CN108699091A - pH响应性荧光化合物和应用其的线粒体自噬检测用组合物及细胞内线粒体自噬的检测方法 - Google Patents
pH响应性荧光化合物和应用其的线粒体自噬检测用组合物及细胞内线粒体自噬的检测方法 Download PDFInfo
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Abstract
本发明公开一种在细胞内线粒体上发生特异性局部化,在溶酶体中的弱酸性pH环境下呈现出强荧光发射,且不容易受到来自自体荧光(self‑fluorescence)和细胞内的其他荧光物质的背景荧光的干扰的新的pH响应性荧光化合物及应用该化合物的线粒体自噬检测用组合物以及细胞内线粒体自噬的检测方法。该pH响应性荧光化合物如下述通式所示,在通式中,L表示接头,X表示药学上允许的阴离子,Y表示可与线粒体蛋白质上的官能团共价结合的反应性官能团。
Description
技术领域
本发明涉及新的pH响应性荧光化合物和应用该化合物的线粒体自噬检测用组合物以及细胞内线粒体自噬的检测方法。
背景技术
在细胞中,存在对于细胞内废弃的蛋白质、细胞器(organelle)等进行再利用或代谢的分解过程,该过程被称为自噬(autophagy:自食)。在该过程中,废弃物质被由脂质双层膜构成的被称为自噬体(autophagosome)的隔离膜所覆盖,并经过与溶酶体(lysosome)融合而被分解。通过自噬进行线粒体(mitochondrion)选择性分解去除的机制,被称为线粒体自噬(mytophagy),起到使生物体对于与线粒体功能障碍相关的疾病产生防御的作用。
作为检测线粒体自噬的通常方法,可以列举如下方法:溶解细胞,从得到的mRNA通过蛋白质免疫印迹法(Western Blot)确认线粒体自噬相关因子的表达。由于该方法会破碎细胞,不能进行活细胞成像(live cell imaging)。
作为细胞内成像的代表性的方法,有如下方法:在线粒体中使被称为Keima的pH响应性荧光蛋白质得以表达,并监控来自Keima的荧光强度的方法(例如,参照非专利文献1)。Keima的激发光谱(excitation spectrum)随着pH发生变化。在中性环境下短波长侧(440nm)呈优势,在酸性环境下长波长侧(550nm)呈优势。对来自这两个激发波长的图像数据所得到的比值(Ratio)(550nm/440nm)图像进行观察,可知在中性环境下Keima的比值变低,在酸性环境下Keima的比值变高。通过利用该现象,能够从荧光图像读取伴有线粒体自噬的线粒体的pH变化,检测出线粒体自噬。但是,由于该方法需要在细胞内进行Keima的表达,因此并不是可适用于所有细胞。
据此,作为能够穿透细胞膜而被导入细胞内,在线粒体上发生特异性局部化(localization),并且发光强度会响应线粒体的pH变化而发生变化的pH响应性荧光色素,有人提出了下述化学式所示的化合物(参照非专利文献2)。该化合物在分子内包括:在线粒体上发生特异性局部化的三苯基膦基(triphenyl phosphonium groups)、具有pH传感器功能的哌嗪(piperazine)环、可与线粒体蛋白质共价结合的反应性官能团的氯甲基(chloromethyl group)、作为荧光生色团的萘酰亚胺(naphthalene imide)。
[化学式1]
现有技术文献
非专利文献
非专利文献1:
Kogure,T.,Karasawa,S.,Araki,T.,Saito,K.,Kinjo,M.,and Miyawaki,A.,Afluorescent variant of a protein from the stony coral Montipora facilitatesdual-color single-laser fluorescence cross-correlation spectroscopy;NatureBiotechnology.24:577-581(2006).
非专利文献2:
Mitochondria-Immobilized pH-Sensitive Off-On Fluorescent Probe,Min HeeLee,Nayoung Park,Chunsik Yi,Ji Hye Han,Ji Hye Hong,Kwang Pyo Kim,Dong HoonKang,Jonathan L.Sessler,Chulhun Kang,and Jong Seung Kim,J.Am.Chem.Soc.2014,136,14136-14142.
