CN111474149B - 一种线粒体的动态评估方法 - Google Patents
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Abstract
本发明公开了一种线粒体的动态评估方法,方法包括:细胞培养及分组、线粒体染色、共聚焦观察、分析计算线粒体运动指数和测细胞系多组细胞的最大氧耗速率、细胞最大呼吸速率、基础呼吸速率、ATP产生值、或备用呼吸能力值。方法通过描述线粒体运动指数,结合已有的描绘线粒体形态的方式,更全面的评估线粒体动态,区分线粒体的适当融合和过度融合,适当分裂和过度分裂。
Description
技术领域
本发明涉及生物技术,尤其涉及一种线粒体的动态评估方法。
背景技术
线粒体是真核动物细胞进行生物氧化和能量转换的主要场所,提供细胞生命活动需要的能量,并参与各种代谢和信号转导功能。线粒体结构与代谢的多样性已在文献中多有报道。线粒体功能异常被证实与人类多种疾病密切相关,包括肥胖、糖尿病、神经退行性病变、肿瘤、心血管疾病等。
线粒体处于不断的融合-分裂的动态变化中,线粒体融合可修复轻微受损的线粒体,而分裂将线粒体中受损的成分选择性分配到子代线粒体中,分为健康的线粒体和严重损伤的线粒体,严重受损的线粒体膜电位发生变化,很难再融合,可以通过线粒体自噬清除。线粒体动态对维持线粒体的大小、形态、数量、功能起着重要作用。
目前评估线粒体动态的方法比较有限。最经典的方法是电镜观察线粒体形态,常用指标包括:线粒体面积、线粒体周长、线粒体长径、线粒体短径、线粒体长宽比(circularity index)、线粒体密度(线粒体数量/视野面积)。上述指标主要针对静止的线粒体形态,对于线粒体动态的评估有局限性,同时也因为电镜标本的制作过程和切面影响统计结果。另一种描述线粒体方式是描述用共聚焦显微镜拍摄的线粒体荧光图片,一般描述每个细胞中线粒体长度分布。也可以通过一段视频观察线粒体在固定时间内发生分裂融合事件的次数,计算方法较繁琐;最新技术分析线粒体形态,可以直接描绘出线粒体的丝网状、点状、棒状、肿胀及碎片状形态。如CN102156988B公开的一种细胞分裂序列检测方法,JP6553046B2公开的的基于细胞的试验中筛选化合物的方法,CN109523577A公开的基于显微图像的亚细胞结构运动轨迹确定方法。
然而,上述方法都只能描述静态情况下线粒体的形态,即处于分裂或者融合状态,且目前对于线粒体过度分裂或者过度融合的界定也不确定,有的以长于10um作为丝状,有的以长于5um作为丝状;或者只有繁杂的方式计算分裂-融合事件的发生次数。但线粒体是一种一直在运动和变化着的细胞器,不断的分裂和融合,不断地与其他细胞器、如内质网、溶酶体、微丝等发生互作,上述方法并不能清楚全面地描绘出线粒体动态,即线粒体运动着的变化。
发明内容
为了克服现有技术的不足,本发明提供一种线粒体的动态评估方法,其能解决无法动态描述线粒体形态的问题。
发明目的:引入线粒体运动指数的概念,从另一个角度描绘线粒体动态,弥补了之前只能描述线粒体静止的形态的缺点;全面的评估线粒体动态与功能的关系,界定过度融合与适当融合,过度分裂与适当分裂的线粒体。
原理说明:适当的线粒体融合可以增强线粒体对应激因素的耐力,即对线粒体是一种保护作用,不容易受到应激因素的损害。但过度融合的线粒体妨碍了线粒体的再循环利用,是线粒体损伤的表现。但适当的线粒体融合和过度的线粒体融合不能区分。同样,适当的线粒体分裂,可以将轻度损伤的线粒体分裂为受损的子代线粒体和健康的子代线粒体,受损的子代线粒体可以被线粒体自噬等方式很快的清除并循环再利用重新合成ATP等,是线粒体的保护形式。而过度分裂出现在很多心脏疾病中,过度分裂导致严重的线粒体功能损伤,导致疾病的进展。
为了区别适当融合和过度融合,适当分裂和过度分裂,为了更全面的描述线粒体功能,引入线粒体运动指数的概念。由于线粒体处于持续的运动中,当疾病因素刺激细胞的早期,线粒体之间及线粒体与其他细胞器之间发生各种物质交换,可以代偿性的增强线粒体对疾病因素的耐力,此时线粒体会出现某种运动的变化。随着刺激的增强或者时间的延长,线粒体功能受损,线粒体与其他细胞器之间的物质或者信号交换发生失代偿,线粒体的运动也会发生变化。这种线粒体运动的变化,可以用线粒体运动指数来描述。
为描述线粒体功能,分别评估在正常情况和病理因素刺激下线粒体的OCR。线粒体与其他细胞器互作,产生各种物质及信号交流,如钙离子、各种小分子等,在细胞的功能中扮演重要角色。结合线粒体运动速度指数从新的角度描述了线粒体形态和功能。
