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正常培养的细胞自噬活性很低,不适于观察,因此,必须对自噬进行人工干预和调节,经报道的工具药有:

一、自噬诱导剂

(1) Bredeldin A / Thapsigargin / Tunicamycin :模拟内质网应激
(2) Carbamazepine/ L-690,330/ Lithium Chloride(氯化锂):IMPase 抑制剂(即Inositol monophosphatase,肌醇单磷酸酶)
(3) Earle's平衡盐溶液:制造饥饿
(4) N-Acetyl-D-sphingosine(C2-ceramide):Class I PI3K Pathway抑制剂
(5) Rapamycin:mTOR抑制剂
(6) Xestospongin B/C:IP3R阻滞剂

二、自噬抑制剂

(1) 3-Methyladenine(3-MA):(Class III PI3K) hVps34 抑制剂
(2) Bafilomycin A1:质子泵抑制剂
(3) Hydroxychloroquine(羟氯喹):Lysosomal lumen alkalizer(溶酶体腔碱化剂)

除了选用上述工具药外,一般还需结合遗传学技术对自噬相关基因进行干预:包括反义RNA干扰技术(Knockdown)、突变株筛选、外源基因导入等。

三、自噬过程进行观察和检测

细胞经诱导或抑制后,需对自噬过程进行观察和检测,常用的策略和技术有:

1、观察自噬体的形成
由于自噬体属于亚细胞结构,普通光镜下看不到,因此,直接观察自噬体需在透射电镜下。Phagophore的特征为:新月状或杯状,双层或多层膜,有包绕 胞浆成分的趋势。自噬体(AV1)的特征为:双层或多层膜的液泡状结构,内含胞浆成分,如线粒体、内质网、核糖体等。自噬溶酶体(AV2)的特征为:单层 膜,胞浆成分已降解。(autophagic vacuole,AV)

2、在荧光显微镜下采用mRFP-GFP-LC3融合蛋白来示踪自噬形成
由于电镜耗时长,不利于监测(Monitoring)自噬形成,人们利用LC3在自噬形成过程中发生聚集的现象开发出了此技术。无自噬时,mRFP-GFP-LC3融合蛋白弥散在胞浆中;自噬形成时,mRFP-GFP-LC3融合蛋白转位至自噬体膜,在荧光显微镜下形成多个明亮的绿色荧光斑点,一个斑点相当于一个自噬体,可以 通过计数来评价自噬活性的高低。
mRFP 用于标记及追踪LC3,GFP 的减弱可指示溶酶体与自噬小体的融合形成自噬溶酶体,即由于GFP荧光蛋白对酸性敏感,当自噬体与溶酶体融合后GFP 荧光发生淬灭,此时只能检测到红色荧光。
我们在显微镜成像后红绿荧光merge后通过merge后出现的黄色斑点即只是自噬体.红色的斑点指示自噬溶酶体,通过不同颜色斑点的计数可以清晰的看出自噬流的强弱。

我们在显微镜成像后红绿荧光merge后通过merge后出现的黄色斑点即只是自噬体.红色的斑点指示自噬溶酶体,通过不同颜色斑点的计数可以清晰的看出自噬流的强弱。
3、利用Western Blot检测LC3-II/I比值的变化以评价自噬形成
自噬形成时,胞浆型LC3(即LC3-I)会酶解掉一小段多肽,转变为(自噬体)膜型(即LC3-II),因此,LC3-II/I比值的大小可估计自噬水平的高低。
(注意:LC3抗体对LC3-II有更高的亲和力,会造成假阳性。方法2和3需结合使用,同时需考虑溶酶体活性的影响。)

4、检测长寿蛋白的批量降解:非特异

5、MDC(Monodansylcadaverine,单丹磺酰尸胺)染色:包括自噬体,所有酸性液泡都被染色,故属于非特异性的。

6、CellTrackerTM Green染色:主要用于双染色,但其能染所有的液泡,故也属于非特异性的。

四、自噬相关蛋白的定位

在研究自噬相关蛋白时,需对其进行定位。由于自噬体与溶酶体、线粒体、内质网、高尔基体关系密切,为了区别,常用到一些示踪蛋白在荧光显微镜下来共定位:

Lamp-2:溶酶体膜蛋白,可用于监测自噬体与溶酶体融合。
LysoTrackerTM 探针:有红或蓝色可选,显示所有酸性液泡。
pDsRed2-mito:载体,转染后表达一个融合蛋白(红色荧光蛋白+线粒体基质定位信号),可用来检测线粒体被自噬掉的程度(Mitophagy)。
MitoTraker探针:特异性显示活的线粒体,荧光在经过固定后还能保留。
Hsp60:定位与线粒体基质,细胞死亡时不会被释放。
Calreticulin(钙网织蛋白):内质网腔
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