发明内容
(发明要解决的课题)
但是,由于非专利文献2中所记载的具有作为荧光生色团的萘酰亚胺(naphthalene imide)的pH响应性荧光化合物的最大激发波长在400nm附近,因此当使用通用的荧光显微镜的488nm激光进行激发(B激发)的情况下,不仅发光强度变低,而且细胞内源性的荧光物质也被激发,进而背景会变高。因此,存在线粒体自噬的检测灵敏度变低的问题。若为了消除灵敏度不足而增加pH响应性荧光化合物的浓度,则对线粒体的损伤加剧,反而导致用于检测细胞内线粒体自噬的pH响应性荧光化合物诱发线粒体自噬,进而无法正确检测细胞内的线粒体自噬的情况。因此优选为尽可能地以低浓度使用,使用在B激发和G激发(546nm)具有高荧光强度的pH响应性荧光化合物。
本发明是为了解决上述问题而进行的,其目的在于提供可在细胞内线粒体上发生特异性局部化的、且在溶酶体中的弱酸性pH环境下呈现强荧光发射、不易受到来自自体荧光和细胞内的其他荧光物质的背景荧光干扰的新的pH响应性荧光化合物和应用该化合物的线粒体自噬检测用组合物,以及细胞内线粒体自噬的检测方法。
(解决课题的方案)
为了达到上述目的,本发明的第一方式,是通过提供下述通式示出的pH响应性荧光化合物来解决上述课题。
[化学式2]
在上述通式中,L表示接头(linker),X表示药学上允许的阴离子,Y表示可与线粒体蛋白质上的官能团共价结合的反应性官能团。
本发明的第二方式是通过提供包含上述通式所示的pH响应性荧光化合物的线粒体自噬检测用组合物,从而解决上述课题。
本发明的第三方式是通过提供细胞内线粒体自噬的检测方法,解决上述课题。所述检测方法包括如下步骤:投入步骤,将上述通式所示的pH响应性荧光化合物投入细胞内;测量步骤,在进行规定时间的培养(Incubation)后测量来自细胞的荧光发射。
在本发明的第一至第三方式中,所述pH响应性荧光化合物也可以是下式9示出的化合物。
[化学式3]
(发明的效果)
上述通式所示的pH响应性荧光化合物,在分子内包含:在线粒体上发生特异性局部化的三苯基膦基、具有pH传感器功能的哌嗪环、可与线粒体蛋白质上共价结合的反应性官能团、作为荧光生色团的疏水性苝酰亚胺(perylene imide)。因此该化合物能够穿透细胞膜被导入细胞内,在线粒体上发生特异性局部化,且与线粒体蛋白质共价结合进而被固定化。
在中性或者碱性条件下,哌嗪环内的氮原子中,因与苝环(perylene ring)的π电子系非共轭的胺的氮原子上的非共用电子对到苝环的π电子系的光诱导电子转移(PET)从而发生消光,从而不产生荧光发射,相对于此,在pH值低于与苝环内的π电子系非共轭的胺的pKa的酸性条件下,由于氮原子被质子化,进而不发生光诱导电子转移(photoinducedelectron transfer),因此发生来自苝环的π电子系的荧光发射。因此,相对于在线粒体存在于细胞质中的情况下不发生荧光发射,在线粒体受到线粒体自噬而转移到溶酶体内的情况下,会“On-Off”的引起荧光发射,所以能够通过荧光成像来区分受到线粒体自噬的线粒体。
另外,由于上述通式所示的pH响应性荧光化合物,具有作为荧光生色团的苝酰亚胺,因此与萘酰亚胺等相比,吸收波长以及荧光波长都出现在长波长侧。因此,不仅是在荧光显微镜中广泛使用的B激发,也可以在G激发下得到激发。据此,即使是在与细胞内的其他细胞器和细胞内源性的其他荧光色素共存的情况下,也能够选择性的进行激发,因此可以降低背景和自体荧光的影响,且能够以高灵敏度检测。进一步,由于上述通式所示的pH响应性荧光化合物具有较宽的π共轭平面,因此同时具有在极性溶剂中自缔合(self-association)而发生浓度猝灭(concentration quenching)的特性。因此在扩散在细胞质中的状态下,不显示荧光发射,能够进一步降低背景。
