CN107228845A - 罗丹明x衍生物在细胞线粒体电势和溶酶体酸度比率检测中的应用 - Google Patents
罗丹明x衍生物在细胞线粒体电势和溶酶体酸度比率检测中的应用 Download PDFInfo
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Abstract
罗丹明X衍生物在细胞线粒体电势和溶酶体酸度比率检测中的应用，涉及罗丹明X衍生物的用途。所述罗丹明X衍生物是一种通过分子内5员酰胺螺环连接二乙烯三胺，再与3‑羧基‑7‑二氨乙基香豆素进行酰胺化反应，最后进行甲基化反应的罗丹明X衍生物。该化合物能同时发出两个波段的荧光，罗丹明X的红色荧光随pH值的升高而减弱，香豆素的蓝色荧光随pH值的升高而增强，该化合物在细胞的溶酶体和线粒体内均有分布，其在溶酶体中主要产生红色荧光，在线粒体中主要产生蓝色荧光，能达到同时检测线粒体电势和溶酶体酸度的作用。
Description
技术领域
本发明涉及罗丹明X衍生物的用途，尤其是涉及罗丹明X衍生物在细胞线粒体电势和溶酶体酸度比率检测中的应用。
背景技术
细胞的正常生长和功能的实现需要多个细胞器的共同协作。线粒体是细胞中进行有氧呼吸的场所，其跨膜电势对于细胞能量代谢至关重要，线粒体还是细胞中感知刺激的重要细胞器，细胞的凋亡，坏死以及自噬过程都涉及线粒体膜电势的变化；溶酶体是细胞中负责消化的细胞器，溶酶体的酸度对细胞分解代谢和稳态的维持有重要意义，细胞的异常(如癌变和溶酶体贮积病等)时常伴随着溶酶体酸度的异常。鉴于线粒体膜电势和溶酶体酸度在调节细胞代谢中的重要作用，在细胞中评估线粒体膜电势和溶酶体酸度的平衡非常必要[1-8]。
参考文献：
[1]J.R.Friedman,J.Nunnari,Nature 2014,505,335-343.
[2]M.Kulawiec,K.M.Owens,K.K.Singh,Cancer biology&therapy 2009,8,1378-1385.
[3]S.Vyas,E.Zaganjor,M.C.Haigis,Cell 2016,166,555-566.
[4]D.R.Green,J.C.Reed,Science 1998,281,1309-1312.
[5]G.Kroemer,M.Jaattela,Nat Rev Cancer 2005,5,886-897.
[6]C.Y.Lim,R.Zoncu,The Journal of cell biology 2016,214,653-664.
[7]R.Scatena,Advances in experimental medicine and biology 2012,942,287-308.
[8]C.Settembre,C.Di Malta,V.A.Polito,M.Garcia Arencibia,F.Vetrini,S.Erdin,S.U.Erdin,T.Huynh,D.Medina,P.Colella,M.Sardiello,D.C.Rubinsztein,A.Ballabio,Science 2011,332,1429-1433.
发明内容
为了解决传统细胞器染料只能单独检测线粒体膜电势或溶酶体酸度，无法同时检测两个细胞器参数的问题，本发明的目的在于提供罗丹明X衍生物在细胞线粒体电势和溶酶体酸度比率检测中的应用。
所述罗丹明X衍生物的化学结构式如下：
所述罗丹明X衍生物在细胞线粒体电势和溶酶体酸度比率检测中的应用
所述细胞为活细胞。
所述细胞线粒体电势和溶酶体酸度比率检测的方法为：共聚焦荧光显微镜法和流式细胞仪法。
本发明的原理如下：
所述罗丹明X衍生物是一种通过分子内5员酰胺螺环连接二乙烯三胺，再与3-羧基-7-二氨乙基香豆素进行酰胺化反应，最后进行甲基化反应的罗丹明X衍生物。该化合物能同时发出两个波段的荧光，罗丹明X的红色荧光随pH值的降低而剧烈增强，香豆素的蓝色荧光随pH值的升高而增强，该化合物在细胞的溶酶体和线粒体内均有分布，其在溶酶体中主要产生红色荧光，溶酶体酸度越高，红色荧光越强，在线粒体中主要产生蓝色荧光，线粒体膜电势越高，蓝色荧光越强，能达到同时检测线粒体电势和溶酶体酸度的作用。