综上,本研究即通过描述线粒体运动指数,结合已有的描绘线粒体形态的方式,更全面的评估线粒体动态,区分线粒体的适当融合和过度融合,适当分裂和过度分裂。
技术方案:本发明的目的采用以下技术方案实现。
一种线粒体的动态评估方法,方法包括:
S1、细胞培养及分组,将细胞系分为多组,按不同组别预处理后放入共聚焦皿中培养相应时间。
S2、线粒体染色,将共聚焦皿中预处理过的细胞通过荧光探针染色,在培养箱中避光静置,然后去除染色工作液并清洗,加入培养基,以待观察。
S3、共聚焦观察,将共聚焦皿置于共聚焦荧光显微镜下,每皿细胞随机选择多个细胞,调整z轴和电压,拍摄视频,每帧间隔t秒,取n帧图片,n取大于1的正整数,共计时间(n-1)t秒。
S4、分析计算线粒体运动指数。
4.1截取共聚焦荧光显微镜从同一个角度下拍摄同一个细胞n帧图片,并进行二值化处理。其中,图片拍摄的时间间隔必须一致,在拍摄某个细胞的过程中,保持镜头角度,镜头参数以及细胞放置位置不变。
4.2计算所有图片中同一细胞的面积,以及相邻两帧图片重叠在一起的重合面积,则相邻两帧的细胞线粒体速度其中1≤i≤n-1(n为大于1的正整数)。
参见图1,同一细胞相邻两帧图片,图1A中时刻细胞面积为Si,在下一帧图片图1B中时刻细胞面积为Si+1,将图1A和图1B重叠,图中交叉重合部分面积为S重,则相邻两帧细胞的运动速率为
其中,那么ki=0就表示图1A和图1B完全重合,即细胞没有任何运动;ki=1就表示图1A与1B中的细胞照片完全不重合,但事实上通过我们拍摄的情况,运动远远达不到这么剧烈,因此ki介于0~1之间,ki值越大表示细胞运动越剧烈。
4.3计算细胞线粒体的平均速度其中1≤i≤n-1,以/>表征线粒运动指数。
优选的,在细胞培养及分组时,用H9C2细胞系开展实验并分为对照组、缺氧组(即control组)、苯肾上腺组(即PE组)进行预处理,其中,对照组采用高糖DMEM培养基培养;缺氧组采用无糖DMEM培养基以及缺氧条件培养4~6h;苯肾上腺组(即PE组)采用高糖DMEM培养基以及30~60uM苯肾上腺素培养20~30h。优选的,在线粒体染色时,将盛有预处理细胞的共聚焦皿中加入绿色荧光探针染色,在30℃~40℃培养箱中避光静置15-30min;随后去除染色工作液,用PBS洗2-3遍后,加入高糖DMEM培养基中以待观察。
优选的,所述荧光探针染色为比率型荧光探针或纳米荧光探针。
优选的,在共聚焦观察时,每皿细胞随机选择6-9个细胞,每帧图片间隔15s,共24帧图片。
优选的,方法还包括:S5、测细胞系多组细胞的最大氧耗速率、细胞最大呼吸速率、基础呼吸速率、三磷酸腺苷(Adenosine triphosphate,ATP)产生值或备用呼吸能力值以观察线粒体功能。
相比现有技术,本发明的有益效果在于:通过引入线粒体运动指数,从线粒体运动的角度描述了线粒体的形态及动态;结合OCR线粒体的最大氧耗速率等参数帮助全面评估线粒体的功能状态,如区别过度融合与适当融合,过度分裂与适当分裂的线粒体。
附图说明
图1为同一细胞相邻两帧图片的运动变化图及重叠图;
图2为相邻两帧细胞的运动速率计算流程图;
图3为具体实施例的细胞线粒体图像处理示意图;
图4为三组细胞的线粒体形态典型图;
图5为线粒体运动指数统计图;
图6为三组细胞OCR代表图;
图7为三组细胞基础呼吸速率统计图;
图8为三组细胞最大呼吸速率统计图;
图9为三组细胞ATP产生值统计图;
图10为三组细胞备用呼吸能力统计图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明的一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例,都属于本发明保护的范围。
S1、细胞培养及分组:用H9C2细胞系开展实验,分为control组、PE组、缺氧组三组,并进行预处理,control组用高糖DMEM培养基培养;缺氧组为细胞用无糖DMEM培养基+缺氧处理4-6h;PE组为用高糖DMEM培养基+50uM苯肾上腺素培养24h。
S2、线粒体染色:按照上述将H9C2细胞种在共聚焦皿,处理培养后加入mito-tracker绿色荧光探针染色(稀释比例1:10000),避光在37°培养箱中15-30min。随后去除染色工作液,用PBS洗2-3遍后,加入DMEM培养基,置于共聚焦荧光显微镜下观察。
S3、共聚焦观察:每皿细胞随机选择6-9个细胞,调整z轴和电压,针对一个细胞的线粒体拍摄视频,每帧间隔15秒,取24帧图片,共计时间(24-1)*15=345秒。参见图3,图3A为一个细胞的线粒体进行15S间隔拍摄的24帧图片,图3B为二值化处理后的相邻图片不重合部分,图3C为相邻图片重合部分。