这些效果结合的结果,本发明提供可在细胞内的线粒体上发生特异性局部化、在溶酶体中的弱酸性pH环境下示出强荧光发射、且不容易受到来自自体荧光和细胞内的其他荧光物质的背景荧光的干扰的新的pH响应性荧光化合物和应用该化合物的线粒体自噬检测用组合物以及细胞内线粒体自噬的检测方法。
附图说明
图1是示出本发明的一个实施方式所涉及的pH响应性荧光化合物的吸收光谱和荧光光谱的图。
图2是示出所述pH响应性荧光化合物的荧光强度的pH变化的图表。
图3是示出导入所述pH响应性荧光化合物的HeLa细胞的自噬诱导前后的荧光显微镜镜像的照片。
图4是示出导入所述pH响应性荧光化合物的Parkin表达HeLa细胞以及Parkin未表达HeLa细胞的荧光显微镜镜像的照片。
图5是示出Parkin表达HeLa细胞以及Parkin未表达HeLa细胞的流式细胞仪测量结果的图表。
具体实施方式
接下来,对本发明的具体实施方式进行说明,以便于理解本发明。
用以下的通式表示本发明的pH响应性荧光化合物。
[化学式4]
在上述通式中,L表示接头,X表示药学上允许的阴离子,Y表示可与线粒体蛋白质上官能团共价结合的反应性官能团。
接头(L)(linker)
作为连接荧光生色团苝酰亚胺、和在线粒体上特异性局部化的三苯基膦基的接头(L),只要不影响苝酰亚胺基的荧光发射特性(发光波长、发光强度、发光强度的pH依存性、激发波长等),以及在线粒体上的局部存在性等,就没有限制,可以使用任意原子团。作为接头L的具体例,可以列举在C-C键间或在侧链中包含氧原子、氮原子、硫磺原子、酯、酰胺、聚氨酯(urethan)、尿素、环烯基(cycloalkylene)、杂芳基(heteroaryl group)等,也可以列举可以具有分支的亚烷基(alkylene)。
阴离子(X)
作为三苯基膦基的抗衡离子(counter ion)的阴离子X,在不影响苝酰亚胺基的荧光发射特性(发光波长、发光强度、发光强度的pH依存性、激发波长等)、在线粒体上的局部存在性、与线粒体蛋白质的反应性等,且不显示细胞毒性等的情况下,没有特殊限制,可以使用药学上允许的任何有机或无机阴离子。作为阴离子X的具体例,可以列举氯离子、溴离子等卤素离子、醋酸根离子、丙酸根离子、乳酸根离子、柠檬酸根离子、酒石酸根离子等的有机酸离子,硝酸根离子、硫酸根离子等的无机酸例子等。
反应性官能团(Y)
作为与线粒体蛋白质上的官能团反应而形成共价键(共价结合)的反应性官能团,在不影响苝酰亚胺基的荧光发射性质(发光波长、发光强度、发光强度的pH依存性、激发波长等),在线粒体上的局部存在性等的情况下,在其一端具有其在线粒体上局部化之前不与其他细胞内蛋白质等发生反应的程度的有适当反应性的原子或原子团,另一端可以不受特殊限制,使用与哌啶环上的氮原子结合的任意官能团。作为反应性官能团Y的具体例,可以列举ω-氯代亚烷基、ω-溴代亚烷基、4-氯甲基苄基等。
作为优选的pH响应性荧光化合物的一个例子,可以列举例如下述式9所示的化合物。
[化学式5]
上述通式所示的化合物,可以通过使用例如后述实施例所示的等任意公知的合成路径(反应以及条件)来进行合成。
由于上述通式所示的化合物(以下有时简称为“所述化合物”)具有对细胞膜的通透性,因此所述化合物的细胞内导入不需要通过使用特别的方法,可以通过单纯地使所述化合物与细胞接触而进行。如此,对导入了所述化合物的细胞进行规定时间的培养,并且使用荧光显微镜等任意公知工具测量细胞的荧光发射,可以对细胞内线粒体自噬进行检测。本发明的实施方式涉及细胞内线粒体自噬的检测方法,所述检测方法包括:投入步骤,将上述通式所示的pH响应性荧光化合物投入细胞内;测量步骤,在进行了规定时间培养后对细胞的荧光发射进行测量。
将所述化合物导入细胞时,可以在将所述化合物以规定浓度溶解或分散于适当的溶剂或者缓冲液的状态下进行。本发明的实施方式涉及以规定浓度在溶剂或缓冲液中溶解或者分散有所述化合物的组合物。
