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Fluorescent tracer method of camptothecine in cell structure

A technology for cell structure and fluorescence tracing, applied in fluorescence/phosphorescence, material analysis through optical means, measurement devices, etc., can solve the loss of camptothecin, affect the accurate observation of camptothecin, and cannot clearly observe the cell structure Arbuscular structure and other problems, to achieve the effect of reducing the degree of loss

Inactive Publication Date: 2009-06-03
NORTHEAST FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Phillips and Hayman in 1970 "Improved procedures for clearing and attaining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection." The microscopic observation of the arbuscular structure in the root cells was reported. The observation of the arbuscular structure in the arbuscular mycorrhizal cells usually requires staining to be clearly observed. Carrying out transparent treatment (soaking with potassium hydroxide solution), and taking this treatment method for camptothecin mycorrhiza will inevitably cause a reaction between camptothecin and strong alkali, resulting in a large loss of camptothecin. and affect the accurate observation of intracellular camptothecin
Pasqua et al reported in "Cellular localization of the anti-cancer drug camptothecin in Camptotheca acuminata Decne (Nyssaceae)." ("Camptotheca in the anti-cancer drug - camptothecin cell localization") in 2004 using camptothecin in the ultraviolet The characteristics of autofluorescence under light can be observed in the cell structure by fluorescence microscopy, but this observation is directly observed without dyeing. Although this method can avoid the loss due to the staining and transparency process during the production process Camptothecin, the distribution of camptothecin in the tissue was observed, but the structure in the cell and the arbuscular structure in the mycorrhizal cells could not be clearly observed

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Select camphoria mycorrhizae, cut into 1 cm long root segments, soak in 10% glycerin, vacuum soak at 25°C and -0.08 MPa for 30 minutes, take them out and place them at 25°C for 3 hours. Transfer to a gelatin embedding agent with a concentration of 8%, that is, dissolve 8 grams of gelatin in 100 ml of a mixed solution of glycerol and water with a volume ratio of 1:1 and embed for 2 minutes. Place the embedded camphor tree root segments in a cryostat, cut longitudinally into slices with a thickness of about 60 microns, and transfer them to glass slides. Use a plastic dropper to draw 0.02% acid fuchsin aqueous solution, that is, 100 ml of a mixed solution of 0.02 g of acid fuchsin dissolved in gelatin, glycerin and water at a volume ratio of 1:1:1, and drop it on the sample slide. 2 drops, after 30 minutes, gently rinse the slides with water until the slices appear light red. The sample slices were observed under a laser confocal microscope, the excitation light wavelengt...

Embodiment 2

[0018] Select the rhizomes of camphoria radiata, cut into 1 cm long root segments, soak them in 15% glycerin, soak them under vacuum at 25°C and -0.08 MPa for 20 minutes, take them out and place them at 25°C for 2 hours. Transfer to a gelatin embedding agent with a concentration of 10%, that is, dissolve 10 grams of gelatin in 100 ml of a mixed solution of glycerol and water at a volume ratio of 1:1 and embed for 1 minute. Place the embedded camphor tree root segments in a cryostat, cut longitudinally into slices with a thickness of about 60 microns, and transfer them to glass slides. Use a plastic dropper to draw 0.05% acid fuchsin aqueous solution, that is, 100 ml of a mixed solution of 0.05 g of acid fuchsin dissolved in gelatin, glycerin and water at a volume ratio of 1:1:1, and drop it on the sample slide. 2 drops, after 20 minutes, gently rinse the slides with water until the slices appear light red. The sample slices were observed under a laser confocal microscope, the...

Embodiment 3

[0020] Select the rhizomes of camphoria radiata, cut into 2 cm long root segments, soak them in 20% glycerin, soak them under vacuum at 25°C and -0.08 MPa for 20 minutes, take them out and place them at 25°C for 2 hours. Transfer to a gelatin embedding agent with a concentration of 12%, that is, dissolve 12 grams of gelatin in 100 ml of a mixed solution of glycerol and water at a volume ratio of 1:1 and embed for 1 minute. Place the embedded camphor tree root segments in a cryostat, cut longitudinally into slices with a thickness of about 90 microns, and transfer them to glass slides. Pipette the configuration with a glue-tipped dropper.

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Abstract

This invention relates to one camptothecine cell structure fluorescence trace method, which uses camptothercine root as materials for vacuum dipping and glue imbedding to process slices, wherein, the acid fuchsine liquid is dyed and observed under laser polymer microscopes on the cluster structure distribution. This invention changes original cluster structure slice process method and dye method to ensure cell cluster structure completeness to lower camptothecine loss degree to get accurate display.

Description

technical field [0001] The invention relates to a method for tracing natural compounds in cell structures, in particular to a method for fluorescently tracing camptothecin in cell structures. Background technique [0002] Mycorrhiza is a common phenomenon of symbiosis between fungi and plants in nature, and the most widely distributed type of mycorrhizal is arbuscular mycorrhizal. Arbuscular mycorrhizae can significantly promote the absorption of water and nutrients by plant roots, enhance plant disease resistance, and thus significantly improve the growth of host plants. [0003] Camptothecin (CPT) is an alkaloid isolated from the unique subtropical tree species in my country—Camptotheca acuminata, which has obvious inhibitory effects on various tumor cells. The content of camptothecin in some camptothecin roots that form arbuscular mycorrhizae is significantly higher than that of camptotheca that does not form arbuscular mycorrhiza. Therefore, the inoculation technology u...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64G01N21/84
Inventor 阎秀峰赵昕谢国恩
Owner NORTHEAST FORESTRY UNIVERSITY