有鉴于此，本发明提供一种应用具有分子内螺环的罗丹明X衍生物来检测细胞线粒体电势和溶酶体酸度的平衡。
本发明的有益效果如下：
1)所述罗丹明X衍生物能够快速、准确地定位在线粒体和溶酶体中；
2)所述罗丹明X衍生物在溶酶体中主要发射红色荧光，在线粒体中主要发射蓝色荧光；
3)同浓度的罗丹明X衍生物孵育细胞同样时间，溶酶体酸度越高，红色荧光越强，线粒体膜电势越高，蓝色荧光越强；
4)所述罗丹明X衍生物在细胞中线粒体和溶酶体的分布和比率可用共聚焦荧光显微镜和流式细胞仪来检测。
附图说明
图1为本发明实施例的罗丹明X衍生物在细胞中与线粒体特异的蛋白共定位的荧光共聚焦显微图。
图2为本发明实施例的罗丹明X衍生物在细胞中与溶酶体特异的蛋白共定位的荧光共聚焦显微图。
图3为本发明实施例的罗丹明X衍生物在细胞受刺激情况下，蓝色荧光和红色荧光的强度变化的共聚焦荧光显微图。
图4为本发明实施例的罗丹明X衍生物在细胞受刺激情况下，蓝色荧光和红色荧光的强度比率变化的流式细胞数据分析图。
具体实施方式
罗丹明X衍生物在细胞中线粒体膜电势和溶酶体酸度比率检测上的应用，所述罗丹明X衍生物的化学结构式如下：
下面将描述本发明的实施例，但本发明的内容完全不局限于此。
以下给出具体实施例。
1)配制含有3μM罗丹明X衍生物的DMEM：
称取25.4mg的罗丹明X衍生物溶于10mL的二甲亚砜中。即得3mmol/L(3mM)的罗丹明X衍生物的标准溶液，取1μL罗丹明X衍生物标准溶液与1mL DMEM混合，得到含有3μmol/L(3μM)罗丹明X衍生物的DMEM。
2)共聚焦荧光显微镜评估罗丹明X衍生物在细胞溶酶体中的分布：
将含有3μM罗丹明X衍生物的DMEM与生长在35mm玻璃底培养皿上的表达有溶酶体相关膜蛋白2融合绿色荧光蛋白的HeLa细胞共同孵育30min，用新鲜的DMEM洗三遍，再用共聚焦荧光显微镜对细胞内荧光信号进行采集。
可以观察罗丹明X衍生物的罗丹明X的红光与溶酶体相关膜蛋白2融合绿色荧光蛋白的绿光定位一致，说明罗丹明X衍生物在溶酶体中富集，并能发射出红色荧光。
3)共聚焦荧光显微镜评估罗丹明X衍生物在细胞线粒体中的分布：
将含有3μM罗丹明X衍生物的DMEM与生长在35mm玻璃底培养皿上的表达有线粒体外膜转位酶20融合绿色荧光蛋白的HeLa细胞共同孵育30min，用新鲜的DMEM洗三遍，再用共聚焦荧光显微镜对细胞内荧光信号进行采集。
可以观察罗丹明X衍生物的香豆素的蓝光与线粒体外膜转位酶20融合绿色荧光蛋白的绿光定位一致，说明罗丹明X衍生物在线粒体中富集，并能发射出蓝色荧光。
4)共聚焦荧光显微镜评估细胞在刺激情况下罗丹明X衍生物的蓝色荧光和红色荧光的强度变化：
将含有3μM罗丹明X衍生物的DMEM与生长在35mm玻璃底培养皿上的野生型HeLa细胞(5盘)共同孵育30min，用新鲜的DMEM洗三遍，再分别用含有二甲亚砜(0.1％)的DMEM，含有巴佛洛霉素A1(100nM)的DMEM，Hank平衡盐溶液，含有尼日利亚菌素(2μM)的DMEM和含有线粒体解偶联剂CCCP(20μM)的DMEM分别孵育野生型HeLa细胞6h，最后利用共聚焦荧光显微镜观察罗丹明X衍生物的红色荧光和蓝色荧光的分布情况和强度变化。
5)流式细胞仪评估细胞在刺激情况下罗丹明X衍生物的蓝色荧光强度和红色荧光强度的比率变化：
将含有3μM罗丹明X衍生物的DMEM与生长在12孔板中的野生型HeLa细胞(5个孔)共同孵育30min，用新鲜的DMEM洗三遍，再分别用含有二甲亚砜(0.1％)的DMEM，含有巴佛洛霉素A1(100nM)的DMEM，Hank平衡盐溶液，含有尼日利亚菌素(2μM)的DMEM和含有线粒体解偶联剂CCCP(20μM)的DMEM分别孵育野生型HeLa细胞6h，最后将细胞消化到流式管中，用流式细胞仪评估罗丹明X衍生物在细胞中的红色荧光和蓝色荧光的强度变化和比率变化。