实施例
接下来,对为了确认本发明的作用效果而进行的实施例进行说明。
实施例1:pH响应性荧光化合物的合成
依照下面所示的反应方案,进行上述式9所示的pH响应性荧光化合物的合成(以下,有时将“以式n(n为1~9的整数)所示的化合物”简称为“化合物n”)。
[化学式6]
[化学式7]
化合物2的合成
在金属高压釜中加入5.5g(14mmol)的苝(perylene)-3,4,9,10-四甲酸二酐(化合物1)、1.57g(7.6mmol)的2,5-二叔丁基苯胺、1.98g(9.0mmol)的醋酸锌二水合物、14g(205.6mmol)的咪唑、12mL的水,用钥勺(spatula)充分混合,在200℃下反应24小时。反应结束后,将内容物移至500mL烧杯中,并用总量200mL左右的乙醇(EtOH)清洗。滴入浓盐酸1mL,在室温下搅拌1小时后,蒸馏去除乙醇(EtOH)。在残留物中添加CHCI3,移至分液漏斗,用水清洗三遍。用Na2SO3将CHCI3层进行干燥,之后浓缩干固。使用硅胶柱,以三氯甲烷/醋酸乙酯=9/1进行纯化(T,Dentani;et al.,Dyes Pigments.,2007,72(3),303-307)。
1H-NMR(400MHz,CDCl3)δ:1.30(s,9H),1.33(s,9H),7.03(s,1H),7.45(d,1H,J=8.4Hz),7.58-7.65(m,3H),7.90(d,2H,J=7.9Hz),8.44(m,3H),8.64(d,2H,J=7.8Hz)。
化合物3的合成
将5g的化合物2(9mmol)投入反应容器中,之后加入10mL的四氢呋喃(THF)进行溶解,加入400mL的叔丁醇(t-BuOH)。添加36g(645mmol)片状(flake)氢氧化钾(KOH),110℃下回流2小时。滴入400mL的醋酸,在室温下搅拌2小时。将析出的黑色结晶取出,进行减压干燥。由于得到的化合物对有机溶剂的溶解性差,无法进行核磁共振(NMR)分析,因此直接将其用于下一个阶段的反应。
化合物4的合成
加入1.5g(4.5mmol)的化合物3、1.86g(5.3mmol)的β-丙氨酸苄酯对甲苯磺酸盐(β-alanine benzyl ester p-toluenesulfonate)、795mg(3.6mmol)的醋酸锌、150ml的喹啉,在氩气氛围下,120℃搅拌过夜。在反应混合物中添加CHCI3,之后用3N HCI清洗三遍。用Na2SO3将CHCI3层进行干燥,之后浓缩干固。使用硅胶柱,以三氯甲烷/醋酸乙酯=9/1进行纯化。
1H-NMR(400MHz,CDCl3)δ:2.85(t,2H,J=7.2Hz),4.53(t,2H,J=7.2Hz),5.14(s,2H),7.26-7.32(m,4H),7.61(t,2H,J=7.6Hz),7.88(d,2H,J=8.0Hz),8.32(d,2H,J=7.4Hz),8.38(d,2H,J=7.1Hz),8.51(d,2H,J=8.0Hz)。
化合物5的合成
将1.0g(2.0mmol)的化合物4溶解于四氢呋喃(THF)80mL-乙醇(EtOH)20ml混合溶液中。加入钥勺一大匙(50mg左右)的10%钯碳催化剂(Pd/C),之后在H2气体环境下,室温搅拌过夜。由于获得的化合物在有机溶剂中的溶解性差,无法进行核磁共振(NMR)分析,因此直接将其用于下一个阶段的反应。
化合物6的合成
将未提纯的化合物5溶解于250ml DMF中。加入981mg(1.0当量、2.54mmol)的溴化(2-氨基乙基)三苯基膦(基于Maryanoff,B.E.;et al.,J.Am.Chem.Soc.1985,107,217-226中所记载的方法合成。)、830mg(1.2当量、3.05mmol)的2-氯-4,6-二甲氧基-1,3,5-三嗪(DMT-MM)、5mL的N,N-二异丙基乙胺(DIEA),在室温下搅拌过夜。确认反应进展后,用蒸发器将溶剂蒸馏去除。使用硅胶柱,以三氯甲烷/甲醇=9/1进行纯化。
