CN114107204B - 脑胶质瘤巨噬细胞类配体的体系构建 - Google Patents
脑胶质瘤巨噬细胞类配体的体系构建 Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明属于生物技术领域，具体涉及一种脑胶质瘤巨噬细胞类配体的体系构建，包括以下步骤：收取肿瘤组织样本，并剪去肿瘤包膜和坏死出血组织；冲洗组织；接着将组织剪至0.5‑1mm3大小；随后重悬后离心；向离心所得沉淀中加入红细胞裂解液裂解，再次离心，吸取上清液并用所述培养基重悬后离心；最后吸取上清液，再用培养基重悬沉淀后加入培养体系继续培养；所述培养体系包括以下组分：不含酚红神经基础的液体培养基、已除去维生素A的B‑27添加剂、2‑羧乙基盐酸磷化氢、L‑丙氨酰‑L‑谷氨酰胺、胰岛素‑转铁蛋白‑硒‑丙酮酸钠添加剂、巨噬细胞集落刺激因子、Ⅱ型干扰素和白细胞介素。采用该方法培养脑胶质瘤巨噬细胞特征的类配体，表型稳定性好。
Description
技术领域
本发明属于生物技术领域，具体涉及一种脑胶质瘤巨噬细胞类配体的体系构建。
背景技术
胶质瘤是中枢神经系统最常见的原发性恶性肿瘤，其死亡率高，易复发，目前缺乏有效的治疗手段(郭鹏超，胶质瘤研究进展[J]，济宁医学院学报，2019；章婷婷，大鼠C6胶质瘤MR显像及靶向治疗的多功能纳米药物载体研究[J]，安徽医科大学，2014)。
现阶段，针对胶质瘤的主流体外细胞培养方案是从胶质瘤中分选肿瘤细胞，进而进行细胞培养或体外刺激，观察细胞表型变化。然而，该细胞培养方式与真实的胶质瘤微环境相差甚远，并不能反映胶质瘤微环境的实际情况。
随着对胶质瘤不断深入的研究，业已证明胶质瘤除了大量肿瘤细胞，微环境中还存在丰富的肿瘤相关巨噬细胞(Tumor associated macrophage,TAM)。在恶性程度最高的胶质母细胞瘤中，巨噬细胞占所有细胞成分的比例可高达30％-40％。TAM是一类有可塑性的细胞群体，在肿瘤组织中以促肿瘤生长和免疫抑制功能的巨噬细胞为主，这些细胞高表达CD163、CD68标记物。这些巨噬细胞还可通过炎性因子分泌、免疫调控等诱导形成胶质瘤特殊的免疫抑制微环境。因此，急需开发新的胶质瘤体外培养方案，建立一种肿瘤细胞与TAM共存，且模拟肿瘤细胞和TAM细胞体内特征(细胞成分、数量比例和标记特征)的体系构建，模拟体内胶质瘤微环境，为胶质瘤的基础和转化治疗研究提供更好模型支持。
然而，目前无专门用于培养脑胶质瘤巨噬细胞类配体的体系构建。现依然通过体外培养单核细胞加入细胞因子进行诱导，每次诱导周期为3到7天，且诱导的巨噬细胞增殖能力差、表型不稳定、无法进行传代。
发明内容
鉴于此，本发明的目的在于提供一种能够提高表型稳定性的脑胶质瘤巨噬细胞类配体的体系构建。
为实现上述目的，本发明的技术方案为：
脑胶质瘤巨噬细胞类配体的体系构建，包括以下步骤：
新鲜手术切除的脑胶质瘤组织置于组织保存液中，并在4℃下立即送往实验室，必须立即对组织进行处理，随即在培养皿中用显微剪剪去肿瘤包膜和坏死出血组织；随后用DPBS冲洗组织三次，接着将组织剪至0.5-1mm3大小；随后转移至离心管中用培养基重悬组织后离心，吸弃上清；向离心所得沉淀中加入红细胞裂解液并混合均匀裂解，再次离心，吸弃上清液并用所述培养液重悬后离心，重复三次；吸取上清液，再用培养基重悬组织后接种到的Transwell小室上，再将小室于37℃、5％二氧化碳培养箱中培养；每隔2～3天更换一次新的培养基，将Transwell小室转移后加入培养体系，继续培养；所述培养体系包括以下组分：不含酚红神经基础的液体培养基、已除去维生素A的B-27添加剂、2-羧乙基盐酸磷化氢、L-丙氨酰-L-谷氨酰胺、胰岛素-转铁蛋白-硒-丙酮酸钠添加剂、巨噬细胞集落刺激因子、Ⅱ型干扰素、白细胞介素4、人表皮生长因子和人碱性成纤维细胞生长因子。
