CN116042724A - 一种通过干扰Slc12a8导致NAD+/NADH水平降低建立衰老细胞模型 - Google Patents
一种通过干扰Slc12a8导致NAD+/NADH水平降低建立衰老细胞模型 Download PDFInfo
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Abstract
本发明涉及通过干扰Slc12a8导致NAD+/NADH水平降低诱导建立衰老细胞模型的新方法，步骤如下：使用小干扰RNA干扰细胞Slc12a8基因48h后，即得衰老细胞模型。本发明方法构建的模型的优势在于缩短建模时间，能同时造成毒性损伤、氧化应激及PARP1、SIRT1蛋白下调，实验条件易控制。该模型体外将小干扰RNA用灭菌的超纯水溶解，使工作液浓度为0.1mmol·L－1，可直接加入NCM460、HT29和HCT116细胞，通过检测细胞毒性、NAD+/NADH水平及细胞氧化应激变化来评价模型。
Description
技术领域
本发明属于细胞工程技术领域，是一种研究Slc12a8水平和NAD+/NADH水平对细胞衰老影响的细胞模型。
背景技术
衰老指机体对环境的生理和心理适应能力进行性降低、逐渐趋向死亡的现象。衰老可分为两类：生理性衰老和病理性衰老。前者指成熟期后出现的生理性退化过程，后者是由于各种外来因素(包括各种疾病)所导致的老年性变化。两者实际很难区分。总之，衰老是许多病理、生理和心理过程的综合作用的必然结果，是个体生长发育最后阶段的生物学心理学过程。
为探究化疗药物作用下，NAD+/NADH水平变化，以及对Slc12a8表达的影响，选用奥沙利铂和阿霉素处理细胞。奥沙利铂是第三代铂类抗癌药，是一种常用的以铂为基础的直肠癌药物，是一种DNA合成抑制剂。它会导致DNA交联损伤，阻止DNA复制和转录并导致细胞死亡。阿霉素是一种抗肿瘤抗生素，可抑制RNA和DNA的合成，对RNA的抑制作用最强，抗瘤谱较广，对多种肿瘤均有作用，属周期非特异性药物，对各种生长周期的肿瘤细胞都有杀灭作用。
细胞模型是微观层面研究衰老的有效方法之一，建立衰老细胞模型来模拟衰老机制是广泛使用的研究手段。瓦尔堡效应指出癌细胞与正常细胞的根本区别便源于两者糖酵解作用与呼吸作用所占比例的差别。认为癌症的主要原因是正常身体细胞的有氧呼吸被糖的酵解所替代了，因此癌细胞需要高水平的烟酰胺腺嘌呤二核苷酸(NAD+)维持高水平的糖酵解。NAD+是氧化还原反应的辅酶，使其成为能量代谢的中心。Slc12a8是合成NAD+前体NMN的转运蛋白，在多种恶性肿瘤中高表达。因为NAD+是细胞物质和能量代谢的重要辅酶，和正常细胞相比，肿瘤细胞对NAD+的需求量更大，所以肿瘤细胞会表达更多的Slc12a8以满足自身对NAD+的高需求，这也使肿瘤细胞更容易受到NAD+生成抑制的影响。
本发明干扰Slc12a8的表达，促进细胞死亡，在此基础上使用奥沙利铂与阿霉素促进DNA损伤药物，降低细胞中NAD+含量，进一步升高癌细胞中活性氧水平，产生DNA的氧化损伤，进而启动癌细胞的凋亡通路，造成癌细胞的死亡。
但NAD+/NADH对结肠癌的影响目前尚知之甚少。用干扰Slc12a8来构建衰老体外细胞模型，将衰老和氧化应激联系起来，探索其癌症发生的潜在病理学作用，目前国内外尚未见相关文献报道。
通过检索，尚未发现与本发明专利申请相关的专利公开文献。
发明内容
本发明的目的在于克服现有技术的不足之处，提供一种通过干扰Slc12a8导致NAD+/NADH水平降低诱导建立衰老细胞模型的新方法，
本发明解决其技术问题所采用的技术方案是：
一种通过干扰Slc12a8导致NAD+/NADH水平降低诱导建立衰老细胞模型的新方法，步骤如下：
使用小干扰RNA干扰细胞Slc12a8基因48h后，即得衰老细胞模型。
而且，所述小干扰RNA工作浓度为0.1mmol·L－1。
而且，所述细胞为NCM460、HT29、HCT116。
而且，所述衰老细胞模型中细胞NAD+/NADH水平降低，细胞死亡率提高；细胞内ROS增加；强化化疗药物刺激细胞ROS水平；细胞PARP1和SIRT1下调。
如上所述的方法制得的衰老细胞模型在药物开发方面中的应用。
本发明取得的优点和积极效果为：