1H-NMR(400MHz,CDCl3)δ:2.65(t,2H,J=7.4Hz),3.73-3.81(m,2H),3.86-3.93(m,2H),4.40(t,2H,J=7.4Hz),7.56(t,2H,J=7.7Hz),7.69-7.86(m,15H),8.20(d,2H,J=8.1Hz),8.29(d,2H,J=7.6Hz),8.38(d,2H,J=8.0Hz),8.80(bt,1H)。
化合物7的合成
在1g化合物6中加入150mL 1,2-二氯乙烷,之后加入539mg(3.0当量、3.9mmol)的K2CO3进行搅拌。将85μL(2.5当量、3.28mmol)溴用5mL1,2-二氯乙烷进行稀释后,滴入反应溶液中,在100℃下搅拌2小时。通过电喷雾质谱法(ESI-MS)分析确认反应进展。由于生成的Br体与原料极性相同,因此难以通过TLC确认反应进展。MS分析的结果,几乎未检测出原料峰值(m/z:761[M+H+]),而检测出了生成物的峰值(m/z:839[M+H+]),因此用蒸发器将溶剂蒸馏去除。使用硅胶柱,以三氯甲烷/甲醇=9/1进行纯化。
1H-NMR(400MHz,CDCl3)δ:2.65(t,2H,J=7.3Hz),2.79(bs,4H),3.24(bs,4H),3.68(s,2H),3.72-3.77(m,2H),3.83-3.88(m,2H),4.41(t,2H,J=7.3Hz),4.6(s,2H),7.66-7.86(m,17H),8.13(d,1H,J=7.3Hz),8.25(d,2H,J=8.3Hz),8.29(d,1H,J=7.3Hz),8.38(d,1H,J=7.4Hz),8.43-8.48(m,2H),9.19(bt,1H)。
化合物8的合成
在500mg的化合物7中添加150mL的2-甲氧基乙醇(2-methoxyethanol)、3.5g(66.6当量、40mmol)哌嗪,在140℃下搅拌过夜。确认反应的进展后,蒸馏去除溶剂。使用硅胶柱,以三氯甲烷/甲醇=8/2进行纯化。
1H-NMR(400MHz,CD3OD)δ:2.56(t,2H,J=6.7Hz),3.15(m,1H),3.50(m,1H),3.52-3.63(m,4H),4.40(t,2H,J=6.8Hz),7.20(d,1H,J=8.4Hz),7.58(t,1H,J=7.9Hz),7.74-7.93(m,15H),8.09-8.19(m,5H,J=8.5Hz),8.30(d,1H,J=7.4Hz),8.36(d,1H)。
化合物9的合成
将300mg的化合物8、1245mg(20当量、7.1mmol)的α,α'-二氯-p-二甲苯(α,α'-dichloro-p-xylene)、50mg(1.0当量、0.35mmol)的K2CO3溶解于150ml乙腈中,回流过夜。通过薄层层析法(TLC)确认反应的进展。为了去除多余的α,α'-二氯-p-二甲苯,将过滤后的反应溶液用旋转蒸馏器去除。使用硅胶柱,以三氯甲烷/甲醇=9/1进行纯化。
1H-NMR(400MHz,CDCl3)δ:2.65(t,2H,J=7.3Hz),2.79(bs,4H),3.24(bs,4H),3.68(s,2H),3.72-3.77(m,2H),3.83-3.88(m,2H),4.41(t,2H,J=7.3Hz),4.6(s,2H),7.16(d,1H,J=7.3Hz),7.37-7.42(m,4H),7.57(t,1H,J=7.9Hz),7.68-7.84(m,15H),8.13-8.28(m,4H),8.33-8.41(m,3H),9.19(bt,1H)。