进一步，所述培养体系还包括胰岛素非必需氨基酸。
进一步，所述培养体系中，B-27添加剂的浓度为0.5wt％-3wt％，2-羧乙基盐酸磷化氢的浓度为1-5mmol/L，L-丙氨酰-L-谷氨酰胺的浓度为2-5mmol/L，非必需氨基酸的浓度为1wt％-5wt％，胰岛素-转铁蛋白-硒-丙酮酸钠添加剂的浓度为1wt％-5wt％，人表皮生长因子的浓度为5-20ng/mL，人碱性成纤维细胞生长因子的浓度为5-20ng/mL，巨噬细胞集落刺激因子的浓度为5-20ng/mL，Ⅱ型干扰素的浓度为10-50ng/mL，白细胞介素4的浓度为10-50ng/mL。
进一步，冲洗组织用的药剂为磷酸盐缓冲液。
进一步，所述离心的转速为300-500转/min，时间为3-10min。
本发明的目的还在于保护用于脑胶质瘤巨噬细胞类配体培养的培养体系，包括以下组分：不含酚红神经基础的液体培养基、已除去维生素A的B-27添加剂、2-羧乙基盐酸磷化氢、L-丙氨酰-L-谷氨酰胺、胰岛素-转铁蛋白-硒-丙酮酸钠添加剂、巨噬细胞集落刺激因子、Ⅱ型干扰素、白细胞介素4、人表皮生长因子和人碱性成纤维细胞生长因子。
进一步，所述培养体系还包括非必需氨基酸。
进一步，B-27添加剂的浓度为0.5wt％-3wt％，2-羧乙基盐酸磷化氢的浓度为1-5mmol/L，L-丙氨酰-L-谷氨酰胺的浓度为2-5mmol/L，非必需氨基酸的浓度为1wt％-5wt％，胰岛素-转铁蛋白-硒-丙酮酸钠添加剂的浓度为1wt％-5wt％，人表皮生长因子的浓度为5-20ng/mL，人碱性成纤维细胞生长因子的浓度为5-20ng/mL，巨噬细胞集落刺激因子的浓度为5-20ng/mL，Ⅱ型干扰素的浓度为10-50ng/mL，白细胞介素4的浓度为10-50ng/mL。
本发明的有益效果在于：
采用本发明的方法培养脑胶质瘤巨噬细胞，表型稳定性好，更能反应脑胶质瘤巨噬细胞微环境状态，为今后脑胶质瘤免疫治疗提供新的模型基础。
附图说明
图1为类配体培养一周后，CD163染色结果图；
图2为胶质瘤巨噬细胞类配体培养八周后SOX2、Ki67、CD68和CD163染色结果图；
图3为HE染色结果图和胶质瘤巨噬细胞类器官培养的光镜结果图。
具体实施方式
所举实施例是为了更好地对本发明的内容进行说明，但并不是本发明的内容仅限于所举实施例。所以熟悉本领域的技术人员根据上述发明内容对实施方案进行非本质的改进和调整，仍属于本发明的保护范围。
实施例1
培养体系，其组成为：不含酚红神经基础的液体培养基(购自Thermo)、B-27添加剂(去除维生素A)0.5wt％(购自Thermo)、2-羧乙基盐酸磷化氢1mmol/L(购自MEC公司)、1mmol/L的L-丙氨酰-L-谷氨酰胺(购自precell公司)、1wt％的非必需氨基酸(购自precell公司)、胰岛素-转铁蛋白-硒-丙酮酸钠添加剂1wt％(购自precell公司)、人表皮生长因子10ng/mL(购自peprotech公司)、人碱性成纤维细胞生长因子10ng/ml(购自peprotech公司)、巨噬细胞集落刺激因子10ng/ml(购自peprotech公司)、Ⅱ型干扰素20ng/mL(购自peprotech公司)和白细胞介素4 20ng/mL(购自peprotech公司)。
