CN113975287A - 重楼皂苷在作为溶酶体靶向抑制剂的应用 - Google Patents
重楼皂苷在作为溶酶体靶向抑制剂的应用 Download PDFInfo
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Abstract
本发明属于医药技术领域，具体涉及重楼皂苷在作为溶酶体靶向抑制剂的应用。本发明采用激光共聚焦实验、电镜观察、mCherry‑GFP‑LC3双荧光系统检测、溶酶体pH测量、蛋白印迹实验确证PD对溶酶体的损伤作用，为研究溶酶体提供新的技术手段。
Description
技术领域
本发明属于医药技术领域，具体涉及重楼皂苷在作为溶酶体靶向抑制剂的应用。
背景技术
重楼是云南重楼或七叶一枝花的干燥根茎，近年来随着对重楼的药理评价不断探究，发现重楼具有抗菌、抗肿瘤的功效。重楼植物根茎中含有许多化合物，包括甾体皂苷、离氨基酸、甾醇级黄酮等，其中甾体皂苷(重楼皂苷)是重楼的主要活性成分1，结构式如下：
溶酶体是膜包被的细胞器，内含有多种水解酶，具有溶解或消化的功能，当组织细胞受到各种理化因素伤害时，溶酶体会通过消化功能来维持细胞的正常功能。溶酶体的功能常常与自噬相关。溶酶体结构和功能被破坏会影响细胞正常结构和功能，在多种疾病进展中起作用。因此，寻找特异性的溶酶体靶向的小分子抑制剂有助于对溶酶体生物学功能进行深入的研究。目前的已经报道的损伤溶酶体小分子药物有巴弗洛霉素A1(BafilomycinA1)和氯喹 (Chloroquine)等，然而这些小分子药物在应用上的作用浓度较高。本发明提供一种破坏溶酶体的试剂，重楼皂苷1(Polyphyllin D，PD)能够在微摩级别实现对溶酶体的特异性损伤，能够作为溶酶体靶向抑制剂的应用。
有相关报道显示，PD可用于治疗乳腺炎、白血病痉挛和呼吸系统、消化道、肝脏、胰腺、膀胱和大脑的肿瘤[2]，对卵巢癌也具有抑制效果[3]。
目前尚未有文章表明重楼皂苷具有溶酶体靶向抑制作用，尚无专利申请。
发明内容
本发明旨在提供本发明目的是展示PD对溶酶体的损伤及功能抑制。
PD对溶酶体的损伤证明：采用激光共聚焦实验，确证PD对溶酶体的损伤作用；接着在电镜下观察PD处理后细胞溶酶体的形态变化；再利用 mCherry-GFP-LC3双荧光系统检测自噬体与自噬溶酶体；以及测量不同浓度的 PD对溶酶体pH影响和蛋白印迹实验检测经PD处理后细胞内自噬蛋白变化情况。
本发明的一个方面提供重楼皂苷1在制备溶酶体靶向抑制剂应用。
本发明的一个方面提供重楼皂苷1在制备溶酶体靶向抑制药物中的应用。
本发明的一个方面提供溶酶体蛋白在筛选抗肿瘤药物中的应用。
本发明的一个方面提供溶酶体在筛选抗肿瘤药物中的应用，其特征在于，所述肿瘤为肝癌。
本发明的一个方面提供溶酶体蛋白作为肝癌的治疗靶点的应用。
本发明主要由通过细胞实验来确证PD对溶酶体的损伤作用。本发明提供重楼皂苷的新应用，为研究溶酶体提供新的技术手段。
附图说明
图1为重楼皂苷对溶酶体膜的影响实验结果；
图2为重楼皂苷对溶酶体大小与自噬溶酶体形成的影响的实验结果；
图3为重楼皂苷对自噬通量的影响的实验结果；
图4为重楼皂苷对LC3和Lamp1共定位的影响的实验结果；
图5为重楼皂苷对溶酶体pH的影响的实验结果；
图6为重楼皂苷对细胞自噬的影响的实验结果。
具体实施方式
以下通过实施例形式的具体实施方式，对本发明的上述内容作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下实施例。
实施例1激光共聚焦实验确证PD对溶酶体的损伤作用
pLVX-Puro-mCherry-Gal3质粒构建：利用同源重组方法，采用 C112-ClonExpressII One Step Cloning Kit试剂盒(诺唯赞，Cat#C112-01)构建，具体如下：
结合ClonExpress II重组反应系统(诺唯赞生物科技有限公司)设计扩增特异引物，
mCherry上游引物：
AGGATCTACCGGTCGCCACCATGGTGAGCAAGGGCGAGGAGG
mCherry下游引物：CTTGTACAGCTCGTCCATGCCG
Gal3上游引物：CTCGAGCTGGATCCATGGCAGACAATTTTTC
Gal3下游引物：
ATCTAGATCCGGTGAATTCTCAAGCGTAATCTGGAACATCGTATGGGTA
选择的空载为pLVX-Puro(addgene#128652，去除GFP),酶切位点ECOR I。