化合物9具有作为荧光生色团的苝酰亚胺。由于苝酰亚胺的最大激发波长为530nm,因此是对应于通用的激光显微镜的荧光分子,是能够以高敏感度检测出的色素。进一步而言,由于激发波长是更长的波长,因此能够避免细胞内的内源性荧光物质被激发,期待其可以降低背景。本化合物除此之外,具有作为pH传感器的哌嗪环,作为线粒体局部化基的三苯基膦基,作为固定化基的氯苄基。本化合物具有如下特征:利用从哌嗪环到苝酰亚胺基的光诱导电子转移(PET)导致的荧光的消光现象,在线粒体附近的pH环境下荧光强度低,而在溶酶体内的弱酸性pH环境下荧光强度高。并且,通过三苯基膦盐膜电位依存性地被诱导至细胞内线粒体,进而通过氯苄基与线粒体蛋白质上的官能团(例如,半胱氨酸残基上的SH基)的反应而形成的共价键,被固定化在聚集的线粒体上。
实施例2:化合物9的荧光特性
我们已经知道由于受共轭平面宽的影响,化合物9具有在极性溶剂中自缔合被促进,进而荧光强度变弱的性质。即,由于在细胞质中扩散的化合物9是无荧光的,因此可以期待背景低且高灵敏度的成像。为了确认化合物9的荧光发射的pH依存性,在含有50%乙腈的缓冲液中进行了pH4.0和7.4下的激发光谱(图1中的“激发”)和荧光光谱(图1中的“荧光”)的测量。图1示出了所得到的激发光谱和荧光光谱。从图1可以看到,化合物9具有斯托克司位移(Stokes shift)大的特征。进一步,可知与pH7.4的情况相比,在pH4.0的情况下荧光强度高,并且最大荧光波长向30nm短波长一侧发生了位移。
在含有50%DMSO的缓冲液中测定了化合物9在各pH下的荧光强度。结果如图2所示。可知,化合物9随着从中性区域变成酸性区域,荧光强度随之变高。这相较于已被报导的萘基酰亚胺(naphthyl imide)型的pH响应性荧光化合物,在酸性侧荧光更加增强,据此,可期待以此作为不容易受在细胞内的其他细胞器的影响的荧光色素。
实施例3:使用HeLa细胞的线粒体自噬检测评价(1)
将HeLa细胞播种在μ-Slide 8孔易必迪(Ibidi)上,在37℃、CO2培养箱中培养过夜。添加以Hanks’HEPES缓冲液稀释的100nmol/L的化合物9,培养30分钟。用Hanks’HEPES缓冲液清洗两遍,添加含有1μM的胰高血糖素(glucagon)和7.5μM的胃酶抑素(pepstatin)A的不含血清Krebs’缓冲液,在37℃培养适当时间,进行饥饿诱导后,通过荧光显微镜进行观察。线粒体自噬诱导前和诱导后的HeLa细胞的观察结果如图3所示。为了确认溶酶体对于化合物9的吸收,将以Hanks’HEPES缓冲液进行稀释的1μM的溶酶体染料(Lyso Dye)在37℃培养30分钟。用Hanks’HEPES缓冲液清洗两遍后,通过荧光显微镜确认共染色的发生。
实施例4:使用HeLa细胞的线粒体自噬检测评价(2)
线粒体自噬诱导,除了上述实施例3所用的饥饿诱导等的大规模(bulk)的方法之外,还可以列举通过Parkin基因和酶PINK1的选择性方法。后者与帕金森病有关,已有启示其与有缺陷的线粒体的品质管理具有关联性(参考例如、Derek Narendra,Atsushi Tanaka,Der-Fen Suen and Richard J.Youle,J.Cell Biol.,2008,183,795-803.)。为了对线粒体选择性自噬的检测进行评价,调制Parkin表达Hela细胞,加入化合物9中,之后添加作为线粒体去极化剂的羰基氰间氯苯腙(carbonyl cyanidem-chlorophenylhydrazone,CCCP),进行培养,进而诱导线粒体自噬。
<使用Parkin表达HeLa细胞的激光共聚焦显微镜成像检测>