S4、分析计算线粒体运动指数,通过软件的运行程序计算相邻图片件的线粒体或细胞运动速率ki及表征线粒体运动指数的ki的平均值。
计算结果:ki值分别为0.4496、0.4506、0.4514、0.4524、0.4522、0.4547、0.4547、0.4535、0.4543、0.4548、0.3970、0.4428、0.4489、0.4481、0.4486、0.4504、0.4487、0.4520、0.4493、0.4441、0.4443、0.4480、0.4492。平均值为0.44789。
S5、测细胞系多组细胞的最大氧耗速率、细胞最大呼吸速率、基础呼吸速率、ATP产生值、或备用呼吸能力值以观察线粒体功能。
具体的,测三组细胞(参见图4)的线粒体最大氧耗速率(Oxygen ConsumptionRate,OCR),观察线粒体功能。使用海马细胞能量代谢测定仪XF96检测三组细胞的糖酵解及氧化磷酸化。分别依次加入寡霉素(Oligomycin)1μmol/L、三氟甲氧基苯腙羰基氰化物(缩写为FCCP)0.5μmol/L、毒鱼藤素(Rotenone)1μmol/L和抗霉素A(antimycin A)1μmol/L。分别计算细胞的基础耗氧量、最大耗氧能力等。
参见图4,待测量的三组细胞的线粒体形态典型图,图5-图10为具体检测的线粒体运动指数统计图、三组细胞OCR代表图、基础呼吸速率、最大呼吸速率、ATP产生值以及备用呼吸能力,结合表征线粒体运动指数的ki的平均值综合评价线粒体的状态。
综上所述,为了更好的描述线粒体,首先,按上述培养及处理细胞,拍摄一段线粒体运动的视频,固定间隔时间和拍摄总帧数。随后,将每一个细胞的线粒体图片进入程序进行分析k值,代表每一个细胞的线粒体运动速度,与其他线粒体功能指标多角度描述线粒体。
最后应说明的是:以上实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
Claims (5)
1.一种线粒体的动态评估方法,其特征在于,方法包括:
S1、细胞培养及分组,将细胞系分为多组,按不同组别预处理后放入共聚焦皿中培养相应时间;
S2、线粒体染色,将共聚焦皿中预处理过的细胞通过荧光探针染色,在培养箱中避光静置,然后去除染色工作液并清洗,加入培养基,以待观察;
S3、共聚焦观察,将共聚焦皿置于共聚焦荧光显微镜下,每皿细胞随机选择多个细胞,调整z轴和电压,拍摄视频,每帧间隔t秒,取n帧图片,n取大于1的正整数,共计时间(n-1)t秒;
S4、分析计算线粒体运动指数,
4.1截取共聚焦荧光显微镜从同一个角度下拍摄同一个细胞n帧图片,并进行二值化处理;
4.2计算所有图片中同一细胞的面积,以及相邻两帧图片重叠在一起的重合面积,则相邻两帧的细胞线粒体速度其中1≤i≤n-1(n为大于1的正整数);
同一细胞相邻两帧图片,某一帧时刻细胞面积为Si,在下一帧时刻图片细胞面积为Si+1,将两帧图片重叠,图中交叉重合部分面积为S重;
其中,ki=0表示相邻两帧图片中的细胞照片完全重合,即细胞没有任何运动;ki=1就表示相邻两帧图片中的细胞照片完全不重合,ki介于0~1之间,ki值越大表示细胞运动越剧烈;
4.3计算细胞线粒体的平均速度其中1≤i≤n-1(n为大于1的正整数),以/>表征线粒运动指数;在细胞培养及分组时,用H9C2细胞系开展实验并分为对照组、缺氧组、苯肾上腺组进行预处理,其中,对照组采用高糖DMEM培养基培养;缺氧组采用无糖DMEM培养基以及缺氧条件培养4~6h;苯肾上腺组采用高糖DMEM培养基以及50uM苯肾上腺素培养20~30h。
2.根据权利要求1所述的方法,其特征在于:在线粒体染色时,将盛有预处理细胞的共聚焦皿中加入绿色荧光探针染色,在37℃培养箱中避光静置15-30min;随后去除染色工作液,用PBS洗2-3遍后,加入无糖DMEM培养基中以待观察。
3.根据权利要求1或2所述的方法,其特征在于:所述荧光探针染色为比率型荧光探针或纳米荧光探针。
4.根据权利要求1所述的方法,其特征在于:在共聚焦观察时,每皿细胞随机选择6-9个细胞,每帧图片间隔15s,共24帧图片。
5.根据权利要求1所述的方法,其特征在于,方法还包括:
S5、测细胞系多组细胞的最大氧耗速率、细胞最大呼吸速率、基础呼吸速率、ATP产生值、或备用呼吸能力值以观察线粒体功能。
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