Method for carrying out lysosome classification by taking lysosome metabolite as marker

A technology for lysosomes and metabolites, applied in the field of lysosome classification, can solve the problem of poor fineness of lysosome classification

Active Publication Date: 2021-08-17
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Aiming at the deficiencies of the prior art, the present invention provides a method for classifying lysosomes using l

Method used

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  • Method for carrying out lysosome classification by taking lysosome metabolite as marker
  • Method for carrying out lysosome classification by taking lysosome metabolite as marker
  • Method for carrying out lysosome classification by taking lysosome metabolite as marker

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Embodiment 1

[0049] This embodiment takes isolated HEK-293T cells as an example for introduction, and of course the present invention can also be used for mouse embryonic fibroblasts (MEF), mouse lung fibroblasts (MLF), bladder cancer cells (T24), human Immortalized bladder epithelial cells (SV-HUC-1), BxPC-3 cells, mouse cardiomyocytes and cardiac fibroblasts, mouse cerebral cortex neurons and glial cells, mouse peritoneal macrophages, mouse skin Cells such as fibroblasts (MEFs).

[0050] This embodiment discloses a method for classifying lysosomes using lysosomal metabolites as markers, comprising the following steps:

[0051] Step 1. Cell culture

[0052] Culture of HEK-293T cells

[0053] HEK-293T cells used for passage were cultured in T25 culture flasks. Aspirate the culture medium in the culture bottle, add 1 mL of preheated trypsin to wash it again, add 1 mL of trypsin, and put it in a 37°C carbon dioxide incubator to digest for 2 minutes; add 4 mL of preheated culture medium to...

Embodiment 2

[0107] Example of tip (electrode tip) and shape

[0108] Such as Figure 2-5 As shown, among them, figure 2 The tip opening diameters of each glass electrode in Tip1-Tip5 are 272nm, 340nm, 298nm, 288nm, 310nm respectively, and the tip length of the glass electrode is selected as 8mm.

[0109] Reproducibility of SLMS using glass electrodes with similar tip size and shape. figure 2 Scanning electron microscope images of five selected glass pipette tips for the geometrical properties of the SLMS glass electrodes. Scale bar 200 μm. image 3 SLMS measures lysine (LYS, relative standard deviation = 7.3%), Figure 4 Histidine (HIS, relative standard deviation = 8.1%) and Figure 5 Reproducibility of arginine (ARG, relative standard deviation = 7.5%).

[0110] Repeated measurements were made at the levels of 5 ppm (34 μM) of LYS, 5 ppm (32 μM) of HIS and 5 ppm (28 μM) of ARG added to the artificial simulated lysosomal fluid (ALF). ALF contains 145mM NaCl, 5mM KCl, 1mM MgCl 2...

Embodiment 3

[0113] In this example, the single lysosome mass spectrometry method established in the above example was used to detect the metabolites of lysosomes and endosomes respectively, and specific marker proteins were used to mark lysosomes and related lysosomes , that is, when performing transfection, endosomes were labeled with Rab5 (ie, EGFP-Rab5A), and lysosomes were labeled with Lamp1 (ie, Lamp1-mCherry) / Lamp2 (ie, Lamp2-EGFP).

[0114] Figure 7 Middle scale bar is 10 μm. Such as Figure 7 Shown in A, showing the distribution of enlarged endosomes and lysosomes in HEK-293T cells. Such as Figure 7 As shown in B, the distribution of enlarged autolysosomes and non-autolysosomes in HEK-293T cells, it can be observed that there are both autolysosomes and non-autolysosomes in the same cell body, indicating that lysosomes in the same cell are also heterogeneous.

[0115] Such as Figure 7 In A: the distribution of enlarged lysosomes and endosomes in HEK-293T cells. Cells were...

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Abstract

The invention discloses a method for carrying out lysosome classification by taking a metabolite of the lysosomes as a marker. The method comprises the following steps: obtaining contents of a single lysosome in a cell by adopting a single lysosome patch clamp technology; performing mass spectrometric detection on the lysosome content to obtain the composition and content of metabolites in the lysosome content; and classifying the lysosomes according to a detection result and differences of metabolite composition and content in a single lysosome content to obtain five different subgroups of lysosomes, including known autophagy lysosomes and endocytosis lysosomes. The lysosomes can be classified more finely, the lysosomes can be divided into five types by adopting the method, and each type of lysosomes has an obvious classification marker. Researchers provide clues for studying the specific functions of various lysosomes in the fields of aging and degenerative diseases such as development of drug metabolism for treating Alzheimer's disease and cancer.

Description

technical field [0001] The invention relates to the field of lysosome classification, in particular to a method for lysosome classification using lysosome metabolites as markers. Background technique [0002] Lysosome is the main degradative organelle in cells and a key site of cell metabolism. Dysfunction of lysosomes is closely related to many diseases, such as lysosomal storage diseases, neurodegenerative diseases, tumors, etc. Therefore, lysosomes are also important targets for disease treatment and drug development. In addition, lysosomes are also involved in the intracellular transport, storage and metabolism of drugs, which are directly related to the resistance of multiple drugs. There are often a large number of lysosomes in cells, and these lysosomes have huge heterogeneity in size, shape, and function. Different lysosomes often play different roles in diseases and drug metabolism. The classification of enzymes and the study of the characteristics and differences...

Claims

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Application Information

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IPC IPC(8): G01N27/62G01N33/487
CPCG01N27/62G01N33/48728
Inventor 熊伟朱洪影仓春蕾
Owner UNIV OF SCI & TECH OF CHINA
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