利用高保真PCR聚合酶扩增目的基因mCherry和Gal3序列。用快切酶 ECOR I(TaKaRa)将空载pLVX-puro切开，将目的基因Gal3和mCherry序列与酶切后的pLVX-puro通过连接酶ExnaseTMⅡ连接，连接条件如下：37℃连接 0.5h，之后立即置冰浴中冷却5min，然后将连接产物与100μL大肠杆菌DH5 α感受态细胞(OD值为0.5)轻轻混合，冰上静置30min；42℃水浴90s，冰水浴孵育2min；加入450μL LB培养基，37℃、200rpm摇瓶培养45min。取100μL菌液均匀涂布在含有Amp的LB培养基平板上，平板倒置，于37℃过夜培养。用无菌的牙签将单个菌落挑至50μL新鲜的液体LB培养基中，混匀，取候选的3个单克隆扩大培养：一部分提取质粒，送公司测序；另一部分保种：-80℃液氮罐中保存。
转染：pLVX-Puro-mCherry-Gal3重组质粒转染到人肝癌细胞Hep3B细胞株。
将上述制备得到的pLVX-Puro-mCherry-Gal3重组质粒用Lip3000按照 Lip3000说明书分别转染到人肝癌Hep3B细胞株(购于中国科学院上海生科院细胞资源中心)，将过表达mCherry-Gal3的Hep3B细胞铺于共聚焦专用皿上，第二天待细胞贴壁后用PD(1.5μM)处理细胞，药物处理24h后，用溶酶体绿色荧光探针(碧云天，C1047S)进行染色，染色方法按照说明书进行在激光共聚焦显微镜下观察溶酶体的破裂情况。
图1显示用PD处理后，溶酶体被破坏，mCherry-Gal3聚集在溶酶体膜外。
实施例2电镜下观察PD处理后细胞溶酶体形态变化
细胞的收集：将经PD处理后的HepG2细胞用胰酶消化下来，收集到1.5mL 的尖底离心管中，以1800r/min离心10min。
固定与清洗：离心后去掉上清，立即加入3％的戊二醛，放入4℃冰箱内固定保存(至少2h以上)。先用0.1mol/L的磷酸盐缓冲液清洗2次，每次10min， 1％的锇酸4℃冰箱内固定1h，0.1mol/L的磷酸盐缓冲液清洗2次，每次10min。在锇酸固定后将细胞包裹在擦镜纸中放入青霉素小瓶中进行后续处理。
脱水与浸透：采用梯度脱水法，30％、50％、70％、90％的乙醇，90％乙醇： 90％丙酮＝1∶1混合液，90％的丙酮、100％丙酮脱水2次，每次10～15min；浸透亦梯度进行，包埋剂：100％丙酮(1∶3、1∶1、3∶1)分别浸透1、 4、12h以上。
包埋与聚合：将擦镜纸打开，用牙签将其中的细胞团块转移到包埋模具中；包埋后在恒温干燥箱中升温聚合。聚合温度与时间分别为：37℃，12h；45℃， 12h和60℃，24～48h。
超薄切片：聚合后的样品包埋块在ULTRCUT E型超薄切片机上制备50～ 70nm的超薄切片。包埋在离心管中的样品，先用单面刀片将离心管切开，取出其中的包埋块，再进行修块、切片。
电子染色：将样品的超薄切片采用滴染法进行醋酸双氧铀、柠檬酸铅双染色，先用醋酸双氧铀铀染30min，双蒸水冲洗干净后，再用柠檬酸铅铅染15min，双蒸水冲洗。
电镜观察。
图2显示用PD(0μM，1.5μM,3μM)处理后在电镜下观察，溶酶体膨胀，加药组溶酶体直径比对照组大，自噬溶酶体增多。
实施例3 mCherry-GFP-LC3双荧光系统检测自噬体与自噬溶酶体
pLVX-Puro-mCherry-GFP-LC3质粒构建：质粒构建方法同上述。
转染：将质粒pLVX-Puro-mCherry-GFP-LC3转染到Hep3B细胞株，转染方法同上述。
将上述得到的Hep3B细胞铺于共聚焦专用皿上，第二天待细胞贴壁后用PD 处理细胞，药物处理24h后，在激光共聚焦显微镜下观察每个像素点的红色绿色荧光情况。
图3显示用PD处理后，在激光共聚焦显微镜下观察到形成黄色荧光光，自噬体与溶酶体的融合在自噬通量中受损。显示用PD处理后，LC3与Lamp1共定位。
图4显示PD处理前LC3-GFP的绿色荧光与Lamp1-mCherrry的红色荧光不重叠，PD处理后LC3与Lamp1共定位，红色荧光与绿色荧光重合出现黄色荧光。
实施例4溶酶体pH测量
用不同浓度的PD(0μM，1.5μM,3μM)处理细胞，溶酶体pH测量方法根据溶酶体pH值检测试剂盒(百奥莱博，Cat#HR8268)说明书进行。
图5显示用PD处理后，溶酶体破裂，溶酶体内pH升高，溶酶体内酶活性丧失。
实施例5蛋白印迹实验检测经PD处理后细胞内自噬蛋白变化情况
用不同浓度的PD(0μM，1.5μM,3μM)处理细胞HepG2和Hep3B，处理24h。