本发明实施例的罗丹明X衍生物与表达有溶酶体相关膜蛋白2融合绿色荧光蛋白的HeLa细胞共同孵育后的共聚焦荧光显微图如图1所示。红色荧光来自罗丹明X，蓝色荧光来自香豆素，绿色荧光来自溶酶体相关膜蛋白2融合绿色荧光蛋白，红色荧光与绿色荧光定位一致，说明罗丹明X衍生物在细胞内的溶酶体中有富集，并且发射出红色荧光。
本发明实施例的罗丹明X衍生物与有线粒体外膜转位酶20融合绿色荧光蛋白的HeLa细胞共同孵育后的共聚焦荧光显微图如图2所示。红色荧光来自罗丹明X，蓝色荧光来自香豆素，绿色荧光来自线粒体外膜转位酶20融合绿色荧光蛋白，蓝色荧光与绿色荧光定位一致，说明罗丹明X衍生物在细胞内的线粒体中有富集，并且发射出蓝色荧光。
本发明实施例的罗丹明X衍生物在野生型HeLa细胞被刺激情况下的荧光比率变化的共聚焦显微图如图3所示，红色荧光来自罗丹明X，蓝色荧光来自香豆素，红色荧光定位于溶酶体，蓝色荧光定位于线粒体。二甲亚砜组为空白组，细胞内可以观察到一定强度的红色荧光与蓝色荧光；巴佛洛霉素A1能使溶酶体丧失酸度，可以观察到在巴佛洛霉素A1孵育后，细胞内红色荧光减弱到接近消失，蓝色荧光维持不变；Hank平衡盐溶液能使细胞发生自噬，进而提高溶酶体酸度，可以观察到Hank平衡盐溶液孵育后，红色荧光明显增强，蓝色荧光维持不变；尼日利亚菌素在使溶酶体丧失酸度的同时，会些微提高线粒体的膜电势，可以观察到尼日利亚菌素孵育后，红色荧光减弱到接近消失，蓝色荧光有了微弱的增强；线粒体解偶联剂(CCCP)能够破坏线粒体膜电势，可以观察到在CCCP孵育后，蓝色荧光大幅度减弱，红色荧光维持不变。以上结果证明罗丹明X衍生物的红色荧光和蓝色荧光可以反映出细胞被刺激后溶酶体酸度和线粒体膜电势的变化。
本发明实施例的罗丹明X衍生物在野生型HeLa细胞被刺激情况下的荧光比率变化的流式细胞分析数据如图4所示，红色荧光来自罗丹明X，蓝色荧光来自香豆素。二甲亚砜组为空白组，细胞内可以观察到一定强度的红色荧光与蓝色荧光，且蓝色荧光强度/红色荧光强度的平均统计值在3.6左右；巴佛洛霉素A1能使溶酶体丧失酸度，可以观察到在巴佛洛霉素A1孵育后，细胞内蓝色荧光强度/红色荧光强度的平均统计值在8.7左右，较二甲亚砜组有所上升；Hank平衡盐溶液能使细胞发生自噬，进而提高溶酶体酸度，可以观察到Hank平衡盐溶液孵育后，细胞内蓝色荧光强度/红色荧光强度的平均统计值在1.1左右，较二甲亚砜组有所下降；尼日利亚菌素在使溶酶体丧失酸度的同时，会些微提高线粒体的膜电势，可以观察到尼日利亚菌素孵育后，细胞内蓝色荧光强度/红色荧光强度的平均统计值在10.1左右，较二甲亚砜组有所上升，并且比巴佛洛霉素A1组更高；线粒体解偶联剂(CCCP)能够破坏线粒体膜电势，可以观察到在CCCP孵育后，细胞内蓝色荧光强度/红色荧光强度的平均统计值在1.9左右，较二甲亚砜组有所下降。以上结果证明罗丹明X衍生物的红色荧光强度/蓝色荧光强度可以反映出细胞被刺激后溶酶体酸度和线粒体膜电势的相对变化。
可以理解，很多细节的变化是可能的，但这并不因此违背本发明的范围和精神，任何所属技术领域的普通技术人员对其所做的适当变化，皆应视为不脱离本发明专利的范畴。
Claims (4)
1.罗丹明X衍生物在细胞中线粒体电势和溶酶体酸度比率检测中的应用，其特征在于所述罗丹明X衍生物的化学结构式如下：
2.罗丹明X衍生物在细胞线粒体电势和溶酶体酸度比率检测中的应用。
3.如权利要求2所述应用，其特征在于所述细胞为活细胞。
4.如权利要求2所述应用，其特征在于所述细胞线粒体电势和溶酶体酸度比率检测的方法为：共聚焦荧光显微镜法和流式细胞仪法。
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