1、与传统的续培养的方法构建衰老细胞模型相比，本发明方法构建的模型的优势在于构建时间大大缩短，快速降低细胞内NAD+/NADH水平水平，促进细胞氧化应激，实验条件易控制。该模型体外将小干扰RNA用灭菌的超纯水溶解，使母液浓度为20mmol·L－1，可直接加入NCM460、HT29和HCT116细胞，通过检测细胞生存率、NAD+/NADH水平及细胞氧化应激变化来评价模型。
2、本发明方法是将小干扰RNA加入NCM460、HT29和HCT116细胞中，诱导48h后，从细胞毒性、SIRT1,PARP1蛋白变化、氧化应激这几方面为评价指标进行检测，旨在为后续研究衰老的发病机制及癌症相关药物开发提供参考。
3、本发明方法构建的模型解决了利用自然衰老构建衰老细胞模型的问题，该模型可以同时引起细胞毒性、氧化应激、NAD+/NADH水平下降。
附图说明
图1为本发明中小干扰RNA、奥沙利铂、阿霉素以及联合作用，干扰NCM460、HT29和HCT116细胞后，MTT法检测细胞毒性图；
图2为本发明中小干扰RNA、奥沙利铂、阿霉素以及联合作用，作用于NCM460、HT29和HCT116细胞48h后，共聚焦显微镜检测细胞内ROS荧光图；
图3为本发明中小干扰RNA、奥沙利铂、阿霉素以及联合作用，作用于NCM460、HT29和HCT116细胞48h后，NAD+试剂盒检测NAD+和NADH水平结果图；
图4为本发明中小干扰RNA、奥沙利铂、阿霉素以及联合作用，作用于NCM460、HT29和HCT116细胞48h后，WesternBlot检测细胞PARP1、SIRT1蛋白表达图。
具体实施方式
下面结合实施例，对本发明进一步说明，下属实施例是叙述性的，不是限定性的，不能以下述实施例来限定本发明的保护范围。
本发明中所使用的原料，如无特殊说明，均为常规市售产品，本发明中所使用的方法，如无特殊说明，均为本领域常规方法，本发明所用各物质质量均为常规使用质量。
一种通过干扰Slc12a8导致NAD+/NADH水平降低诱导建立衰老细胞模型的新方法，步骤如下：
使用小干扰RNA干扰细胞Slc12a8基因48h后，即得衰老细胞模型。
较优地，所述小干扰RNA工作浓度为0.1mmol·L－1。
较优地，所述细胞为NCM460、HT29、HCT116
较优地，所述衰老细胞模型中细胞NAD+/NADH水平降低，细胞死亡率提高；细胞内ROS增加；强化化疗药物刺激细胞ROS水平；细胞PARP1和SIRT1下调。
如上所述的方法制得的衰老细胞模型在药物开发方面中的应用。
具体地，相关制备及检测如下：
(1)小干扰RNA、奥沙利铂、阿霉素以及联合作用，干扰NCM460、HT29和HCT116细胞48h后，MTT法检测细胞毒性图，结果如图1所示干扰Slc12a8促进细胞死亡，且干扰Slc12a8促进奥沙利铂和阿霉素对细胞的致死率；
(2)小干扰RNA、奥沙利铂、阿霉素以及联合作用，干扰NCM460、HT29和HCT116细胞48h后，探针法检测细胞内ROS，结果如图2所示发现细胞内ROS大量增加，可能引起氧化应激。
(3)小干扰RNA干扰NCM460、HT29和HCT116细胞48h后，结果如图3所示，细胞内NAD+和NADH水平显著降低；
(4)小干扰RNA干扰NCM460、HT29和HCT116细胞48h后，结果如图4所示，细胞内PARP1和SIRT1水平下降。
综上所述，本发明提供了一种新的衰老细胞模型，并发现其导致细胞毒性并引起氧化应激，可为衰老和癌症治疗机制提供新的思路。
更具体地，相关制备及检测如下：
1、实验试剂准备：
(1)制备DMEM、DF12、1640培养基
取1袋DMEM高糖培养基粉末(13.4g/pkg)，分别倒入装有800mL去离子水的1000mL烧杯中，放置于磁力搅拌器上搅拌溶解，再分别加入3.7gNaHCO3搅拌溶解。充分溶解后调整pH值至7.2后定容至1000mL，无菌0.22μm孔径的滤器对培养基进行过滤除菌，分装于无菌的玻璃瓶中，密封后放于4℃冰箱保存。
(2)制备cellPBS磷酸盐缓冲液
将氯化钠8.00g、氯化钾0.20g、磷酸二氢钾0.24g、磷酸氢二钠1.44g依次倒入装有800mL高温高压灭菌后的去离子水的1000mL烧杯中，放置于磁力搅拌器上搅拌溶解，定容至1000mL，用无菌0.22μm孔径的滤器进行过滤，分装储存后，置于4℃保存。
(3)制备0.25％胰蛋白酶溶液