裂解细胞：用预冷的PBS(0.01M、pH＝7.4)洗三遍，加入100μL的RIPA 细胞裂解液(碧云天，P0013B)，冰上裂解30min，每隔10min轻轻颠倒混匀一次；4℃、13200rpm离心30min，取上清进行蛋白浓度测定；
制样：各取30μg的蛋白加入20 L 1×蛋白上样缓冲液(碧云天，P0015)，混匀，在沸水浴中煮10min，得到样品；
制胶：配制10％的分离胶(5mL)：1.5mM Tris-HCl(pH8.8)1.3mL，ddH2O 1.9mL，30％丙烯酰胺1.7mL，10％SDS(十二烷基硫酸钠)50μL，10％过硫酸胺50μL，TEMED(四甲基乙二胺)3μL(加入后混匀，快速制胶)；配制 5％浓缩胶(3mL)：1.5mM Tris-HCl(pH8.8)0.38mL，ddH2O 2.1mL，30％丙烯酰胺0.5mL，10％SDS(十二烷基硫酸钠)30μL，10％过硫酸胺30μL，TEMED (四甲基乙二胺)6μL(加入后混匀，快速制胶)；
电泳：装好电泳仪(湖南湘仪实验室仪器开发有限公司)，加入电泳缓冲液并且将上述③的煮好的样品冷却上样；上样完后，先使用80V电压跑30min，再使用120V电压，直至溴酚蓝条带接近末端，结束电泳；
转膜：裁剪相应大小的PVDF膜，将PVDF膜用甲醇浸润活化至变色，按照滤纸-凝胶-PVDF膜-滤纸的顺序摆好，接入转膜电源，235mA恒电流冰上转膜150min；
封闭：转膜完后，取出PVDF膜，用TBST润洗去除残留的转膜缓冲液，然后用5％的脱脂牛奶室温封闭2h；
一抗孵育：封闭完后用TBST润洗去除残留的牛奶，根据蛋白指示带剪膜，加入稀释1:1000的相应的一抗:ATG5(Cell Signaling Technology)，LC3(Cell SignalingTechnology)，Lamp1(Abclonal)，p62(Cell Signaling Technology)，β-Actin(Bioworld)，4℃孵育过夜(16h)；
二抗孵育：回收一抗，用TBST洗膜30min，每隔十分钟换一次液，加入稀释1:4000的相应二抗(HS101-01、HS201-01，全式金公司)，室温孵育2h；
显影：回收二抗，用TBST洗膜30min，每隔十分钟换一次液；合理控制曝光时间显影。
图6显示用PD(0μM，1.5μM,3μM，6μM)处理后，自噬标志蛋白ATG5， LC3,LAMP1和p62随着药物浓度增加而增加。
参考文献：
1.武珊珊,高文远,段宏泉,贾伟.重楼化学成分和药理作用研究进展.中草药,2004(03):344-347.
2.Pharmacopoeia Commission of the People’s Republic of China:ThePharmacopoeia of the People’s Republic of China.Beijing:People’s MedicalPublishing House,Chemical Industry Press,1990.
3.Xiao X,Zou J,Bui-Nguyen TM,et al.Paris saponin II of RhizomaParidis--a novel inducer of apoptosis in human ovarian cancer cells.BiosciTrends. 2012；6(4):201-211.doi:10.5582/bst.2012.v6.4.201 4.
Li M,Yang J,Zhou W,et al.Activation of an AKT/FOXM1/STMN1 pathwaydrives resistance to tyrosine kinase inhibitors in lung cancer.Br J Cancer.2017；117(7):974-983.doi:10.1038/bjc.2017.292.
上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。
Claims (5)
1.重楼皂苷1在制备溶酶体靶向抑制剂应用。
2.重楼皂苷1在制备溶酶体靶向抑制药物中的应用。
3.溶酶体蛋白在筛选抗肿瘤药物中的应用。
4.溶酶体在筛选抗肿瘤药物中的应用，其特征在于，所述肿瘤为肝癌。
5.溶酶体蛋白作为肝癌的治疗靶点的应用。
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Application publication date: 20220128 |
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