对比例1
培养体系，其组成为：50％DMEM/F12培养基，50％神经基础培养基Neurobasal，1wt％的细胞培养添加剂Glutamax，1wt％非必需氨基酸NEAAs，0.5wt％不含维生素A的细胞培养添加剂B-27，1wt％青霉素-链霉素和2.5ng/mL胰岛素。
实施例2
脑胶质瘤免疫微环境特征的类配体的体系构建，其以下按照具体步骤进行：
取同一例脑肿瘤标本，将组织转移至培养皿中，用显微剪剪去肿瘤包膜和坏死出血组织；随后用预冷的DPBS冲洗组织，将组织放入1.5ml EP管中剪至0.5-1mm3大小；用培养基重悬组织，转移至15ml离心管中；400转/min下离心5min；吸取上清液，并向上清液中加入3ml红细胞裂解液，混合均匀，然后重悬组织，室温下裂红，400转/min下离心3min，吸弃上清液再用培养基重悬组织，400转/min下离心5分钟，再次吸弃上清液，均分为两组，分别加入实施例1(实验组)和对比例1(对照组)的培养基，置于37℃、5％二氧化碳培养箱中培养；每隔三天更换一次新的培养体系。
将类配体培养一周后进行CD163免疫荧光染色，并统计阳性细胞占总细胞数的百分比；结果如图1所示结果如1所示；
将采用实施例2的培养体系培养的类配体培养1周后、2周后及4周后，进行免疫荧光染色，具体过程为，将培养的类配体转移至离心管中，于300转/min转速下离心5min，弃上清液；再将离心所得沉淀转移至冰冻切片机探头上，加入OCT包埋剂，快速冷冻，然后进行切片，切片厚度6um；防脱载玻片标片；冰丙酮固定15min；室温晾干；染色前PBS洗三次，每次5min，用含0.3％Triton X-100的免疫荧光封闭液室温封闭1h；用1ml移液器小心吸出封闭液，加入配置好的一抗加到载玻片上，4℃孵育过夜；第二天PBS洗三次，每次5min；加入对应的荧光二抗，室温孵育1h；PBS洗三次，每次5min；加入含DAPI的抗荧光淬灭剂进行封片，荧光显微镜拍照；分别对肿瘤干细胞标记物SOX2、细胞增殖标记物Ki67、巨噬细胞标记物CD68和巨噬细胞标记物CD163进行染色；统计阳性细胞占总细胞数的百分比；结果如图2所示；
将实施例2中的类配体培养前、培养1周后、2周后、4周及8周后，进行HE染色，将前期的切片，75％酒精1s、85％酒精1s、95％酒精1s、100％酒精1s、苏木素染色5min、水洗1min、1％盐酸酒精分化1s、水冲洗2min、伊红染色1s、75％酒精1秒、85％酒精1秒、95％酒精1s、100％酒精1s、二甲苯1s、晾干后中性树胶封片，并用光镜观察类配体的形态，结果如图3所示。
由图1可知，与对比例1相比，实施例1的类配体的阳性细胞比例得到了显著提高，由此表明，采用本发明的方法培养巨噬细胞类配体，表型稳定性得到了显著提高。
由图2可知，培养四周后，巨噬细胞维持亲本肿瘤表型特性。由此表明，采用本发明的方法培养巨噬细胞类配体，能够维持亲本肿瘤的特征。
由图3HE染色结果可知，经8周的培养，巨噬细胞维持亲本肿瘤表型特性。由此表明，采用本发明的方法培养巨噬细胞类配体，能够维持亲本肿瘤的特征。
由图3光镜结果可知，经8周的培养，类配体具有一定增殖能力。由此表明，采用本发明的方法培养巨噬细胞类配体，能够维持亲本肿瘤的增殖能力。