准确称取胰蛋白酶粉末0.25g，加入4℃预冷400mLPBS溶液中并置于磁力搅拌器上搅拌溶解，充分溶解后定容至500mL。无菌0.22μm滤膜过滤，分装，封口后4℃储存。
2、NCM460、HT29、HCT116细胞培养、传代和冻存：
(1)细胞培养
将细胞放置于37℃，5％CO2，饱和湿度的细胞培养箱中进行培养；每天于倒置显微镜下观察细胞形态及数量，每1～3天换液，当培养基颜色转为橙黄色时进行换液处理；将新鲜的完全培养基与磷酸盐缓冲液PBS于超净工作台中紫外照射30min。吸去旧培养基，用PBS清洗一遍，弃去cellPBS。小心沿培养皿壁加入完全培养基10ml。将细胞放回CO2培养箱，继续培养。
(2)细胞传代
实验前将细胞用完全培养基、胰酶、cellPBS于超净工作台中紫外照射30min。吸去旧培养基，用cellPBS清洗两遍，弃去cellPBS加入3ml0.25％胰酶消化，显微镜下观察，消化至细胞边缘收缩变圆，弃去胰酶，加3ml完全培养基终止消化。将细胞吹悬至单个细胞，根据细胞生长速度，按1:3-1:5比例传入新的培养皿内，补加培养基到10ml。将细胞放回37℃，5％CO2培养箱继续培养。相隔24h更换新鲜的培养基，以使其正常生长。
(3)细胞冻存
将胎牛血清与二甲基亚砜(DMSO)按体积比9:1(即900μL胎牛血清：100μL二甲基亚砜(DMSO))的比例混匀配制成备用冻存液。取对数生长期的细胞，用cellPBS洗两次，再加入3ml0.25％胰蛋白酶，将培养瓶置37℃、5％CO2孵箱2-3min，观察细胞胞体回缩变圆时终止消化，用细胞培养液将细胞吹悬，转移至玻璃离心管中，1200r/min离心3min，去上清。将之前配好的细胞冻存液加入到玻璃离心管中，轻轻吹悬细胞。将细胞悬液分装在2ml内旋冻存管中，密封后标记细胞名称和冷冻日期。将冷冻管先置于4℃放30min，然后-20℃停留1-2h，再放到-80℃冷冻冰箱冷冻过夜，最终放入-150℃冷冻冰箱中或液氮罐中保存。
3、DCFH-DA探针法检测ROS：
将对数期细胞用胰酶消化后制成细胞悬浮液，接种于24孔板上，培养过夜后。将细胞用药物处理细胞一定时间对应培养基中培养24、48h，吸弃培养基，用PBS洗2-3遍，加入含DCFH-DA(10μM)的无血清培养基在37℃下避光培养20min，每隔5-10min摇晃孔板一次，之后用无血清培养基洗涤2-3遍。使用488nm激发波长，525nm发射波长，激光共聚焦显微镜观察并拍照。
4、WST-8检测NAD+/NADH含量
(1)细胞样品的准备：对于贴壁细胞，约1×106个细胞(大约相当于6孔板一个孔长满的细胞数量)，吸净培养液，用移液器加入200μl的NAD+/NADH提取液，并轻轻吹打，以促进细胞的裂解；对于悬浮细胞，约1×106个细胞，600g离心5分钟，吸净培养液，用移液器加入200μl冰浴预冷的NAD+/NADH提取液，并轻轻吹打，以促进细胞的裂解；裂解过程在室温或冰上操作均可。随后12,000g，4℃离心5-10分钟，取上清作为待测样品备用。
样品中NAD+和NADH的总量的测定：吸取20μl待测样品至96孔板中,后续如果发现样品中的NAD+和NADH的总量过高，超出标准曲线的范围，则需要用NAD+/NADH提取液将样品适当稀释后再进行检测；总量过低时则需要加大细胞或组织样品的用量。
(2)样品中NAD+、NADH的含量或者NAD+/NADH比值的测定：吸取50-100μl待测样品于离心管中，60℃水浴或PCR仪上加热30分钟以分解NAD+。如果加热后产生不溶物，则需10,000g，室温或4℃离心5分钟，吸取20μl上清液作为待测样品至96孔板中，为了减少实验误差建议设置样品的重复孔。
(3)37℃避光孵育10分钟。此孵育步骤的目的是将样品中的NAD+转化为NADH；在加入乙醇脱氢酶工作液的过程中须轻柔操作，以免产生气泡。若不慎出现气泡，可使用细小的吸头或针头戳破。
(4)适当混匀显色液，然后每孔加入10μl显色液，混匀，37℃避光孵育30分钟，此时会形成橙黄色的formazan,测量450nm处的吸光度。
尽管为说明目的公开了本发明的实施例，但是本领域的技术人员可以理解：在不脱离本发明及所附权利要求的精神和范围内，各种替换、变化和修改都是可能的，因此，本发明的范围不局限于实施例所公开的内容。
