CN117024554A - 一种Aβ1-42寡聚体存储液的制备方法及其应用 - Google Patents
一种Aβ1-42寡聚体存储液的制备方法及其应用 Download PDFInfo
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Abstract
本发明提供了一种Aβ1‑42寡聚体存储液的制备方法，该方法为：将提前冷却的六氟异丙醇加入至装有Aβ1‑42单体粉末的离心管中，室温孵育60min，得到Aβ1‑42寡聚体‑HFIP，放置在冰上10min后移至通风橱内，打开离心管盖，室温静置9h，在管底得到Aβ1‑42寡聚体膜，加入二甲基亚矾，将离心管置于超声浴中，在25℃、200W下超声10min，得到Aβ1‑42寡聚体‑DMSO溶液，加入无酚红的F12/DMEM培养基，用移液器吹打混匀后，置于4℃孵育12h，得到Aβ1‑42寡聚体存储液。还提供了用于检测Aβ1‑42寡聚体对小鼠海马神经元HT‑22细胞系的毒性、脂质过氧化损伤的方法。本发明制备的Aβ1‑42寡聚体存储液可用于研究其对小鼠海马神经元HT‑22细胞系的毒性、脂质过氧化损伤情况，亦可用其制备阿尔兹海默病细胞或小鼠模型。
Description
技术领域
本发明属于Aβ1-42寡聚体技术领域，具体涉及一种Aβ1-42寡聚体存储液的制备方法及其应用。
背景技术
阿尔兹海默病(AD)是老年人最常见的痴呆症形式，占痴呆病例的50-80％，其特征是痴呆、慢性进行性神经退行性变和认知损害，包括记忆丧失、行为改变和细胞外斑块的存在。β-淀粉样蛋白(Aβ)形成细胞外斑块，其诱导的神经毒性与AD发病机制密切。Aβ是一种38-43个氨基酸残基肽，通过β-和γ-分泌酶连续裂解淀粉样前体蛋白(APP)而产生，循环于血液、脑脊液和脑间质液中，大多与伴侣蛋白分子结合，少数以游离状态存在。人体内Aβ最常见的亚型是Aβ1-40与Aβ1-42，其中Aβ1-42具有更强的毒性，更容易聚集形成Aβ寡聚体(AβOs)，从而形成Aβ沉淀的核心，所以我们重点关注Aβ1-42寡聚体的细胞毒性作用。
AβOs是Aβ的可溶性聚集体，在神经毒性、突触毒性、触发有害的级联反应、导致AD的特征性病理改变过程中发挥重要作用。AβOs为3至10nm大小不等的小球形结构。大量体外和体内研究共同证明了AβOs诱导神经元脂质过氧化、突触缺陷、凋亡等各种病理改变。对AD患者大脑的病理研究显示，AβOs的细胞外积累导致神经元丢失。
海马神经元位于大脑颞叶内侧的海马体中，与人的记忆、认知密切相关，AD多数会导致海马神经元损伤，因此患者会出现记忆障碍、认知障碍、痴呆等症状。有研究报道，AβOs对海马神经元具有直接毒性，通过导致突触丢失，破坏神经元突触传递功能，这是AD发生的重要原因。
发明内容
本发明所要解决的技术问题在于针对上述现有技术的不足，提供一种Aβ1-42寡聚体存储液的制备方法及其应用，该Aβ1-42寡聚体存储液可用于研究其对小鼠海马神经元HT-22细胞系的毒性、脂质过氧化损伤情况，亦可用其制备阿尔兹海默病细胞或小鼠模型。
为解决上述技术问题，本发明采用的技术方案是：一种Aβ1-42寡聚体存储液的制备方法，该方法为：
S1、将提前冷却的六氟异丙醇加入至装有Aβ1-42单体粉末的离心管中，室温孵育60min，得到Aβ1-42寡聚体-HFIP；
S2、将S1中得到的Aβ1-42寡聚体-HFIP放置在冰上10min后移至通风橱内，打开离心管盖，室温静置9h，在管底得到Aβ1-42寡聚体膜；
S3、向S2中管底得到Aβ1-42寡聚体膜的离心管中加入二甲基亚矾，将离心管置于超声浴中，在温度为25℃，功率为200W的条件下超声10min，得到Aβ1-42寡聚体-DMSO溶液；
