Bioengineering
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Chapters
Summary May 17th, 2021
Please note that all translations are automatically generated.
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该协议通过使用丰富的脂肪酸、胆固醇、低浓度的细胞因子和缺氧来模拟骨髓微环境，使静默的人类造血干细胞在体外得以维持。
Transcript
细胞周期静止是造血干细胞的一个关键特征。此协议有助于了解在接近生理条件下，静默的人类 HSC 在体外的行为。利用此协议，研究人员可以测试各种化合物、营养物质或蛋白质的影响，这些化合物、营养物质或蛋白质可以调节细胞循环状态，并以可扩展的方式区分 HSC，而无需使用动物模型。
抗癌药物对静止的HSC和循环祖先的生存有不同的影响。该协议使得有可能找到可以备用静止的HSC或有选择地损害静止白血病干细胞的代理。示范程序的将是小林弘一，来自大宝实验室的高级研究员。
首先，在玻璃管中溶解每毫升棕榈酸钠16毫克，每毫升酸钠30毫克，在玻璃管中溶解甲醇中每毫升胆固醇4毫克。将脂质溶液储存在负30摄氏度，并在使用前解冻。在新鲜的玻璃管中，混合脂质溶液，获得每毫升棕榈酸盐的最终浓度为100微克，油酸盐为100微克，胆固醇为每毫升20微克。
通过脂质溶液传递氮气蒸发甲醇。在37摄氏度的水浴中加热玻璃管，完全蒸发剩余的甲醇。准备DMEM/F-12介质与 HEPES 和谷氨酰胺。
加入青霉素和硫酸链霉素，最终浓度分别为每毫升50单位和50微克。介质可储存在摄氏四度至少两个月。将 4% 的 BSA 添加到 DMEM/F-12 介质中，加入 HEPES 和谷氨酰胺，然后使用氢氧化钠溶液将介质的 pH 度调整到 7.6。
将介质加入带脂质的玻璃管中。通过声波完全溶解脂质。如果介质在声波化后不透明，请延长声波时间。
当BSA和脂质溶解时，样品应储存在负80摄氏度，并在两个月内使用。将胰岛素、转移素、塞莱尼特钠和乙醇胺混合物加入 DMEM/F-12，然后使用 0.22 微米过滤器过滤混合介质。在使用之前，在培养介质中加入人体干细胞因子或SCF以及人类血栓素或TPO，最终浓度为每毫升3毫微克。
将先前准备的文化介质中的 200 微升与细胞因子转移到平底 96 井板。为了避免介质蒸发，用 100 到 200 微升 PBS 填充所有未使用的油井。在培养介质中以每微升60个细胞的细胞因子，在培养介质中恢复分类造血干细胞或HSC。
将600个细胞加入到每个井中。不到300个细胞将导致更大的技术变异，由于营养剥夺或积累不利的细胞因子或化疗因子，在一口井中培养超过1000个细胞应避免。在37摄氏度的潮湿多气体孵化器中培养细胞，在5%的二氧化碳和1%的氧气大气中培养细胞。
经过七天的纯化HSC培养，高达80%的细胞显示标记CD34为正型，CD38为负表型。总细胞数取决于细胞因子浓度。SCF 和 TPO 的较高浓度诱导进入细胞周期、增殖和分化。
以标记 CD34 正、CD38 阴性、CD90 正和 CD45 RA 负表型为特征的表型 HSC 数量与 SCF 或 TPO 浓度成正比增加，而总细胞中的频率则有所下降。经过三个月的培养成人骨髓HSC移植后，重组可以评估为其在人体CD45阳性穆林、CD45阴性Ter119阴性细胞周围血液中频率的函数。在用刚解冻或培养的HSC移植的NOG小鼠中重组了三个血统，包括CD19正B细胞、CD13负细胞、CD33正骨髓细胞和CD3正骨髓细胞。
遵循文化，HSC 可以进行基因表达分析，如实时 PCR 和 RNA 测序。也可以使用移植到免疫缺陷小鼠身上进行功能验证。研究人员可以通过调整细胞因子浓度，直接比较在规定条件下的循环和静止的HSC。
这将有助于了解静默 HSC 特定的自我更新计划、抗压机制和代谢特性，这些在体内环境中很难测试。
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