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正常培養的細胞自噬活性很低,不適于觀察,因此,必須對自噬進行人工干預和調節,經報道的工具藥有:

一、自噬誘導劑

(1) Bredeldin A / Thapsigargin / Tunicamycin :模擬內質網應激
(2) Carbamazepine/ L-690,330/ Lithium Chloride(氯化鋰):IMPase 抑制劑(即Inositol monophosphatase,肌醇單磷酸酶)
(3) Earle's平衡鹽溶液:制造饑餓
(4) N-Acetyl-D-sphingosine(C2-ceramide):Class I PI3K Pathway抑制劑
(5) Rapamycin:mTOR抑制劑
(6) Xestospongin B/C:IP3R阻滯劑

二、自噬抑制劑

(1) 3-Methyladenine(3-MA):(Class III PI3K) hVps34 抑制劑
(2) Bafilomycin A1:質子泵抑制劑
(3) Hydroxychloroquine(羥氯喹):Lysosomal lumen alkalizer(溶酶體腔堿化劑)

除了選用上述工具藥外,一般還需結合遺傳學技術對自噬相關基因進行干預:包括反義RNA干擾技術(Knockdown)、突變株篩選、外源基因導入等。

三、自噬過程進行觀察和檢測

細胞經誘導或抑制后,需對自噬過程進行觀察和檢測,常用的策略和技術有:

1、觀察自噬體的形成
由于自噬體屬于亞細胞結構,普通光鏡下看不到,因此,直接觀察自噬體需在透射電鏡下。Phagophore的特征為:新月狀或杯狀,雙層或多層膜,有包繞 胞漿成分的趨勢。自噬體(AV1)的特征為:雙層或多層膜的液泡狀結構,內含胞漿成分,如線粒體、內質網、核糖體等。自噬溶酶體(AV2)的特征為:單層 膜,胞漿成分已降解。(autophagic vacuole,AV)

2、在熒光顯微鏡下采用mRFP-GFP-LC3融合蛋白來示蹤自噬形成
由于電鏡耗時長,不利于監測(Monitoring)自噬形成,人們利用LC3在自噬形成過程中發生聚集的現象開發出了此技術。無自噬時,mRFP-GFP-LC3融合蛋白彌散在胞漿中;自噬形成時,mRFP-GFP-LC3融合蛋白轉位至自噬體膜,在熒光顯微鏡下形成多個明亮的綠色熒光斑點,一個斑點相當于一個自噬體,可以 通過計數來評價自噬活性的高低。
mRFP 用于標記及追蹤LC3,GFP 的減弱可指示溶酶體與自噬小體的融合形成自噬溶酶體,即由于GFP熒光蛋白對酸性敏感,當自噬體與溶酶體融合后GFP 熒光發生淬滅,此時只能檢測到紅色熒光。
我們在顯微鏡成像后紅綠熒光merge后通過merge后出現的黃色斑點即只是自噬體.紅色的斑點指示自噬溶酶體,通過不同顏色斑點的計數可以清晰的看出自噬流的強弱。

我們在顯微鏡成像后紅綠熒光merge后通過merge后出現的黃色斑點即只是自噬體.紅色的斑點指示自噬溶酶體,通過不同顏色斑點的計數可以清晰的看出自噬流的強弱。
3、利用Western Blot檢測LC3-II/I比值的變化以評價自噬形成
自噬形成時,胞漿型LC3(即LC3-I)會酶解掉一小段多肽,轉變為(自噬體)膜型(即LC3-II),因此,LC3-II/I比值的大小可估計自噬水平的高低。
(注意:LC3抗體對LC3-II有更高的親和力,會造成假陽性。方法2和3需結合使用,同時需考慮溶酶體活性的影響。)

4、檢測長壽蛋白的批量降解:非特異

5、MDC(Monodansylcadaverine,單丹磺酰尸胺)染色:包括自噬體,所有酸性液泡都被染色,故屬于非特異性的。

6、CellTrackerTM Green染色:主要用于雙染色,但其能染所有的液泡,故也屬于非特異性的。

四、自噬相關蛋白的定位

在研究自噬相關蛋白時,需對其進行定位。由于自噬體與溶酶體、線粒體、內質網、高爾基體關系密切,為了區別,常用到一些示蹤蛋白在熒光顯微鏡下來共定位:

Lamp-2:溶酶體膜蛋白,可用于監測自噬體與溶酶體融合。
LysoTrackerTM 探針:有紅或藍色可選,顯示所有酸性液泡。
pDsRed2-mito:載體,轉染后表達一個融合蛋白(紅色熒光蛋白+線粒體基質定位信號),可用來檢測線粒體被自噬掉的程度(Mitophagy)。
MitoTraker探針:特異性顯示活的線粒體,熒光在經過固定后還能保留。
Hsp60:定位與線粒體基質,細胞死亡時不會被釋放。
Calreticulin(鈣網織蛋白):內質網腔
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