S4、向S3中得到的Aβ1-42寡聚体-DMSO溶液中加入无酚红的F12/DMEM培养基，用移液器吹打混匀后，置于4℃孵育12h，得到Aβ1-42寡聚体存储液；所述Aβ1-42寡聚体存储液于－80℃冰箱保存、备用。
优选地，S1中所述提前冷却的六氟异丙醇和Aβ1-42单体粉末的用量比为220μL:1mg。
优选地，S3中所述Aβ1-42寡聚体-DMSO溶液的浓度为5mmol/L。
优选地，S4中所述Aβ1-42寡聚体存储液的浓度为212.2μmol/L。
本发明还提供了上述制备方法制备的Aβ1-42寡聚体存储液的应用，所述Aβ1-42寡聚体存储液中的Aβ1-42寡聚体用于小鼠海马神经元HT-22细胞系的毒性、脂质过氧化损伤的检测。
优选地，用原子力显微镜鉴定寡聚体、用光学显微镜结合CCK-8检测Aβ1-42寡聚体对HT-22细胞的毒性、经脂质过氧化荧光探针标记后，通过荧光显微镜结合荧光酶标仪检测Aβ1-42寡聚体对HT-22细胞的脂质过氧化损伤。
本发明还提供了上述制备方法制备的Aβ1-42寡聚体存储液的应用，所述Aβ1-42寡聚体存储液用于制备阿尔兹海默病细胞或小鼠模型。
本发明与现有技术相比具有以下优点：
1、本发明中的Aβ1-42寡聚体的制备方法，通过超声助溶使Aβ1-42寡聚体薄膜充分溶解；不含酚红的F12/DMEM培养液稀释，4℃过夜孵育后，不进行14000r/min的离心操作，避免了Aβ1-42寡聚体溶液浓度降低；不用负压抽吸机、0.22μm低蛋白吸附过滤器，简化了制备流程，操作简单便捷，同时降低了所得溶液中Aβ1-42寡聚体的损失。
2、本发明中用原子力显微镜鉴定寡聚体：可提供寡聚体真正的三维表面图；无需对样品进行任何的特殊处理，比如镀铜或碳等，避免对样品造成不可逆转的伤害；原子力显微镜不管是在常压下，还是在液体环境下，都可以很好的运行；
用光学显微镜结合CCK-8检测Aβ1-42寡聚体对HT-22细胞的毒性：形态学结合吸光度值，更加全面的反应细胞损伤情况；检测快速，且灵敏度高，甚至可以测定较低的细胞密度；易于重复，且细胞毒性小；
经脂质过氧化荧光探针标记后，通过荧光显微镜结合荧光酶标仪检测Aβ1-42寡聚体对HT-22细胞的脂质过氧化损伤：形态学结合荧光值，更加全面的体现细胞脂质过氧化情况；荧光探针可以同时反应细胞膜脂质的氧化、还原状态；适用于活细胞检测，检测迅速、灵敏度高、对细胞毒性小。
本发明制备的Aβ1-42寡聚体存储液可用于研究其对小鼠海马神经元HT-22细胞系的毒性、脂质过氧化损伤情况，亦可用其制备阿尔兹海默病细胞或小鼠模型。
下面结合附图和实施例对本发明作进一步详细说明。
附图说明
图1是本发明实施例1的原子力显微镜下的Aβ1-42寡聚体图。
图2是本发明实施例1的不同浓度Aβ1-42寡聚体处理HT-22细胞后光学显微镜下的形态图。
图3是本发明实施例1的CCK-8检测不同浓度组HT-22细胞的存活率。
图4是本发明实施例1的不同浓度Aβ1-42寡聚体对HT-22细胞脂质过氧化的影响图。
图5是本发明实施例1的不同浓度Aβ1-42寡聚体对HT-22细胞氧化型脂质荧光值/还原型脂质荧光值的影响图。
具体实施方式
实施例1
本实施例的Aβ1-42寡聚体存储液的制备方法，该方法为：
S1、将220μL的提前冷却的六氟异丙醇(HFIP)加入至装有1mg的Aβ1-42单体粉末的离心管中，室温孵育60min，得到Aβ1-42寡聚体-HFIP；
所述Aβ1-42单体粉末，市购，由杭州丹港生物科技有限公司合成；
S2、将S1中得到的Aβ1-42寡聚体-HFIP放置在冰上10min后移至通风橱内，打开离心管盖，室温静置9h，在管底得到Aβ1-42寡聚体膜；
