Method for evaluating antifungal effect of traditional Chinese medicine monomer through candida albicans cell wall reconstruction

A technology of Candida albicans and Candida albicans, applied in instruments, measuring devices, scientific instruments, etc., can solve the problems of bacterial resistance and endangering the life safety of patients.

Pending Publication Date: 2020-09-25
ANHUI UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, with the long-term use of traditional antifungal drugs, more and more serious strains of drug resistance have emerged in clinical practice, which has seriously endangered the lives of patients

Method used

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  • Method for evaluating antifungal effect of traditional Chinese medicine monomer through candida albicans cell wall reconstruction
  • Method for evaluating antifungal effect of traditional Chinese medicine monomer through candida albicans cell wall reconstruction
  • Method for evaluating antifungal effect of traditional Chinese medicine monomer through candida albicans cell wall reconstruction

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Embodiment 1

[0023] Example 1: A method for evaluating the antifungal effect of traditional Chinese medicine monomers by remodeling the cell wall of Candida albicans

[0024] The exposure of β-glucan in the cell wall of Candida albicans was achieved by inoculum (=1×10 3 CFU / mL) and THMs at its MICs concentration were incubated in a 4ml EP tube for 12h, then washed once with sterile PBS, and blocked with 2% BSA (Albumin Fraction V) prepared in sterile PBS for 1 hour (Cat. no. 9048-46-8, Biofroxx, Shanghai, China), and then wash once with sterile PBS; then add 100ul of β-glucan antibody (β-glucan antibody: sterile PBS=1:300, Cat . no. 400-2, Bioscience Supplies, Australia) placed in 4°C refrigerator for staining for 4 hours, after staining, washed twice with sterile PBS, and then cy3 monoclonal antibody mouse IgG (cy3: sterile PBS=1 :100, Cat. no 143702A, Abbkine, Shanghai, China) at room temperature for 1 hour in the dark, then washed with sterile PBS for 3 times in the dark, then resuspen...

Embodiment 2

[0028] Example 2: A method for evaluating the antifungal effect of traditional Chinese medicine monomers by remodeling the cell wall of Candida albicans

[0029] 1. Preparation of bacterial solution

[0030] C. albicans SC5314 was donated by Professor Jiang Yuanying, School of Pharmacy, Second Military Medical University, Shanghai, China. All the strains were routinely preserved on Sabouraud agar, and activated and cultured in liquid sabouraud medium (Hope Biotechnology, Qingdao, Shandong) at 37°C for 12-16 hours until the strains reached the exponential growth stage. Fungal cells were collected after centrifugation at 3000r / min. After washing twice with PBS (Leagene, Beijing, China), the combined bacterial cells were recovered in RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA), and the pH of RPMI-1640 medium was adjusted to 7.5 with a concentration of 1 M NaOH.

[0031] 2. Drug susceptibility test of traditional Chinese medicine monomer

[0032] The initial concentration ...

Embodiment 3

[0042] Example 3: A method for evaluating the antifungal effect of traditional Chinese medicine monomers by remodeling the cell wall of Candida albicans

[0043] A method for evaluating the antifungal effect of traditional Chinese medicine monomers through Candida albicans cell wall remodeling, comprising the following steps:

[0044] Step 1: Activate and cultivate the preserved Candida albicans strain for 12-16 hours until the Candida albicans strain reaches the exponential growth stage, then collect the fungal cells after centrifugation, wash with PBS, and place the Candida albicans strain cells in RPMI-1640 medium Recovery, and then adjust the pH value of RPMI-1640 medium to 7.5, and adjust the concentration of Candida albicans cells to 1×10 3 CFU / mL, to obtain the evaluation bacterial solution;

[0045] Step 2: Add the Chinese medicine monomer to the evaluation bacteria solution, incubate for 12 hours, wash once with sterile PBS, then block with BSA with a mass fraction o...

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Abstract

A method for evaluating the antifungal effect of a traditional Chinese medicine monomer through candida albicans cell wall reconstruction comprises the following steps: adding the traditional Chinesemedicine monomer into an evaluation bacterial solution, incubating for 12 hours, washing once by using sterile PBS, sealing for 1 hour by using BSA with the mass fraction of 2% prepared by using the sterile PBS, and washing once by using the sterile PBS; then adding 100 [mu]l of a beta-glucan antibody; dyeing in a refrigerator at 4 DEG C for 4 hours; after dyeing is finished, washing twice with sterile PBS, dyeing IgG for 1 hour at room temperature in a dark place by using a cy3 monoclonal antibody mouse, washing for 3 times with sterile PBS in a dark place, resuspending in a dark place by using 1ml of sterile PBS, shaking in a dark place, uniformly mixing, and detecting the fluorescence exposure intensity of glucan by using a flow cytometer; or detecting the fluorescence exposure intensity of chitin by using a flow cytometer; or recording fluorescence images of beta-glucan and chitin by using an inverted fluorescence microscope. Candida albicans cell wall reconstruction is used for evaluating the antifungal effect of the traditional Chinese medicine monomer, and clinical significance is achieved.

Description

technical field [0001] The invention relates to a method for evaluating the antifungal effect of a traditional Chinese medicine monomer by remodeling the cell wall of Candida albicans. Background technique [0002] Candida albicans is a common opportunistic pathogenic fungus belonging to the genus Candida in the family Saccharomyces. Candida albicans ranks first in the genus Candida in terms of morbidity and mortality. Drug resistance in Candida albicans mainly comes from long-term / repeated drug use and / or internal biofilm formation. It can cause superficial disturbances as well as deep intrusions. However, with the long-term use of traditional antifungal drugs, more and more serious strains of drug resistance have appeared clinically, which has seriously endangered the lives of patients. [0003] The cell wall of C. albicans is composed of β-glucan, mannoprotein and a small amount of chitin (chitin). The cell wall proteins of Candida albicans are involved in the adhesio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/50
CPCG01N33/5044
Inventor 邵菁刘娟娟李倩倩潘敏陈梦丽汪天明吴大强马克龙颜贵明汪长中
Owner ANHUI UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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