此外，应当理解，虽然本说明书按照实施方式加以描述，但并非每个实施方式仅包含一个独立的技术方案，说明书的这种叙述方式仅仅是为清楚起见，本领域技术人员应当将说明书作为一个整体，各实施例中的技术方案也可以经适当组合，形成本领域技术人员可以理解的其他实施方式。
Claims (3)
1.脑胶质瘤巨噬细胞类配体的体系构建方法，其特征在于，包括以下步骤：
新鲜手术切除的脑胶质瘤组织置于组织保存液中，并在4℃下立即送往实验室，必须立即对组织进行处理，随即在培养皿中用显微剪剪去肿瘤包膜和坏死出血组织；随后用DPBS冲洗组织三次，接着将组织剪至0.5-1mm3大小；随后转移至离心管中用培养基重悬组织后离心，吸弃上清；向离心所得沉淀中加入红细胞裂解液并混合均匀裂解，再次离心，吸弃上清液并用所述培养液重悬后离心，重复三次；吸取上清液，再用培养基重悬组织后接种到Transwell小室上，再将小室于37℃、5％二氧化碳培养箱中培养；每隔2～3天更换一次新的培养基，将Transwell小室转移后加入培养体系，继续培养；所述培养体系包括以下组分：不含酚红的神经基础的液体培养基、已除去维生素A的B-27添加剂、2-羧乙基盐酸磷化氢、L-丙氨酰-L-谷氨酰胺、胰岛素-转铁蛋白-硒-丙酮酸钠添加剂、巨噬细胞集落刺激因子、Ⅱ型干扰素、白细胞介素4、人表皮生长因子和人碱性成纤维细胞生长因子；
其中，所述培养体系中，B-27添加剂的浓度为0.5wt％-3wt％，2-羧乙基盐酸磷化氢的浓度为1-5mmol/L，L-丙氨酰-L-谷氨酰胺的浓度为2-5mmol/L，胰岛素-转铁蛋白-硒-丙酮酸钠添加剂的浓度为1wt％-5wt％，人表皮生长因子的浓度为5-20ng/mL，人碱性成纤维细胞生长因子的浓度为5-20ng/mL，巨噬细胞集落刺激因子的浓度为5-20ng/mL，Ⅱ型干扰素的浓度为10-50ng/mL，白细胞介素4的浓度为10-50ng/mL。
2.根据权利要求1所述的体系构建方法，其特征在于，所述培养体系还包括非必需氨基酸。
3.据权利要求1或2所述的体系构建方法，其特征在于，所述离心的转速为300-500转/min，时间为3-10min。
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| JPH05130863A (ja) * | 1991-10-17 | 1993-05-28 | Japan Tobacco Inc | サイトカイン活性化マクロフア−ジ |
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| JPH05130863A (ja) * | 1991-10-17 | 1993-05-28 | Japan Tobacco Inc | サイトカイン活性化マクロフア−ジ |
| CN102286426A (zh) * | 2011-08-03 | 2011-12-21 | 深圳市北科生物科技有限公司 | 单个核细胞培养液 |
| CN106244546A (zh) * | 2016-08-24 | 2016-12-21 | 中国人民解放军第三军医大学第附属医院 | 一种m2型肿瘤相关巨噬细胞模型的构建方法及应用 |
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