【文章內容簡介】 侶后被轉運到溶酶體腔中，然后被溶酶體酶消化。 Autophagy, a membrane trafficking process leading to lysosomal degradation, is implicated in cancer, neurodegeneration, and aging. ? 在形態(tài)學上，即將發(fā)生 自噬的細胞胞漿中出現(xiàn)大量 游離的膜性結構 ，稱為 前自噬泡（自噬發(fā)生的鐵證之一） 。前自噬泡逐漸發(fā)展成為由雙層膜結構形成的 自噬泡（ 自噬發(fā)生的鐵證之二 ） ，其中包裹著變性壞死的細胞器和部分細胞漿 。 ? 自噬泡的外膜與溶酶體膜融合，內膜及其包裹的物質進入溶酶體腔，被溶酶體中的酶水解。此過程使進入溶酶體中的物質分解為其組成成分，并被細胞再利用，這種吞噬了細胞內成分的溶酶體被稱為 自噬溶酶體 。 自噬泡 Autolysosomes 自噬溶酶體 細胞的自噬過程 Ryter,et al. Proc Am Thorac Soc Vol 7. pp 40–47, 2022 自噬泡 (phagophore)或自噬體 (autophagosome)形成方面的穩(wěn)態(tài)監(jiān)測方法和細胞自噬的過程 (Flux)監(jiān)測。 ?檢測自噬的 金指標 ：透射電鏡檢測。 ?自噬體膜 標志性蛋白質 的檢測：利用 Western Blot檢測 LC3II／ I比值的變化以評價自噬形成或在熒光顯微鏡下采用 GFPLC3融合蛋白來示蹤自噬形成。 研究 Beclin1(哺乳動物自噬的標記蛋白，參與自噬體膜的形成 ) 單丹 (磺 )酰戊二胺 (MDC)染色法：包括自噬體，所有酸性液泡都被染色，故屬于非特異性的。 ?對自噬溶酶體及其殘體的檢測有 吖啶橙染色法 ；殘體的檢測主要是對 脂褐素顆粒 的顯微觀察。 自噬體的研究和檢測方法 ?電鏡 ? 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） ?無自噬 時 ，GFPLC3融合蛋白彌散在胞 漿 中； ?自噬 形成 時 ，GFPLC3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(shù)來 評 價自噬活性的高低。 ?熒光顯微鏡下： 采用 GFPLC3融合蛋白 示蹤自噬形成 ?Western Blot檢測 LC3II/I比值的變化 正常培養(yǎng)的細胞自噬活性很低，不適于觀察，可對自噬進行人工干預和調節(jié)。 （一）自噬誘導劑 1） Bredeldin A / Thapsigargin / Tunicamycin ：模擬內質網應激 2） Carbamazepine/ L690， 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） NAcetylDsphingosine（ C2ceramide）： 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） 3Methyladenine（ 3MA）：（ 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） Autophagy in chronically ischemic myocardium ? 動物模型的制作 ? 自噬形態(tài)學的電鏡觀察 ? 蛋白的提取 ? 雙相電泳分離 ? MALDI TOF MS分析 ? 數(shù)據庫搜索 ? 差異表達蛋白的 WB驗證與定位研究 動物模型的制作 ? Chronically instrumented pigs were studied with repetitive myocardial ischemia produced by one, three, or six episodes of 90 min of coronary stenosis(冠狀動脈狹窄 ) [30% reduction in baseline coronary flow followed by reperfusion(再灌注 ) every 12 h] with the nonIS region as control. ? In this model, wall thickening in the IS region was chronically depressed by ≈37%. Electron micrographs of different types of AVs observed in the sixepisode chronically IS region (A–C) and sixepisode NI region (D). ? (A) Autophagic vacuoles(Avs,自體吞噬泡 ) containing remnants of mitochondria are demonstrated. ? (B) Doublemembrane AVs containing recognizable cytoplasmic contents are displayed. ? (C) AVs containing multivesicular bodies surrounded by a sequestering membrane are demonstrated. ? (D) These AVs were not observed in the sixepisode ? NI. Arrows indicate AVs. (Magnifications: 1,400–2,000, A–D。 5,000, A and B Insets.) Alteration of cathepsin D in chronically IS myocardium. ? (Upper) 2D gel map of protein extracts from chronically IS region after six episodes. ? (Lower)A magnified gel region from the NI region (Left) and the IS after either three (Center) or six (Right) episodes of CS. ? Arrows indicate the spots after six episodes that were all later identified as cathepsin D. MW, molecular weight. ? Each lower image is a 6fold magnification from the original gel. Comparison of expression level of cathepsin B and D in chronically IS myocardium at different episodes ? Comparison of expression level of cathepsin B and D in chronically IS myocardium at different episodes. (A and B) Western blot analysis of cathepsin D (A) and cathepsin B (B) at six episodes chronically IS vs. NI regions (Upper) and three episodes (Lower). The expression of cathepsins B and D significantly increased at six episodes (**, P 。 *, P vs. NI). (C) The direct parison of levels of cathepsin B (CB) and cathepsin D (CD) in chronically IS in one, three, and six episodes, again showing marked increased expression of both proteins after six episodes. (D) The elevated expression of cathepsins B and D was confirmed to occur in myocytes from the chronically IS myocardium. ADU, arbitrary densitometric units. Enzyme activity assays of cathepsin B (A), cathepsin D (B), and βhexosaminidase (C) ? Enzyme activity assays of cathepsin B (A), cathepsin D (B), and βhexosaminidase (C). Activity of both cathepsin B and D and βhexosaminidase was much higher at six episodes in chronically IS region vs. NI region pared with three episodes vs. NI. **, P vs. NI。 *, P vs NI. Western blot analysis of Hsc73 at six episodes chronically IS vs. NI(nonIS) myocardium (A) and at three episodes in isolated myocytes (B) and paring effects in necrotic tissue after myocardial infarction (IF) vs. NI regions (C). ? The expression of Hsc73 increased significantly in the chronically IS of six episodes (**, P vs. NI), but not in the IS after three episodes pared with NI. The elevation of Hsc73 after six episodes was confirmed in isolated myocytes. In contrast, the expression of Hsc73 decreased in the infarcted region. ADU, arbitrary densitometric units. Western blot analysis of beclin 1 at three and six episodes (chronically IS regions) ? Western blot analysis of beclin 1 at three and six episodes (chronically IS vs. NI regions) (A) and in isolated myocytes from the chronically IS (B) and paring effects in necrotic tissue after myocardial infarction (IF) vs. NI regions (C). The expression of beclin 1 was increased significantly in the chronically IS after six episodes (*, P vs. NI), but not after three episodes. The elevation o