S3、向S2中管底得到Aβ1-42寡聚体膜的离心管中加入44μL二甲基亚矾(DMSO)，将离心管置于超声浴中，在温度为25℃，功率为200W的条件下超声10min，得到浓度为5mmol/L的Aβ1-42寡聚体-DMSO溶液；
本实施例中通过超声助溶使Aβ1-42寡聚体薄膜充分溶解；
S4、向S3中得到的Aβ1-42寡聚体-DMSO溶液中加入无酚红的F12/DMEM培养基，用移液器吹打混匀后，置于4℃孵育12h，得到浓度为212.2μmol/L的Aβ1-42寡聚体存储液；所述Aβ1-42寡聚体存储液于－80℃冰箱保存、备用；
所述F12/DMEM培养基市购，购于赛默飞世尔科技(中国)有限公司，包括以下成分的原料制成：15mMN-2羟乙基哌嗪-N-2-乙烷磺酸(HEPES)，3151mg/LD-葡萄糖，55mg/L丙酮酸钠，365mg/LL-谷氨酰胺，1200mg/L碳酸氢钠以及微量元素，pH值7.0-7.4。
本实施例用不含酚红的F12/DMEM培养基稀释，4℃过夜孵育后，不进行离心操作，避免了Aβ1-42寡聚体溶液浓度降低。
本实施例不用负压抽吸机、0.22μm低蛋白吸附过滤器，简化了制备流程，满足所有实验室的条件，减少所得溶液中Aβ1-42寡聚体的损失。
本实施例还提供了上述制备方法制备的Aβ1-42寡聚体存储液的应用，所述Aβ1-42寡聚体存储液中的Aβ1-42寡聚体用于海马神经元HT-22细胞系的毒性、脂质过氧化损伤的检测。
(一)原子力显微镜检测Aβ1-42寡聚：
将制备好的Aβ1-42寡聚体滴在云母片上，干燥后用原子力显微镜进行测试，测试条件为：美国布鲁克DimensonICON原子力显微镜tapping模式，扫描速度1KHZ，像素256。检测图像图1所示，图1中：原子力显微镜检测Aβ1-42寡聚体二维平面图，结合右侧图例可以看出寡聚体粒径大致在3至15nm范围内；B：Aβ1-42寡聚体三维图；C：Aβ1-42寡聚体三维峰图。由图1可知颜色越浅颗粒直径越大，颜色越深直径越小。结合图1A右侧标尺可知，呈白色的颗粒直径大约为3-15nm，从粒径大小判断属于Aβ1-42寡聚体。
(二)Aβ1-42寡聚体对小鼠海马神经元HT-22细胞的毒性：
HT-22细胞传代至96孔板培养12h后，分别用浓度为(0,0.5,1,2.5,5,10,20)μmol/L的Aβ1-42寡聚体处理各组细胞12h；在光学显微镜下观察各组细胞的形态，采集图像；然后弃去旧培养基，向每孔加入100μl新鲜培养基，同时各加入10μlCCK-8溶液，置入细胞培养箱继续培养2h；取出避光，用酶标仪测定在450nm处的吸光度，计算各组细胞活性。图2中A-G：浓度分别为(0,0.5,1,2.5,5,10,20)μmol/L的Aβ1-42寡聚体处理各组HT-22细胞12h后，光学显微镜下的细胞形态，标尺＝50μm。
Aβ1-42寡聚体对HT-22细胞的毒性作用呈浓度依赖性，Aβ1-42寡聚体浓度越高，HT-22细胞损伤越明显。Aβ1-42寡聚体浓度增加到2.5μmol/L时，HT-22细胞出现明显损伤(图2D)。
如图3所示，CCK-8检测不同浓度组HT-22细胞的存活率，各浓度的Aβ1-42寡聚体均使HT-22细胞存活率显著下降，当Aβ1-42寡聚体浓度高于2.5μmol/L时，HT-22细胞存活率的降低呈浓度依赖性。图3中*P<0.05,**P<0.01,***P<0.001vs0μmol/L；#P<0.05,###P<0.001vs2.5μmol/L；&&P<0.01,&&&P<0.001vs5.0μmol/L；$$P<0.01vs10.0μmol/L.
