致神經退化性疾病，故我們利用內毒素LPS刺激BV2細胞誘導其產生發炎介質（例如iNOS, COX-2），證實PPL給予並不會加重其發炎反應，且能抑制COX-2蛋白的表現。此外，檢測發現PPL含有1 × 10 12 MP/mL，其平均大小為160 nm。
綜觀上述可知，對於細胞治療和再生醫學，我們的研究提供了製備安全的客製化血小板裂解物技術的可行性，特別是可作為中樞神經系統的神經保護製劑。
Ensuring optimal quality and safety of blood products for regenerative medicine is mandatory. Recently, platelet lysates (PL) rich in growth factors (GFs) have emerged as a powerful clinical-grade supplement of growth media for ex vivo expansion of mesenchymal stromal cells (MSC) and as a therapeutic product to promote healing in trauma, degenerative pathologies, bone reconstruction, some metabolic diseases, and, possibly, neurodegenerative disorders. The interest in platelet extracellular vesicles, also called platelet-derived microparticles (PMPs), as physiological delivery system of GFs, has emerged. However, the presence of phosphatidylserine on their bilayer lipid membrane may cause thrombotic and inflammatory side effects in a blood medium. Another transfusional risk is the transmission of pathogenic viruses, including hepatitis C virus (HCV), requiring the implementation of dedicated viral inactivation or removal methods such as solvent/detergent (S/D) or heat treatments, or nanofiltration.
Our thesis first evaluated technologies to improve the safety of therapeutic blood products including those for regenerative medicine: (a) impact of removal of MPs in plasma by 75nm-nanofiltration and (b) capacity of S/D or 56°C heat treatments, or nanofiltration to inactivate/remove HCV. These results were used for developing and improving a customized clinical-grade platelet pellet lysate (PPL) for specific applications in neuroregeneration. We hypothesized that a tailor-made PPL enriched in multiple neurotrophic growth factors and depleted of plasma proteins could be a potent neuroprotective agent and exert neuroprotection of dopaminergic neurons for brain administration as disease modifying strategy of neurodegenerative disorders.
Our data show that 75 nm-nanofiltration preserved protein and lipoprotein profile, coagulation factor content and global coagulation activity of plasma. Specialized biophysical methods showed the capacity of this nanofilter to remove MPs and to avoid an in vitro MP-associated generation of thrombin in plasma. Second, our data using luciferase-tagged infectious cell culture-derived HCV (HCVcc) particles spiked to human plasma revealed the capacity of the S/D treatment to fully inactivate HCVcc within 30 minutes of treatment at 31°C, as shown by the baseline level of reporter signals, total loss of viral infectivity and absence of viral protein NS5A. Taken together, plasma nanofiltration and S/D treatment are valuable techniques to improve the safety of plasma for transfusion with regards to thrombogenicity and risks of HCV transmission, respectively.
We used these results to prepare and characterize a tailor-made, virally-inactivated, heat-treated platelet pellet lysate (HPPL) depleted of plasma proteins (in particular fibrinogen), and enriched in a physiological pleiotropic mixture of neurotrophins for brain administration. ELISA analysis and proteomics studies revealed that heat-treatment at 56°C for 30 min decreased the total protein content, modified the relative protein content, and influenced favorably the balance of platelet components with neuroprotective activity. This heat-treatment, which was found to unexpectedly improve the neuroprotective activity, also contributes to reduce HCV infectivity, as do the S/D and 20-nm-nanofiltration. PPL exerted strong neuroprotective effects in Parkinson’s disease (PD) models (a) in vitro, using LUHMES cells exposed to MPP+ neurotoxin, and (b) in vivo, in mice intoxicated by MPTP neurotoxin. In addition, we show that brain delivery of HPPL by intranasal (i.n.) administration induces a diffusion of growth factors and protect the expression of tyrosine hydroxylase (TH) expression in the substantia nigra pars compacta and striatum in the mice/MPTP model. Since neuro-inflammation can be detrimental in neurodegenerative disorders, we verified that the PPL did not stimulate the release of inflammatory markers (e.g. COX-2, iNOS) by BV2 microglial cells in culture, and could restrict COX-2 expression when cells were exposed to LPS. In addition, the PPL was found to contain 1 x 1012 MP/mL with a mean size of 160 nm. Interestingly, these MPs were found not to exert a thrombogenic impact in vitro when spiked to cerebro-spinal fluid and, furthermore, induced a neuroprotective activity in LUHMES cells exposed to neurotoxin.
Altogether, our data demonstrate the technical feasibility of developing virally-safe customized platelet lysate preparations for use in regenerative medicine, and most specifically for potential disease-modifying strategy in the treatment of neurodegenerative disorders of the central nervous system, such as PD. |
