| 摘要: | 背景
隨著世界人口老化，神經退化性疾病越來越受到關注。 這些疾病會影響人類的情緒、語言和身體行為，並造成巨大的社會成本。 目前，大多數神經退化性疾病是不可逆和無法治療的，需要更多的研究來早期發現和治癒。 為了治療這些疾病，除了保護原始神經幹細胞（NSC）外，刺激大腦中的幹細胞增殖並分化為神經細胞（包括能夠整合到神經元網路中的成熟神經元）也很重要。 我們實驗室的臨床前研究發現，經過熱處理的人類血小板顆粒裂解物（「HPPL」）含有神經營養和血管生成生長因子以及各種其他營養因子，可以在臨床前細胞和細胞中發揮神經保護和神經恢復作用。神經退化性疾病和創傷的動物模型。 我們現在想了解 HPPL 是否也能促進 NSC 的增生和分化，進而改善記憶和認知。
目標
使用小鼠的室下區 (SVZ) 和齒狀回 (DG) 組織建立離體神經球測定，以評估 HPPL 刺激 NSC 或神經祖細胞 (NPC) 增殖和分化的能力。
材料與方法
為了建立離體神經球測定，從 7-8 週齡 C57BL6/JNARL 小鼠的大腦中分離出 SVZ 和 DG。 將組織解離並在含有EGF和b-FGF的神經基礎培養基中培養。 我們將細胞培養 SVZ 6-8 天，DG 培養 10-14 天，並使用倒置螢光顯微鏡透過觀察新形成的神經球的數量和大小來評估增殖。 然後在 PDL/層粘連蛋白包被的玻璃蓋玻片上分化神經球，並用特異性抗體和 DAPI 標記細胞核、神經元和分化細胞。 從台北血液中心獲得的人類血小板濃縮液（PC）以300×g離心除去殘留的紅血球，然後以3000×g離心以獲得血小板。 為了製備 HPPL，將來自臨床級血小板濃縮物的血小板顆粒懸浮在 PBS 中，凍融 (-80°C/37°C) 3 次，澄清 (4500 x g)，熱處理（56°C，30 分鐘），離心兩次（6000×g），上清液-80℃保存。 為了對 HPPL 的神經發生活性進行初步評估，在添加或不添加不同濃度的 HPPL（0.5%、1%、2.5% 和 5%）的情況下進行離體神經球測定。
結果
我們可以使用 SVZ 和 DG 組織成功實施離體神經球測定，並定義允許產生可計數和測量的神經球的條件。 允許神經球分化的條件也被成功定義。 初步評估表明，與對照條件相比，HPPL 有助於神經球的增殖，且與 HPPL 濃度的增加呈線性趨勢。 使用 5% HPPL，從 DG 產生的神經球的平均大小約為 68μm，從 SVZ 部分產生的神經球的平均大小約為 129μm。 在 1% HPPL 存在下，DG 的神經球大小約為 91.82 ?m，SVZ 的神經球大小約為 159 ?m，顯示 HPPL 可以刺激神經球大小。 目前的數據還表明，隨著 HPPL 濃度的增加，神經球大小的增加與其數量的減少有關。
結論
我們可以使用 7-8 週齡小鼠的 SVZ 和 DG 組織建立離體神經球測定。 初步數據顯示 HPPL 具有刺激 SVZ 和 DG 幹細胞增殖神經球的潛力。 這些數據需要透過進一步的體外和體內臨床前研究來證實，支持 HPPL 在治療與神經退化性疾病和腦外傷相關的認知和記憶缺陷方面的轉化潛力。
Background
As the world population ages, neurodegenerative diseases are receiving increasing attention. These diseases can affect human emotions, language, and physical behavior, and cause huge social costs. Currently, most neurodegenerative diseases are irreversible and untreatable, requiring more research for early detection and cure. To treat these diseases, in addition to protecting the original neural stem cells (NSCs), it is important to stimulate the stem cells in the brain to proliferate and differentiate into neural cells including mature neurons capable to integrate into neuronal networks. Pre-clinical studies in our laboratory have found that a heat-treated human platelet pellet lysate ("HPPL"), which contains neurotrophic and angiogenic growth factors, and various other trophic factors, can exert neuroprotective and neurorestorative effects in pre-clinical cellular and animal models of neurodegenerative disorders and trauma. We now want to understand whether HPPL can also promote the proliferation and differentiation of NSCs to improve memory and cognition.
Aims
To establish an ex vivo neurosphere assay using subventricular zone (SVZ) and dentate gyrus (DG) tissues from mice to assess the capacity of HPPL to stimulate NSCs or neural progenitor cells (NPCs) to proliferate and differentiate.
Material and Methods
To establish the ex vivo neurosphere assay, SVZ and DG were isolated from the brain of 7-8 weeks-old C57BL6/JNARL mice. The tissues were dissociated and cultured in neural basal medium containing EGF and b-FGF. We cultured the cells for 6-8 days for SVZ, and for 10-14 days for DG, and we used an inverted fluorescence microscope to assess proliferation by observing the number and size of newly formed neurospheres. Neurospheres were then differentiated on PDL/laminin-coated glass coverslips and nuclei, neurons, and differentiated cells were labeled with specific antibodies and DAPI. Human platelet concentrates (PC) obtained from the Taipei Blood Center were centrifuged at 300 × g to remove residual red blood cells, then at 3000 × g to obtain platelets. To prepare HPPL, platelet pellets from clinical grade platelet concentrates were suspended in PBS, frozen-thawed (-80°C/37°C) three times, clarified (4500 x g), heat-treated (56 °C, 30 min), centrifuged twice (6000 × g), and the supernatant stored at ?80 °C. In order to perform preliminary assessment of the neurogenesis activity of HPPL the ex vivo neurosphere assay was perfomed with or without the addition of different concentrations of HPPL (0.5%, 1%, 2.5% and 5%).
Results
We could successfully implement the ex vivo neurosphere assay using SVZ and DG tissues and define conditions allowing the generation of neurospheres that could be counted and measured. The conditions allowing the differentiation of the neurospheres were also successfully defined. Preliminary assessment indicated that, compared to the control conditions, HPPL contributed to the proliferation of neurospheres with a linear trend associated with increasing concentrations of HPPL. The mean size of neurospheres generated from the DG was about 68?m, and from the SVZ part about 129?m using 5% HPPL. The size of the neurospheres from the DG was about 91.82 ?m and that from the SVZ was about 159 ?m in the presence of 1% HPPL, indicating that HPPL could stimulate neurosphere size. Current data also suggest that with increasing HPPL concentration, the increase in the neurosphere size is associated with a decrease in their number.
Conclusions
We could establish the ex vivo neurosphere assay using the SVZ and DG tissues from 7-8 weeks old mice. Preliminary data suggest the potential of HPPL to stimulate the proliferation of neurospheres from the stem cells present in SVZ and DG. These data, which will need to be confirmed by further ex vivo and in vivo preclinical studies, support the translational potential of HPPL for the treatment of cognitive and memory defects associated with neurodegenerative diseases and brain trauma. |