(三)Aβ1-42寡聚体对HT-22细胞的脂质过氧化损伤：
HT-22细胞传代至96孔板培养12h后，分别用浓度为(0,0.5,1,2.5,5,10,20)μmol/L的Aβ1-42寡聚体处理各组细胞12h；弃去旧培养基，向每孔加入100μl新鲜培养基，同时向各孔加入一定量的懋康生物的C11BODIPY581/591脂质过氧化荧光探针存储液，使其终浓度为5μM，37℃孵育30min；弃去培养基，用PBS清晰3次，荧光酶标仪检测各组荧光值(还原态用Ex/Em＝570/610nm；氧化态用Ex/Em＝488/525nm)，用氧化态/还原态比值反应各组细胞脂质过氧化水平；之后用荧光显微镜采集各组细胞氧化态、还原态的图像。
图4中绿色荧光为氧化型脂质，红色荧光为还原型脂质，用浓度为(0,0.5,1,2.5,5)μmol/L的Aβ1-42寡聚体处理HT-22细胞12h，绿色荧光逐渐增强，即Aβ1-42寡聚体对HT-22细胞的脂质过氧化作用呈浓度依赖性。浓度为(10,20)μmol/L的Aβ1-42寡聚体处理HT-22细胞12h，绿色荧光强度和对照组相比无明显增强，这可能和细胞膜的损伤有关。
如图5所示，不同浓度Aβ1-42寡聚体对HT-22细胞氧化型脂质荧光值/还原型脂质荧光值的影响。分别用浓度为(0,0.5,1,2.5,5,10,20)μmol/L的Aβ1-42寡聚体处理各组细胞12h，用氧化型脂质荧光值/还原型脂质荧光值反应细胞脂质过氧化水平。浓度为(2.5,5)μmol/L的Aβ1-42寡聚体处理HT-22细胞后，脂质过氧化水平较其余各组均显著增高，其中5μmol/L浓度组高于2.5μmol/L浓度组。图5中***P<0.001vs0μmol/L；###P<0.001vs0.5μmol/L；&&&P<0.001vs1.0μmol/L；$$P<0.01,$$$P<0.001vs2.5μmol/L；！！！P<0.001vs5.0μmol/L。
本实施例用原子力显微镜鉴定寡聚体：可提供寡聚体真正的三维表面图；无需对样品进行任何的特殊处理，比如镀铜或碳等，避免对样品造成不可逆转的伤害；原子力显微镜不管是在常压下，还是在液体环境下，都可以很好的运行；
用光学显微镜结合CCK-8检测Aβ1-42寡聚体对HT-22细胞的毒性：形态学结合吸光度值，更加全面的反应细胞损伤情况；检测快速，且灵敏度高，甚至可以测定较低的细胞密度；易于重复，且细胞毒性小；
经脂质过氧化荧光探针标记后，通过荧光显微镜结合荧光酶标仪检测Aβ1-42寡聚体对HT-22细胞的脂质过氧化损伤：形态学结合荧光值，更加全面的体现细胞脂质过氧化情况；荧光探针可以同时反应细胞膜脂质的氧化、还原状态；适用于活细胞检测，检测迅速、灵敏度高、对细胞毒性小。
本实施例中制备的Aβ1-42寡聚体能诱导小鼠海马神经元脂质过氧化损伤。
本实施例中制备的Aβ1-42寡聚体存储液用于制备阿尔兹海默病细胞或小鼠模型。阿尔兹海默病主要是由于AβOs在神经元中聚集，对神经元产生细胞毒性，导致神经元发生脂质过氧化、凋亡等损伤。AβOs尤其常积聚于海马部位，引起记忆、认知、意识障碍。我们已经证实Aβ1-42寡聚体可以引起小鼠海马神经元细胞毒性，发生脂质过氧化损伤，这充分说明Aβ1-42寡聚体可以用来制备阿尔兹海默病细胞模型。对于动物模型的制备，可以将Aβ1-42寡聚体通过立体定位注射入小鼠海马中，通过水迷宫、旷场实验、高架迷宫等动物行为学实验检测模型小鼠的认知、记忆，证实模型小鼠认知、记忆发生障碍；通过分子生物学实验检测脑组织细胞损伤指标、脂质过氧化损伤水平，证实Aβ1-42寡聚体可以在体内损伤小鼠海马神经元，进而可以用来制备阿尔兹海默病小鼠模型。
