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Application of polyphyllin I in lysosome detection

A technology of papaya saponin and lysosome, which is applied in the direction of measuring devices, instruments, analytical materials, etc., and can solve the problems that there is no papaya saponin Ⅰ and so on

Active Publication Date: 2021-07-13
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] It was reported earlier that saponin Ⅰ has potential antitumor activity, but so far, there is no report that saponin Ⅰ can target lysosomes

Method used

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  • Application of polyphyllin I in lysosome detection
  • Application of polyphyllin I in lysosome detection
  • Application of polyphyllin I in lysosome detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] The synthesis of embodiment 1Polyphyllin I-Cy3

[0050] 1.1 Experimental steps

[0051] Synthetic steps of Polyphyllin I-Cy3: Weigh 5mg polyphyllin I and dissolve it in 0.5mL dimethylformamide (DMF, Shanghai Titan Technology Co., Ltd.), add 3.45mgCy3-COOH (Ruixi Biology) , 1.34mg1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, Bi De Pharmaceutical) and 71.47μg 4-dimethylaminopyridine (DMAP, Adamas Reagent Co., Ltd.) Dissolved completely (painous saponin Ⅰ: Cy3-COOH: EDC: DMAP = 1: 1.2: 1.2: 0.1 (molar ratio). Stir the reaction at 50°C for 6 hours, and pass through a rotary evaporator (type YRE2000E, Yuhua Instrument, Gongyi City) Co., Ltd.) under the condition of 40 ℃ and 120r / min, the solvent was removed by rotary evaporation under reduced pressure, then washed 3 times with acetone, recrystallized with ethanol, and dried in vacuo to obtain the product. Using high performance liquid chromatography (HPLC, Hangzhou Xinqiao Biotechnology Co., Ltd.) to ve...

Embodiment 2

[0057] Embodiment 2Polyphyllin I-Cy3 targeting lysosome

[0058] 2.1 Experimental steps:

[0059] 2.1.1 Cell culture

[0060] Liver cancer HepG2 cells were purchased from the American Type Culture Collection (ATCC). At 37°C, 5% CO 2 Cultured in DMEM medium (Invitrogen) containing 10% (v / v) fetal bovine serum (Gibco) and 1% (w / v) penicillin / streptomycin (Invitrogen) in an incubator. When the cells are subcultured, first remove the original medium, wash it twice with PBS buffer, add trypsin (Gibco company) to digest the cells, and leave it in the cell incubator for about 1 min. If the cells have fallen off, add the medium to stop the digestion . The cells were blown down gently, centrifuged at 216g for 3min, the supernatant was removed, and the cells were resuspended in fresh DMEM medium containing 10% (v / v) fetal bovine serum and 1% (w / v) penicillin / streptomycin, The passage ratio was 1:4. The cells used for confocal observation were all cultured in confocal culture dishe...

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Abstract

The invention discloses application of polyphyllin I in lysosome detection. Experiments prove that the polyphyllin I has a specific lysosome targeting effect; and in order to facilitate observation and detection, the polyphyllin I and a fluorescent dye are further synthesized into a red fluorescent probe which is named as Polyphyllin I-CY3, and the structural formula of the polyphyllin I-CY3 is shown in (II) that is described in the specification. The polyphyllin I-CY3 red fluorescent probe synthesized by the invention can permeate cell membranes, can be used for specific fluorescent staining of living cell lysosome, and has the advantages of good targeting property, high detection speed, good repeatability, convenience in use, no cytotoxicity and the like.

Description

technical field [0001] The invention belongs to the field of biological detection, and in particular relates to the application of saponin I in the detection of lysosomes. Background technique [0002] Lysosomes (lysosomes) are single-membrane-coated sac-like structures with a diameter of about 0.025 to 0.8 microns, which contain a variety of hydrolytic enzymes. They are organelles with certain morphological structures and important physiological functions in biological cells. exogenous and endogenous macromolecules. Since Belgian scholar Cristian de Duve and others discovered lysosomes in rat liver cells in 1955, people have studied lysosomes in all aspects, and found that lysosomes are related to many diseases such as metabolic diseases, inflammation, tumors and immunity, etc. occurrence is closely related. Therefore, it is of great significance to develop probes that can accurately label and visualize lysosomes. [0003] Cy3 dye is an amino-reactive dye with a molecula...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07J71/00C09K11/06G01N21/64
CPCC07J71/0005C09K11/06G01N21/6428G01N21/6458C09K2211/1029C09K2211/1088G01N2021/6439
Inventor 汪洋张静何庆瑜
Owner JINAN UNIVERSITY
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