5、WesternBlot检测细胞PARP1和SIRT1蛋白表达：
将对数期细胞胰酶消化后制成细胞悬浮液，接种于6孔板上，培养24h后。小干扰RNA处理48h，终止培养，每孔加入蛋白裂解液150μL，收集蛋白，将经过煮沸、冷却后离心的总蛋白，进行SDSPAGE泳之后转移到硝酸纤维素膜(NC膜)上，用质量百分数为5％的脱脂奶粉溶液将转好的NC膜封闭1h，TBS溶液洗涤3次，4℃条件下与相应一抗过夜孵育。之后回收一抗，PBS洗涤3次，加入二抗(按照体积比例1：5000稀释)5mL于常温条件下避光孵育2h，重复洗涤，将NC膜平铺于Odyssey红外激光成像系统中，设置程序，进行数据分析。
尽管为说明目的公开了本发明的实施例，但是本领域的技术人员可以理解：在不脱离本发明及所附权利要求的精神和范围内，各种替换、变化和修改都是可能的，因此，本发明的范围不局限于实施例所公开的内容。
Claims (5)
1.一种通过干扰Slc12a8导致NAD+/NADH水平降低诱导建立衰老细胞模型的新方法，其特征在于：步骤如下：
使用小干扰RNA干扰细胞Slc12a8基因48h后，即得衰老细胞模型。
2.根据权利要求1所述的通过干扰Slc12a8导致NAD+/NADH水平降低诱导建立衰老细胞模型的新方法，其特征在于：所述小干扰RNA工作浓度为0.1mmol·L－1。
3.根据权利要求1所述的通过过干扰Slc12a8导致NAD+/NADH水平降低诱导建立衰老细胞模型的新方法，其特征在于：所述细胞为NCM460、HT29、HCT116。
4.根据权利要求1至4任一项所述的通过干扰Slc12a8导致NAD+/NADH水平降低诱导建立衰老细胞模型的新方法，其特征在于：所述衰老细胞模型中细胞NAD+/NADH水平降低，细胞死亡率提高；细胞内ROS增加；强化化疗药物刺激细胞ROS水平；细胞PARP1和SIRT1下调。
5.如权利要求1至4任一项所述的方法制得的衰老细胞模型在药物开发方面中的应用。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110996967A (zh) * | 2017-08-10 | 2020-04-10 | 华盛顿大学 | 使用烟酰胺单核苷酸进行治疗的组合物和方法 |
| CN112156105A (zh) * | 2020-10-15 | 2021-01-01 | 天津科技大学 | 一种抑制剂联合小干扰rna抑制结肠癌细胞活性的新方法 |
| CN113662954A (zh) * | 2021-08-27 | 2021-11-19 | 上海英库商务咨询有限公司 | 修复线粒体磨损、dna损伤，抗衰老的膳食配方 |
-
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110996967A (zh) * | 2017-08-10 | 2020-04-10 | 华盛顿大学 | 使用烟酰胺单核苷酸进行治疗的组合物和方法 |
| CN112156105A (zh) * | 2020-10-15 | 2021-01-01 | 天津科技大学 | 一种抑制剂联合小干扰rna抑制结肠癌细胞活性的新方法 |
| CN113662954A (zh) * | 2021-08-27 | 2021-11-19 | 上海英库商务咨询有限公司 | 修复线粒体磨损、dna损伤，抗衰老的膳食配方 |
Non-Patent Citations (2)
| Title |
|---|
| ALESSIA GROZIO等: "Slc12a8 is a nicotinamide mononucleotide transporter", 《NAT METAB . 》, vol. 1, no. 1, 31 January 2019 (2019-01-31), pages 47 - 57 * |
| 刘心怡: "NAD 补救合成途径及 SLC12A8 转运蛋白影响结肠癌细胞药物敏感性及耐药性的机制研究", 《天津科技大学》, 13 December 2022 (2022-12-13), pages 1 - 4 * |
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