将HeLa细胞播种在μ-Slide 8孔易必迪(Ibidi)上,在37℃、CO2培养箱中培养过夜。用HilyMax(同仁化学研究所制造)将Parkin质粒导入细胞,并培养过夜。用含血清培养基清洗后,添加以Hanks’HEPES缓冲液稀释的100nmol/L的化合物9的溶液,培养30分钟。用含血清培养基清洗,之后添加包含10μmpl/L的CCCP的培养基,培养24小时。使用荧光显微镜确认线粒体自噬的发生,之后将1μM溶酶体染料(Lyso Dye)在37℃培养箱培养30分钟。用Hanks’HEPES缓冲液清洗两遍,之后用荧光显微镜确认共染色的发生。
从来自共聚焦显微镜的荧光图像(参照图4)观察到,在Parkin未表达HeLa细胞(Parkin(-))中,来源于化合物9的荧光微弱。另一方面,可知在Parkin表达HeLa细胞(Parkin(+))中,观察到了来源于化合物9的荧光,进而与溶酶体染色剂(Lyso dye)发生共染色(参照图4)。这表示线粒体与溶酶体融合,这揭示了化合物9是即使在线粒体选择性诱导条件下也可检测出线粒体自噬的色素。
<使用Parkin表达HeLa细胞的流式细胞计数(FCM)分析>
将HeLa细胞播种在24孔板(plate)上,在37℃、CO2培养箱中培养过夜。用HilyMax(同仁化学研究所制造)将Parkin质粒导入细胞中,并培养过夜。用含血清培养基清洗后,添加以Hanks’HEPES缓冲液稀释的100nmol/L的化合物9的溶液,培养30分钟。用含血清培养基清洗后,以含有10μmpl/L的CCCP的培养基培养24小时。用PBS清洗后,用胰蛋白酶(trypsin)以及EDTA分离细胞。离心回收细胞,之后用0.5mL的HBSS使之悬浮,之后用FCM(BD FACS cant2)进行解析。
使用流式细胞计数(FCM)进行定量分析的结果,在羰基氰间氯苯腙(CCCP)诱导线粒体自噬下观测到了约1.3倍的荧光增强(图5)。根据以上的结果,能够确认:化合物9是能够通过使用荧光成像以及FCM检测出线粒体自噬现象的荧光色素。
Claims (6)
1.一种pH响应性荧光化合物,其特征在于,
由以下通式所示,
[化学式1]
在上述通式中,
L表示接头,
X表示药学上允许的阴离子,
Y表示可与线粒体蛋白质上的官能团共价结合的反应性官能团。
2.根据权利要求1所述的pH响应性荧光化合物,其特征在于,
所述pH响应性荧光化合物是下述式9所示的化合物,
[化学式2]
3.一种线粒体自噬检测用组合物,其特征在于,包含由下述通式所示的pH响应性荧光化合物,
[化学式3]
在上述通式中,
L表示接头,
X表示药学上允许的阴离子,
Y表示可与线粒体蛋白质上的官能团共价结合的反应性官能团。
4.根据权利要求3所述的线粒体自噬检测用组合物,其特征在于,
所述pH响应性荧光化合物是下述式9所示的化合物,
[化学式4]
5.一种细胞内线粒体自噬的检测方法,其特征在于,包括如下步骤:
投入步骤,将由下述通式所示的pH响应性荧光化合物投入细胞内;
测量步骤,进行规定时间的培养后,测量来自细胞的荧光发射;
[化学式5]
在上述通式中,
L表示接头,
X表示药学上允许的阴离子,
Y表示可与线粒体蛋白质上的官能团共价结合的反应性官能团。
6.根据权利要求5所述的细胞内线粒体自噬的检测方法,其特征在于,
所述pH响应性荧光化合物是下述式9所示的化合物,
[化学式6]
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CN108699091B (zh) | 2020-10-02 |
US20190062356A1 (en) | 2019-02-28 |
US10787473B2 (en) | 2020-09-29 |
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