以上所述，仅是本发明的较佳实施例，并非对本发明作任何限制。凡是根据发明技术实质对以上实施例所作的任何简单修改、变更以及等效变化，均仍属于本发明技术方案的保护范围内。
Claims (7)
1.一种Aβ1-42寡聚体存储液的制备方法，其特征在于，该方法为：
S1、将提前冷却的六氟异丙醇加入至装有Aβ1-42单体粉末的离心管中，室温孵育60min，得到Aβ1-42寡聚体-HFIP；
S2、将S1中得到的Aβ1-42寡聚体-HFIP放置在冰上10min后移至通风橱内，打开离心管盖，室温静置9h，在管底得到Aβ1-42寡聚体膜；
S3、向S2中管底得到Aβ1-42寡聚体膜的离心管中加入二甲基亚矾，将离心管置于超声浴中，在温度为25℃，功率为200W的条件下超声10min，得到Aβ1-42寡聚体-DMSO溶液；
S4、向S3中得到的Aβ1-42寡聚体-DMSO溶液中加入无酚红的F12/DMEM培养基，用移液器吹打混匀后，置于4℃孵育12h，得到Aβ1-42寡聚体存储液；所述Aβ1-42寡聚体存储液于－80℃冰箱保存、备用。
2.根据权利要求1所述的一种Aβ1-42寡聚体存储液的制备方法，其特征在于，S1中所述提前冷却的六氟异丙醇和Aβ1-42单体粉末的用量比为220μL:1mg。
3.根据权利要求1所述的一种Aβ1-42寡聚体存储液的制备方法，其特征在于，S3中所述Aβ1-42寡聚体-DMSO溶液的浓度为5mmol/L。
4.根据权利要求1所述的一种Aβ1-42寡聚体存储液的制备方法，其特征在于，S4中所述Aβ1-42寡聚体存储液的浓度为212.2μmol/L。
5.一种如权利要求1-4的所述制备方法制备的Aβ1-42寡聚体存储液的应用，其特征在于，所述Aβ1-42寡聚体存储液中的Aβ1-42寡聚体用于小鼠海马神经元HT-22细胞系的毒性、脂质过氧化损伤的检测。
6.一种如权利要求5所述的应用，其特征在于，用原子力显微镜鉴定寡聚体、用光学显微镜结合CCK-8检测Aβ1-42寡聚体对HT-22细胞的毒性、经脂质过氧化荧光探针标记后，通过荧光显微镜结合荧光酶标仪检测Aβ1-42寡聚体对HT-22细胞的脂质过氧化损伤。
7.一种如权利要求1-4的所述制备方法制备的Aβ1-42寡聚体存储液的应用，其特征在于，所述Aβ1-42寡聚体存储液用于制备阿尔兹海默病细胞或小鼠模型。
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| 王宝萍: "Aβ42及CD40L对原代小胶质细胞活化及神经元的作用研究", 《中国优秀硕士学位论文全文数据库（医药卫生科技辑）》, vol. 2013, no. 4, 15 April 2013 (2013-04-15), pages 13 - 14 * |
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