Laser confocal microscrope method for analyzing aristolochic acid nephropathy biomarker

A technique for aristolochic acid and an analysis method, which is applied in the biological technology and medical fields, and can solve the problems of complex processing methods, inability to intuitively reflect the actual distribution state of adducts, and the like.

Inactive Publication Date: 2013-11-13
PEKING UNIV
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  • Abstract
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  • Application Information

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Problems solved by technology

[0004] The technical problem to be solved by the present invention is to provide a laser confocal method for in situ analysis of AA-DNA adducts, to overcome the complexity of the sample pretreatment methods in the prior art and the inability to directly reflect the adducts in vivo and in cells. Disadvantages of the actual distribution state

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  • Laser confocal microscrope method for analyzing aristolochic acid nephropathy biomarker
  • Laser confocal microscrope method for analyzing aristolochic acid nephropathy biomarker
  • Laser confocal microscrope method for analyzing aristolochic acid nephropathy biomarker

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Embodiment 1

[0031] Take the renal tubular epithelial cells cultured in vitro, culture them with DMEM medium containing no more than 0.5% fetal bovine serum, inoculate the cells into a 35mm glass-bottom dish, and when they grow to 80% confluence, use 0.5 μg / ml The culture medium of AA-I was stimulated for 24h. AA-DNA adducts can be detected in the nucleus using confocal laser microscopy using an excitation wavelength of 300 nm and an emission wavelength of 580 nm. The fluorescence intensity of the adduct was significantly stronger than the autofluorescence intensity in HK-2 cells of the blank control group ( image 3 ), the same below.

Embodiment 2

[0033] HK-2 cells cultured in vitro were stimulated with AA-I at a concentration of 1 μg / ml for 24 hours, and AA-DNA adducts could be detected in the nucleus by laser confocal microscopy. The excitation wavelength is 310nm and the emission wavelength is 550nm.

Embodiment 3

[0035] HK-2 cells cultured in vitro were stimulated with AA-I at a concentration of 5 μg / ml for 6 hours, and AA-DNA adducts could be detected in the nucleus by laser confocal microscopy. The excitation wavelength is 340nm and the emission wavelength is 520nm.

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Abstract

The invention relates to a laser confocal microscrope method for analyzing an aristolochic acid nephropathy biomarker. The method is a rapid and sensitive in-situ analysis method for qualitatively and semi-quantitatively analyzing the aristolochic acid-DNA adduct in a biological sample without complex sample pretreatment, and the biological sample comprises stimulated aristolochic acid compound in-vitro cultured cells, experiment animal histocytes giving medicines containing the aristolochic acid components, cast-off cells in urine, and histocytes of the aristolochic acid nephrosis patient. The method can be used for the rapid clinic diagnosis and relevant researches of the aristolochic acid nephrosis.

Description

[technical field] [0001] The invention belongs to the field of biotechnology and medicine, and specifically relates to a laser confocal microscopy method for analyzing biomarkers of aristolochic acid nephropathy, which can be used for rapid clinical diagnosis and related research work of aristolochic acid nephropathy. [technical background] [0002] Aristolochic acid nephropathy (AAN) caused by Chinese herbal medicines containing aristolochic acid (AA) has attracted great attention at home and abroad. The main features of AAN are chronic renal failure, renal interstitial fibrosis, and concomitant urothelial neoplasia [1] , the early symptoms are not obvious, and more importantly, there is no clear index (golden index) for the diagnosis of AAN in clinical practice. Although Guan Mutong and other traditional Chinese medicines containing a large amount of aristolochic acid have been banned in my country and many countries, there are still some medicines (such as cinnabar lotus...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
Inventor 王璇高志李晓玫蔡少青袁兰
Owner PEKING UNIV
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